DNA SEQUENCINGMETHODS COLLECTION
An overview of recent DNA-seq publications featuring Illumina® technology

TABLE OF CONTENTS
06

Introduction

07

Sequence Rearrangements

08

RAD and PE RAD-Seq: Restriction-Site Associated DNA Sequencing

11

ddRADseq: Double Digest Restriction-Site Associated DNA Marker Generation

13

2b-RAD: RAD With Type IIB Restriction Endonucleases

14

SLAF-seq: Specific Locus Amplified Fragment Sequencing

16

hyRAD: Hybridization RAD for Degraded DNA

17

Rapture: Restriction-Site Associated DNA Capture

18

Digenome-seq: Cas9-Digested Whole-Genome Sequencing

19

CAP-seq: CXXC Affinity Purification Sequencing

20

CPT-seq: Contiguity-Preserving Transposition Sequencing

21

RC-Seq: Retrotransposon Capture Sequencing

22

Tn-Seq: Transposon Sequencing INSeq: Insertion Sequencing

24

TC-Seq: Translocation-Capture Sequencing

25	Rep-Seq: Repertoire Sequencing Ig-seq: DNA Sequencing of Immunoglobulin Genes MAF: Molecular
Amplification fingerprinting
26

EC-seq: Excision Circle Sequencing

27

Bubble-Seq: Libraries of Restriction Fragments that Contain Replication Initiation Sites (Bubbles)28

NSCR: Nascent Strand Capture and Release29

Repli-Seq: Nascent DNA Replication Strand Sequencing

30

NS-Seq: Nascent Strand Sequencing

31

DNA break mapping

32

Map DNA Single-Strand Breaks (SSB-Seq)

33

BLESS: Breaks labeling and Enrichment on Streptavidin and Sequencing

34

DSB-Seq: Map DNA Double-Strand Breaks

35

Break-seq: Double-Stranded Break Labeling

36

GUIDE-seq: Genome-Wide, Unbiased Identification of DSBs Enabled by Sequencing

38

HTGTS: High-Throughput Genome-Wide Translocation Sequencing

39

LAM-HTGTS: Linear Amplification-Mediated High-Throughput Genome-Wide Sequencing

40

Low-Level DNA Detection

41

smMIP: Single-Molecule Molecular Inversion Probes

43

MIPSTR: Targeted Capture of STR Loci by smMIPs

44

MDA: Multiple Displacement Amplification

46	MIDAS: Microwell displacement amplification system IMS-MDA: Immunomagnetic Separation for Targeted
Bacterial Enrichment for MDA ddMDA: Digital Droplet MDA

2

DNA Sequencing Methods Collection

48

MALBAC: Multiple Annealing and Looping–Based Amplification Cycles

50

nuc-seq: Single G2/M Nucleus Sequencing of Cells in S Phase SNES: Single Nucleus Exome Sequencing

52

OS-Seq: Oligonucleotide-Selective Sequencing

53

Safe-SeqS: Safe-Sequencing System is a Unique Molecular Identifier (UMI) Approach to Detect Rare Variants

55

Duplex-Seq: Duplex Sequencing

57

DR-Seq: DNA-mRNA Sequencing

58

G&T-Seq: Genome and Transcriptome Sequencing

59

scM&T-Seq: Single-Cell Methylome and Transcriptome Sequencing

60

scTrio-seq: Single-Cell Triple Omics Sequencing

61

scBS-Seq: Single-Cell Bisulfite Sequencing

62

scAba-seq: Detect 5hmC Marks in Single Cells With AbaSI Nuclease

63

Single-cell RC-Seq: Single-Cell Retrotransposon Capture Sequencing

64

scATAC-Seq (Cell index variation): Single-Cell Assay for Transposase-Accessible Chromatin

65

scATAC-Seq (Microfluidics variation): Single-Cell Assay for Transposase-Accessible Chromatin

66

Drop-ChIP or scChIP-seq: Droplet-Based Single-Cell Chromatin Immunoprecipitation Sequencing

67

SMDB: Single-Molecule Droplet Barcoding

68

Epigenetics

70

BS-Seq, Bisulfite-seq, and WGBS: Whole-Genome Bisulfite Sequencing (WGBS)72

PBAT: Post-bisulfite Adapter Tagging

73

BSPP: Bisulfite Sequencing With Padlock Probes

74

RRBS-Seq: Reduced-Representation Bisulfite Sequencing

76

BSAS: Bisulfite Amplicon Sequencing

77

MRE-Seq and Methyl-Seq: Methylation-Sensitive Restriction Enzyme Sequencing

78	EpiRADseq: Double-Digest Restriction Site–Associated DNA Marker Generation with a Methylation-Sensitive
Restriction Enzyme
79

T-WGBS: Tagmentation-Based Whole-Genome Bisulfite Sequencing

80

JBP1-seq: J-Binding Protein 1 Sequencing

81

Aba-seq: AbaSI Coupled with Sequencing

82

TAmC-Seq: Tet-Assisted 5-Methylcytosine Sequencing

83

fC-Seal: A 5-Formylcytosine-Selective Chemical Labeling84

fC-CET: 5fC Chemical Labeling and C-to-T Transition During PCR

85

CAB-Seq: Chemical Modification–Assisted Bisulfite Sequencing

86

oxBS-Seq: Oxidative Bisulfite Sequencing

88	redBS-Seq: Reduced Bisulfite Sequencing caMAB-seq: 5-Carboxylcytosine Methylase-Assisted
Bisulfite Sequencing
89

fCAB-seq: 5-Formylcytosine Chemical Modification–Assisted Bisulfite Sequencing

90

MAB-seq: M.SssI Methylase-Assisted Bisulfite Sequencing

91

RRMAB-seq: Reduced-Representation M.SssI Methylase-Assisted Bisulfite Sequencing

92

TAB-Seq: Tet-Assisted Bisulfite Sequencing

94

MIRA: Methylated-CpG Island Recovery Assay (MIRA)

An overview of recent DNA-seq publications featuring Illumina technology395	MeDIP-Seq: Methylated DNA Immunoprecipitation DIP-seq: DNA Immunoprecipitation Followed by HighThroughput Sequencing
97

hMeDIP-seq: Hydroxymethylated DNA Immunoprecipitation and Sequencing

99	MBDCap-seq: Methyl-CpG Binding Domain (MBD)–Based Capture and Sequencing MethylCap-Seq:
Capture of Methylated DNA Using the Methyl-CpG Binding Domain MBD domain of MeCP2 MiGS: MethylCpG Binding Domain–Isolated Genome Sequencing
101	BisChIP-seq: Bisulfite-Treated Chromatin-Immunoprecipitated DNA ChIP-BS-seq: ChIP of Bisulfite-treatedchromatin-Bisulfite Sequencing ChIP-BMS: Chromatin Immunoprecipitation with Bisulfite Methylation
Sequencing Assay

103 DNA-Protein Interactions
104	ChIP-Seq: Chromatin Immunoprecipitation Sequencing HT-ChIP: High-Throughput ChIP ChIP-exo:
Exonuclease trimmed ChIP Mint-ChIP: Multiplexed ChIP
106

DNaseI Seq or DNase-Seq: DNase l Hypersensitive Sites Sequencing

108

DNase I SIM: DNase I Simplified in-Nucleus Method for plants

109	MNase-Seq: Micrococcal Nuclease Sequencing MAINE-Seq: Micrococcal Nuclease–Assisted Isolation of
Nucleosomes Nucleo-Seq: Isolated Nucleosome Sequencing Nuc-seq: Isolated Nucleosome Sequencing
111

FiT-Seq: Fixed-Tissue Chromatin Immunoprecipitation Sequencing

112

PAT-ChIP: Pathology Tissue Chromatin Immunoprecipitation

113

X-ChIP-seq: High-Resolution Crosslinking Chromatin Immunoprecipitation Sequencing

114

ORGANIC: Occupied Regions of Genomes from Affinity-Purified Naturally Isolated Chromatin

115	ATAC-Seq: Assay for Transposase-Accessible Chromatin Sequencing Fast-ATAC: ATAC-seq Optimized forBlood Cells
118

THS-seq: Transposome Hypersensitive Sites Sequencing

119

CATCH‑IT: Covalent Attachment of Tags to Capture Histones and Identify Turnover120

MINCE-seq: Mapping in Vivo Nascent Chromatin with EdU

121	FAIRE-seq: Formaldehyde-Assisted Isolation of Regulatory Elements Sono-Seq: Sonication of
Crosslinked Chromatin
123

NOMe-Seq: Nucleosome Occupancy Methylome-Sequencing

124

ChIPmentation: Chromatin Immunoprecipitation with Sequencing Library Preparation by Tn5 Transposase125

ChIA-PET: Chromatin Interaction Analysis by Paired-End Tag Sequencing

127

3-C, Capture-C and Hi-C: Chromatin Conformation Capture Sequencing

129

NG Capture-C: Next-Generation Capture-C

130

4C-seq: Circular Chromatin Conformation Capture132

UMI-4C: Circular Chromosome Conformation Capture with Unique Molecular Identifiers

133

5C: Chromatin Conformation Capture Carbon Copy

134

Pu-seq: Polymerase Usage Sequencing

135

PB–seq: Protein/DNA Binding Followed by High-Throughput Sequencing

136	SELEX or SELEX-seq: Systematic Evolution of Ligands by Exponential Enrichment HT-SELEX: HighThroughput Systematic Evolution of Ligands by Exponential Enrichment
1384

HiTS-FLIP: High-Throughput Sequencing With Fluorescent Ligand Interaction Profiling

DNA Sequencing Methods Collection

139

DamID: DNA Adenine Methyltransferase Interaction Detection141

MPE-seq: Methidiumpropyl Ethylenediaminetetraacetic Acid (EDTA) Sequencing

142

Chem-seq: Identify Sites Bound by Small Chemical Molecules

143 Protein-Protein Interaction
144

PD-Seq: Candidate Cellular Protein Target Identification

145

ProP-PD: Proteomic Peptide-Phage Display PDZ-Seq: PDZ Domains Sequencing

An overview of recent DNA-seq publications featuring Illumina technology

5INTRODUCTION
This publication is a collection of next-generation sequencing (NGS) methods for DNA sequencing, compiled from the scientific
literature. It is both a tribute to the creativity of the users and the versatility of the technology. We hope it will inspire researchers to
use these methods or to develop new ones to address new scientific challenges.
A method refers to the processing steps between extracting the nucleic acids (sample preparation) and the addition ofoligonucleotide adapters for sequencing (library preparation). With a few extra processing steps, a wide range of scientificquestions can be addressed by this technology.
When using this document, consider the following points:• New methods are continually being developed. For the most up-to-date list of methods, visit: www.illumina.com/science/
sequencing-method-explorer.html• Only the most recent back references are provided, typically for the past 2 years.
• With few exceptions the capitalization, dashes and special characters in the method name are exactly the same as in theoriginal, referenced paper. This is important, because methods such as CapSeq1 and CAP-seq2 are quite different and refer to
RNA and DNA methods, respectively.
• The methods are arranged according to their similarity, so the most similar methods should be adjacent. To visually compare
the methods, refer to http://www.illumina.com/content/dam/illumina-marketing/documents/applications/ngs-library-prep/
ForAllYouSeqMethods.pdf• When methods are essentially identical, and can be represented by a single diagram, they are grouped together, such as GROseq3 and BRIC-Seq.4• The diagrams are stylized depictions of the methods. They may not include every detail of the method.
• These methods were developed by users, so readers should refer to the original publications for detailed descriptions andprotocols.
Have we missed anything? Contact us if you are aware of a protocol that should be listed.

6

1.

Gu W., Lee H. C., Chaves D., et al. CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C. elegans piRNA precursors. Cell.
2012;151:1488-1500.

2.

Illingworth R. S., Botting C. H., Grimes G. R., Bickmore W. A. and Eskeland R. PRC1 and PRC2 are not required for targeting of H2A.Z to developmental genes
in embryonic stem cells. PLoS One. 2012;7:e34848.

3.

Core L. J., Waterfall J. J. and Lis J. T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science.
2008;322:1845-1848.

4.

Tani H., Mizutani R., Salam K. A., et al. Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals.
Genome Res. 2012;22:947-956.

DNA Sequencing Methods CollectionSEQUENCE REARRANGEMENTS
A growing body of evidence suggests that somatic genomic rearrangements, such as retrotransposition and copy-number variants(CNVs), are relatively common in healthy individuals.5,6,7 Cancer genomes also contain numerous complex rearrangements.8 While
many of these rearrangements can be detected during routine NGS, specific methods are available to study rearrangements suchas transposable elements.
Transposable genetic elements (TEs) comprise a vast array of DNA sequences with the ability to move to new sites in genomes,
either directly by a cut-and-paste mechanism (transposons) or indirectly through an RNA intermediate (retrotransposons).9
TEs make up approximately 66–69% of the human genome10 and play roles in ageing, cancers, brain function, development,
embryogenesis, and phenotypic variation in populations and evolution.11 Along with sequence rearrangements by TEs,
chromosome and centromere rearrangements can lead to multiple diseases and disorders.12

Genomic rearrangements in somatic cells can lead to the metastasis of cancerous cells.

5.

O’Huallachain M., Weissman S. M. and Snyder M. P. The variable somatic genome. Cell Cycle. 2013;12:5-6.

6.

Macosko E. Z. and McCarroll S. A. Genetics. Our fallen genomes. Science. 2013;342:564-565.

7.

McConnell M. J., Lindberg M. R., Brennand K. J., et al. Mosaic copy number variation in human neurons. Science. 2013;342:632-637.

8.

Malhotra A., Lindberg M., Faust G. G., et al. Breakpoint profiling of 64 cancer genomes reveals numerous complex rearrangements spawned by homologyindependent mechanisms. Genome Res. 2013;23:762-776.

9.

Fedoroff N. V. Presidential address. Transposable elements, epigenetics, and genome evolution. Science. 2012;338:758-767.

10.

Grandi F. C. and An W. Non-LTR retrotransposons and microsatellites: Partners in genomic variation. Mob Genet Elements. 2013;3:e25674.

11.

Gschwend A. R., Weingartner L. A., Moore R. C. and Ming R. The sex-specific region of sex chromosomes in animals and plants. Chromosome Res.
2012;20:57-69.

12.

Chiarle R. Translocations in normal B cells and cancers: insights from new technical approaches. Adv Immunol. 2013;117:39-71.

An overview of recent DNA-seq publications featuring Illumina technology

7RAD and PE RAD-Seq: Restriction-Site Associated DNA Sequencing
RAD-seq is a protocol for genotyping and discovery of single-nucleotide polymorphisms (SNPs).13 This approach is particularly
useful for genotyping when a reference genome is not available, such as in ecological studies.14 PE RAD-seq, also called RAD-PE,
is the same protocol as RAD but uses paired-end sequencing for improved alignments.15 Several variations, such as ddRADseq,162b-RAD,17 SLAF-seq,18 and hyRAD19 have been developed to address specific applications, and multiple software packages areavailable to analyze RAD data.20,21
In this method, genomic DNA (gDNA) is first digested with a restriction enzyme and a barcoded P1 adapter is ligated to thefragments. The adapter-ligated fragments from different samples are combined, if samples are multiplexed, and the DNA issheared. The fragments are size-selected and purified. The P2 adapter-primers are ligated and the fragments are amplified to
produce the sequencing library.
Restriction sites

Restriction
digestion

Add barcoded adapters

Shear andsize selectAdd P2
adapter

AmplifyDNA

A schematic overview of RAD-seq.

8

Advantages

Disadvantages• No reference genome required 22• Relatively inexpensive, compared to whole-genome sequencing• The degree of genome coverage can be adjusted by selecting
various restriction enzymes and fragment sizes• There can be gaps in the genome coverage• Requires high-quality DNA (see hyRAD23 for low-quality DNA)• Sequence polymorphism at the DNA restriction sites causes
a progressive loss of shared restriction sites among
diverging clades 24

13.

Baird N. A., Etter P. D., Atwood T. S., et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One. 2008;3:e3376.

14.

Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics.
Nat Rev Genet. 2016;17:81-92.

15.

Willing E. M., Hoffmann M., Klein J. D., Weigel D. and Dreyer C. Paired-end RAD-seq for de novo assembly and marker design without available reference.
Bioinformatics. 2011;27:2187-2193.

16.

Peterson B. K., Weber J. N., Kay E. H., Fisher H. S. and Hoekstra H. E. Double digest RADseq: an inexpensive method for de novo SNP discovery andgenotyping in model and non-model species. PLoS One. 2012;7:e37135.

17.

Wang S., Meyer E., McKay J. K. and Matz M. V. 2b-RAD: a simple and flexible method for genome-wide genotyping. Nat Methods. 2012;9:808-810.

18.

Sun X., Liu D., Zhang X., et al. SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing.
PLoS One. 2013;8:e58700.

19.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

20.

Fan W., Zong J., Luo Z., et al. Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in RiceMarker-Assisted Breeding. PLoS One. 2016;11:e0147187.

21.

Catchen J., Hohenlohe P. A., Bassham S., Amores A. and Cresko W. A. Stacks: an analysis tool set for population genomics. Mol Ecol. 2013;22:3124-3140.

22.

Reitzel A. M., Herrera S., Layden M. J., Martindale M. Q. and Shank T. M. Going where traditional markers have not gone before: utility of and promise for RAD
sequencing in marine invertebrate phylogeography and population genomics. Mol Ecol. 2013;22:2953-2970.

23.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

24.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

DNA Sequencing Methods Collection

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet.
2016;17:81-92.
da Fonseca R. R., Albrechtsen A., Themudo G. E., et al. Next-generation biology: Sequencing and data analysis approaches for non-model organisms. Mar Genomics.
2016;30:3-13.
Kagale S., Koh C., Clarke W. E., et al. Analysis of Genotyping-by-Sequencing (GBS) Data. Methods Mol Biol. 2016;1374:269-284.
Kim C., Guo H., Kong W., et al. Application of genotyping by sequencing technology to a variety of crop breeding programs. Plant Sci. 2016;242:14-22.
Manel S., Perrier C., Pratlong M., et al. Genomic resources and their influence on the detection of the signal of positive selection in genome scans. Mol Ecol.
2016;25:170-184.
Sanders I. R. and Rodriguez A. Aligning molecular studies of mycorrhizal fungal diversity with ecologically important levels of diversity in ecosystems. ISME J.
2016;10:2780-2786.

References
Ren P., Peng W., You W., et al. Genetic mapping and quantitative trait loci analysis of growth-related traits in the small abalone Haliotis diversicolor using
restriction-site-associated DNA sequencing. Aquaculture. 2016;454:163-170.
The authors used RAD-seq to construct a high-resolution linkage map of the abalone Haliotis diversicolor for growth-related quantitative trait locus (QTL) analysis.
They were able to build a reduced-representation library with 3756 loci in more than 95% of the offspring. Based on this map, they identified 15 QTLs for 6 growthrelated traits.
Illumina Technology: HiSeq 2000 System

Wang J., Xue D. X., Zhang B. D., et al. Genome-Wide SNP Discovery, Genotyping and Their Preliminary Applications for Population Genetic Inference inSpotted Sea Bass (Lateolabrax maculatus). PLoS One. 2016;11:e0157809.
The researchers used PE RAD-seq on 30 individuals from 2 populations to discover 22,648 SNPs across the genome of L. maculatus. The results showed shallow, but
significant, genetic differentiation between the 2 populations.
Illumina Technology: HiSeq 2500 System

He T., D’Agui H., Lim S. L., Enright N. J. and Luo Y. Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime insouthwestern Australia, a global biodiversity hotspot. Sci Rep. 2016;6:26315.
This study applied RAD-seq and environmental association analysis to 80 plants and found candidate genes associated with rainfall gradients, temperatures, and fireintervals. The authors discovered that overall population adaptive genetic variation was affected significantly by shortened fire intervals, whereas declining rainfall andrising temperature did not have a detectable influence. Gene annotation further revealed 4 genes with functions in stress tolerance, the regulation of stomatal opening
and closure, energy use, and morphogenesis with adaptation to climate and fire intervals.
Illumina Technology: HiSeq 2000 System

Paun O., Turner B., Trucchi E., et al. Processes Driving the Adaptive Radiation of a Tropical Tree (Diospyros, Ebenaceae) in New Caledonia, a Biodiversity
Hotspot. Syst Biol. 2016;65:212-227.
The authors used RAD-seq to resolve phylogenetic relationships among 21 diploid Diospyros species that radiated recently in New Caledonia. The dataset contained
84 individuals from 39 populations. The 8400 filtered SNPs generally confirmed species delimitations and produced a well-supported phylogenetic tree.
Illumina Technology: HiSeq 2000

Bian C., Hu Y., Ravi V., et al. The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts. Sci Rep.
2016;6:24501.
Clark L. V. and Sacks E. J. TagDigger: user-friendly extraction of read counts from GBS and RAD-seq data. Source Code Biol Med. 2016;11:11, Hou Y., Nowak M. D.,
Mirre V., et al. RAD-seq data point to a northern origin of the arctic-alpine genus Cassiope (Ericaceae). Mol Phylogenet Evol. 2015;95:152-160.
Diaz-Arce N., Arrizabalaga H., Murua H., Irigoien X. and Rodriguez-Ezpeleta N. RAD-seq derived genome-wide nuclear markers resolve the phylogeny of tunas.
Mol Phylogenet Evol. 2016;102:202-207.
Diaz-Arce N., Arrizabalaga H., Murua H., Irigoien X. and Rodriguez-Ezpeleta N. RAD-seq derived genome-wide nuclear markers resolve the phylogeny of tunas.
Mol Phylogenet Evol. 2016;102:202-207.
Fan W., Zong J., Luo Z., Chen M., Zhao X., et al. Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Applicationin Rice Marker-Assisted Breeding. PLoS One. 2016;11:e0147187.
Fernandez R., Schubert M., Vargas-Velazquez A. M., et al. A genomewide catalogue of single nucleotide polymorphisms in white-beaked and Atlantic white-sided
dolphins. Mol Ecol Resour. 2016;16:266-276.

An overview of recent DNA-seq publications featuring Illumina technology

9Herrera S. and Shank T. M. RAD sequencing enables unprecedented phylogenetic resolution and objective species delimitation in recalcitrant divergent taxa.
Mol Phylogenet Evol. 2016;100:70-79.
Jiang N., Zhang F., Wu J., et al. A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large plant
populations. Theor Appl Genet. 2016;129:1739-1757.
Manthey J. D. and Robbins M. B. Genomic insights into hybridization in a localized region of sympatry between pewee sister species (Contopus sordidulus × C. virens)
and their chromosomal patterns of differentiation. Avian Research. 2016;7:.
Qiu S., Bergero R., Guirao-Rico S., et al. RAD mapping reveals an evolving, polymorphic and fuzzy boundary of a plant pseudoautosomal region. Mol Ecol.
2016;25:414-430.
Ravinet M., Westram A., Johannesson K., et al. Shared and nonshared genomic divergence in parallel ecotypes of Littorina saxatilis at a local scale. Mol Ecol.
2016;25:287-305.
Takahashi T. and Sota T. A robust phylogeny among major lineages of the East African cichlids. Mol Phylogenet Evol. 2016;100:234-242.
Yang H., Wei C. L., Liu H. W., et al. Genetic Divergence between Camellia sinensis and Its Wild Relatives Revealed via Genome-Wide SNPs from RAD Sequencing.
PLoS One. 2016;11:e0151424.
Yang J., Guo B., Shikano T., Liu X. and Merila J. Quantitative trait locus analysis of body shape divergence in nine-spined sticklebacks based on high-density SNPpanel. Sci Rep. 2016;6:26632.
Zhao Y., Zhang C., Chen H., et al. QTL mapping for bacterial wilt resistance in peanut (Arachis hypogaea L.). Mol Breed. 2016;36:13.
Combosch D. J. and Vollmer S. V. Trans-Pacific RAD-Seq population genomics confirms introgressive hybridization in Eastern Pacific Pocillopora corals. Mol Phylogenet
Evol. 2015;.
DaCosta J. M. and Sorenson M. D. ddRAD-seq phylogenetics based on nucleotide, indel, and presence-absence polymorphisms: Analyses of two avian genera with
contrasting histories. Mol Phylogenet Evol. 2015;.
Demos T. C., Kerbis Peterhans J. C., Joseph T. A., et al. Comparative Population Genomics of African Montane Forest Mammals Support Population Persistence
across a Climatic Gradient and Quaternary Climatic Cycles. PLoS One. 2015;10:e0131800.
Guo B., DeFaveri J., Sotelo G., Nair A. and Merila J. Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks. BMC Biol.
2015;13:19.
Liu S., Clark L. V., Swaminathan K., et al. High-density genetic map ofMiscanthus sinensisreveals inheritance of zebra stripe. GCB Bioenergy. 2016;8:616-630.
Longo G. and Bernardi G. The evolutionary history of the embiotocid surfperch radiation based on genome-wide RAD sequence data. Mol Phylogenet Evol.
2015;88:55-63.
Marubodee R., Ogiso-Tanaka E., Isemura T., et al. Construction of an SSR and RAD-Marker Based Molecular Linkage Map of Vigna vexillata (L.) A. Rich. PLoS One.
2015;10:e0138942.
Mathers T. C., Hammond R. L., Jenner R. A., et al. Transition in sexual system and sex chromosome evolution in the tadpole shrimp Triops cancriformis. Heredity(Edinb). 2015;.
Shao C., Niu Y., Rastas P., et al. Genome-wide SNP identification for the construction of a high-resolution genetic map of Japanese flounder (Paralichthys olivaceus):
applications to QTL mapping of Vibrio anguillarum disease resistance and comparative genomic analysis. DNA Res. 2015;.
Takahashi T. and Moreno E. A RAD-based phylogenetics for Orestias fishes from Lake Titicaca. Mol Phylogenet Evol. 2015;.
Tennessen J. A., Bonner K. M., Bollmann S. R., et al. Genome-Wide Scan and Test of Candidate Genes in the Snail Biomphalaria glabrata Reveal New Locus
Influencing Resistance to Schistosoma mansoni. PLoS Negl Trop Dis. 2015;9:e0004077.
Yu S., Chu W., Zhang L., et al. Identification of Laying-Related SNP Markers in Geese Using RAD Sequencing. PLoS One. 2015;10:e0131572.
Zhou Z., Liu S., Dong Y., et al. High-Density Genetic Mapping with Interspecific Hybrids of Two Sea Urchins, Strongylocentrotus nudus and S. intermedius, by RAD
Sequencing. PLoS One. 2015;10:e0138585.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

10

DNA Sequencing Methods Collection

ddRADseq: Double Digest Restriction-Site Associated DNA Marker Generation
ddRADseq,25 also called ddRAD, is a variation on the RAD sequencing protocol,26 which is used for SNP discovery andgenotyping.27,28 In this variation, the fragment shearing is replaced with a second restriction digestion to improve the tunability
and accuracy of the size-selection step. The protocol also includes a second index to allow combinatorial indexing. Several RAD
variations, such as 2b-RAD,29 SLAF-seq,30 and hyRAD,31 have been developed to address specific applications, and multiple
software packages are available to analyze RAD data.32,33
In this method, gDNA is first digested with a restriction enzyme, and a barcoded P1 adapter is ligated to the fragments.
The adapter-ligated fragments from different samples are combined, if samples are multiplexed, and the DNA is digested by asecond restriction enzyme. The fragments are size-selected and purified. The P2 adapter-primers are ligated, and the fragments
are amplified to produce the sequencing library.
Restriction sites

Restriction
digestion

Add barcoded adapters

Add P2
adapter

Second restriction
digestAmplifyDNA

A schematic overview of ddRADseq.

Advantages

Disadvantages• No reference genome required 34• Relatively inexpensive, compared to whole-genome sequencing• The degree of genome coverage can be adjusted by selecting
various restriction enzymes••

There can be gaps in the genome coverage
Requires high-quality DNA (see hyRAD35 for low-quality DNA)

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet.
2016;17:81-92.
Kagale S., Koh C., Clarke W. E., Bollina V., Parkin I. A., et al. Analysis of Genotyping-by-Sequencing (GBS) Data. Methods Mol Biol. 2016;1374:269-284.

25.

Peterson B. K., Weber J. N., Kay E. H., Fisher H. S. and Hoekstra H. E. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping inmodel and non-model species. PLoS One. 2012;7:e37135.

26.

Baird N. A., Etter P. D., Atwood T. S., et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One. 2008;3:e3376.

27.

Baird N. A., Etter P. D., Atwood T. S., et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One. 2008;3:e3376.

28.

Willing E. M., Hoffmann M., Klein J. D., Weigel D. and Dreyer C. Paired-end RAD-seq for de novo assembly and marker design without available reference.
Bioinformatics. 2011;27:2187-2193.

29.

Wang S., Meyer E., McKay J. K. and Matz M. V. 2b-RAD: a simple and flexible method for genome-wide genotyping. Nat Methods. 2012;9:808-810.

30.

Sun X., Liu D., Zhang X., et al. SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing.
PLoS One. 2013;8:e58700.

31.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

32.

Fan W., Zong J., Luo Z., et al. Development of a RAD-Seq Based DNA Polymorphism Identification Software, AgroMarker Finder, and Its Application in RiceMarker-Assisted Breeding. PLoS One. 2016;11:e0147187.

33.

Catchen J., Hohenlohe P. A., Bassham S., Amores A. and Cresko W. A. Stacks: an analysis tool set for population genomics. Mol Ecol. 2013;22:3124-3140.

34.

Reitzel A. M., Herrera S., Layden M. J., Martindale M. Q. and Shank T. M. Going where traditional markers have not gone before: utility of and promise for RAD
sequencing in marine invertebrate phylogeography and population genomics. Mol Ecol. 2013;22:2953-2970.

35.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

An overview of recent DNA-seq publications featuring Illumina technology

11References
DaCosta J. M. and Sorenson M. D. ddRAD-seq phylogenetics based on nucleotide, indel, and presence-absence polymorphisms: Analyses of two avian
genera with contrasting histories. Mol Phylogenet Evol. 2016;94:122-135.
This study used ddRADseq for phylogenetic analyses of the Lagonosticta firefinches (family Estrildidae) and the Vidua brood parasitic finches (family Viduidae). The
researchers obtained > 1000 homologous loci despite ~20 million years divergence. In addition to nucleotide polymorphisms, the ddRADseq data yielded large sets ofindel and locus presence–absence polymorphisms, all of which had higher consistency indices than mitochondrial DNA (mtDNA) sequence data.
Illumina Technology: HiSeq 2000 System

Lal M. M., Southgate P. C., Jerry D. R. and Zenger K. R. Fishing for divergence in a sea of connectivity: The utility of ddRADseq genotyping in a marine
invertebrate, the black-lip pearl oyster Pinctada margaritifera. Mar Genomics. 2016;25:57-68.
The authors applied ddRADseq to Pinctada margaritifera and detected 5243 high-quality, genome-wide SNP markers. They were able to assess population structure,
genome diversity, and perform association testing in 156 individuals belonging to 3 wild populations and 1 hatchery-produced population from the Fiji Islands. They also
found shallow, but significant, population structure among the wild populations.
Illumina Technology: HiSeq 2000 System

Brown J. K., Taggart J. B., Bekaert M., et al. Mapping the sex determination locus in the hapuku (Polyprion oxygeneios) using ddRAD sequencing. BMC Genomics.
2016;17:448.
Clark L. V. and Sacks E. J. TagDigger: user-friendly extraction of read counts from GBS and RAD-seq data. Source Code Biol Med. 2016;11:11.
Hou Y., Nowak M. D., Mirre V., et al. RAD-seq data point to a northern origin of the arctic-alpine genus Cassiope (Ericaceae). Mol Phylogenet Evol. 2016;95:152-160.
Shirasawa K., Hirakawa H. and Isobe S. Analytical workflow of double-digest restriction site-associated DNA sequencing based on empirical and in silico optimization in
tomato. DNA Res. 2016;23:145-153.
Wu Z., Wang B., Chen X., et al. Evaluation of Linkage Disequilibrium Pattern and Association Study on Seed Oil Content in Brassica napus Using ddRAD Sequencing.
PLoS One. 2016;11:e0146383.
Yang G. Q., Chen Y. M., Wang J. P., et al. Development of a universal and simplified ddRAD library preparation approach for SNP discovery and genotyping inangiosperm plants. Plant Methods. 2016;12:39.
Cai G., Yang Q., Yi B., et al. A bi-filtering method for processing single nucleotide polymorphism array data improves the quality of genetic map and accuracy ofquantitative trait locus mapping in doubled haploid populations of polyploid Brassica napus. BMC Genomics. 2015;16:409.
Davik J., Sargent D. J., Brurberg M. B., et al. A ddRAD Based Linkage Map of the Cultivated Strawberry, Fragaria xananassa. PLoS One. 2015;10:e0137746.
Leache A. D., Banbury B. L., Felsenstein J., de Oca A. N. and Stamatakis A. Short Tree, Long Tree, Right Tree, Wrong Tree: New Acquisition Bias Corrections forInferring SNP Phylogenies. Syst Biol. 2015;64:1032-1047.
Leache A. D., Chavez A. S., Jones L. N., et al. Phylogenomics of phrynosomatid lizards: conflicting signals from sequence capture versus restriction site associated
DNA sequencing. Genome Biol Evol. 2015;7:706-719.
Meik J. M., Streicher J. W., Lawing A. M., Flores-Villela O. and Fujita M. K. Limitations of climatic data for inferring species boundaries: insights from speckled
rattlesnakes. PLoS One. 2015;10:e0131435.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

12

DNA Sequencing Methods Collection

2b-RAD: RAD With Type IIB Restriction Endonucleases
2b-RAD is similar to ddRadseq but uses type IIB restriction enzymes (BsaXI or AlfI), which will cleave upstream and downstream ofa recognition site. This shears the target genome into a large number of DNA fragments with a constant length of 33 bp (BsaXI) or
36 bp (AlfI). These short DNA fragments can be sequenced to determine genetic variants.
In this method, gDNA is first digested with a restriction enzyme (BsaXI), and adapters with partial (NNN) overhangs are ligated to
the fragments. The adapter-ligated fragments from different samples are combined, and the fragments are amplified to produce
the sequencing library.
BsaXI restriction sites

Restriction digestion with type IIB
restriction endonucleases eg, BsaXI

Amplify

Add restrictionsite-specific adapters

Add sample-specificadapters

AmplifyDNA

A schematic overview of 2b-RAD.

Advantages

Disadvantages

•

•
•

•
•

Highly reduced 2b-RAD libraries require much less sequencing for
accurate genotyping
High density of markers
No interim purification steps, reducing losses and processing time

Requires a reference genome
Short tags may not be long enough for efficient locus discriminationin complex genomes

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;.
da Fonseca R. R., Albrechtsen A., Themudo G. E., Ramos-Madrigal J., Sibbesen J. A., et al. Next-generation biology: Sequencing and data analysis approaches fornon-model organisms. Mar Genomics. 2016;30:3-13.
Jiang N., Zhang F., Wu J., Chen Y., Hu X., et al. A highly robust and optimized sequence-based approach for genetic polymorphism discovery and genotyping in large
plant populations. Theor Appl Genet. 2016;129:1739-1757.
Kagale S., Koh C., Clarke W. E., Bollina V., Parkin I. A., et al. Analysis of Genotyping-by-Sequencing (GBS) Data. Methods Mol Biol. 2016;1374:269-284.
Manel S., Perrier C., Pratlong M., Abi-Rached L., Paganini J., et al. Genomic resources and their influence on the detection of the signal of positive selection in genome
scans. Mol Ecol. 2016;25:170-184.

References
Pecoraro C., Babbucci M., Villamor A., et al. Methodological assessment of 2b-RAD genotyping technique for population structure inferences in yellowfin
tuna (Thunnus albacares). Mar Genomics. 2016;25:43-48.
The global population genetic structure of yellowfin tuna (Thunnus albacares) is poorly understood. The authors used 2b-RAD to show shallow but significant population
structure among oceans (fixation index (FST) = 0.0273; P < 0.01). Discriminant analysis of principal components supported the presence of genetically discrete yellowfin
tuna populations among 3 oceanic pools.
Illumina Technology: HiSeq 2500 System
Fu B., Liu H., Yu X. and Tong J. A high-density genetic map and growth related QTL mapping in bighead carp (Hypophthalmichthys nobilis). Sci Rep.
2016;6:28679.
The authors used 2b-RAD sequencing of 117 individuals in an F1 family to construct a map with 3121 SNP markers. Based on this genetic map, they identified 1
genome-wide significant and 37 suggestive QTLs for 5 growth-related traits in 6 linkage groups (LG3, LG11, LG15, LG18, LG19, LG22).
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

13

SLAF-seq: Specific Locus Amplified Fragment Sequencing
SLAF-seq is an optimized version of ddRADseq, specifically intended for large-scale genotyping experiments.36
The enzymes and the sizes of the restriction fragments are optimized with training data to ensure even distribution and avoid
repeats. The fragments are also selected over a tight range, to optimize PCR amplification. The protocol is similar to ddRAD, witha first digestion with MseI, heat inactivation, and a second digestion with AluI. The resulting fragments are PCR-amplified, adapters
are added, and the fragments are purified to produce the sequencing library.
Restriction sites

Digest with MseI

Add MseI
adapters

Digest with AluI

PCR with barcoded
MseI primer

Purify andpool

Add sequencing
adapters

PCR

DNA

A schematic overview of SLAF-seq.

Advantages

Disadvantages

•
•
•

•

•

Deep sequencing for genotyping accuracy
Reduced-representation strategy to reduce sequencing costs
Predesigned reduced-representation scheme to optimize marker
efficiency
Double barcode system for large populations

Does not cover the whole genome

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;.

References
Zhao X., Huang L., Zhang X., et al. Construction of high-density genetic linkage map and identification of flowering-time QTLs in orchardgrass using SSRs
and SLAF-seq. Sci Rep. 2016;6:29345.
The authors developed 447,177 markers based on SLAF-seq and used them to perform a comparative genomics analysis between perennial ryegrass andorchardgrass. They found 11 potentially significant QTLs for 2 target traits—heading date (HD) and flowering time (FT).
Illumina Technology: HiSeq 2500 System
Geng X., Jiang C., Yang J., et al. Rapid Identification of Candidate Genes for Seed Weight Using the SLAF-Seq Method in Brassica napus. PLoS One.
2016;11:e0147580.
The researchers used SLAF-seq with association analysis and bulked segregant analysis to identify candidate genes in 1000-seed weight (TSW) data. They found a
total of 1933 high-quality polymorphic SLAF markers and 4 TSW-associated markers.
Illumina Technology: HiSeq 2500

Zhang J., Yuan H., Li M., et al. A High-Density Genetic Map of Tetraploid Salix matsudana Using Specific Length Amplified Fragment Sequencing (SLAFseq). PLoS One. 2016;11:e0157777.
The authors created an intraspecific F1 hybrid population by crossing the salt-sensitive “Yanjiang” variety of an arbor tree species (Salix matsudana) as the female
parent with the salt-tolerant “9901” variety as the male parent. They genotyped this population, along with its parents. Both the parents and offspring were tetraploid,
but the authors were able to construct a genetic map with 6737 SLAF markers. Their data will be used to map quantitative trait loci that modulate salt tolerance andresistance in Salix.
Illumina Technology: HiSeq 2500 System

36.

14

Sun X., Liu D., Zhang X., et al. SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing. PLoS One.
2013;8:e58700.

DNA Sequencing Methods Collection

Wei Q. Z., Fu W. Y., Wang Y. Z., et al. Rapid identification of fruit length loci in cucumber (Cucumis sativus L.) using next-generation sequencing (NGS)-based QTL
analysis. Sci Rep. 2016;6:27496.
Xu X., Chao J., Cheng X., et al. Mapping of a Novel Race Specific Resistance Gene to Phytophthora Root Rot of Pepper (Capsicum annuum) Using Bulked Segregant
Analysis Combined with Specific Length Amplified Fragment Sequencing Strategy. PLoS One. 2016;11:e0151401.
Ye Y., Cai M., Ju Y., et al. Identification and Validation of SNP Markers Linked to Dwarf Traits Using SLAF-Seq Technology in Lagerstroemia. PLoS One.
2016;11:e0158970.
Zhang H., Yi H., Wu M., et al. Mapping the Flavor Contributing Traits on “Fengwei Melon” (Cucumis melo L.) Chromosomes Using Parent Resequencing and Super
Bulked-Segregant Analysis. PLoS One. 2016;11:e0148150.
Cai C., Cheng F. Y., Wu J., Zhong Y. and Liu G. The First High-Density Genetic Map Construction in Tree Peony (Paeonia Sect. Moutan) using Genotyping by SpecificLocus Amplified Fragment Sequencing. PLoS One. 2015;10:e0128584.
Ma J. Q., Huang L., Ma C. L., et al. Large-Scale SNP Discovery and Genotyping for Constructing a High-Density Genetic Map of Tea Plant Using Specific-Locus
Amplified Fragment Sequencing (SLAF-seq). PLoS One. 2015;10:e0128798.
Qin D., Dong J., Xu F., et al. Characterization and fine mapping of a novel barley Stage Green-Revertible Albino Gene (HvSGRA) by Bulked Segregant Analysis based on
SSR assay and Specific Length Amplified Fragment Sequencing. BMC Genomics. 2015;16:838.
Shan T., Pang S., Li J., Li X. and Su L. Construction of a high-density genetic map and mapping of a sex-linked locus for the brown alga Undaria pinnatifida(Phaeophyceae) based on large scale marker development by specific length amplified fragment (SLAF) sequencing. BMC Genomics. 2015;16:902.
Wang J., Zhang K., Zhang X., et al. Construction of Commercial Sweet Cherry Linkage Maps and QTL Analysis for Trunk Diameter. PLoS One. 2015;10:e0141261.
Wang W., Zhang T., Zhang G., et al. Genome-wide association study of antibody level response to NDV and IBV in Jinghai yellow chicken based on SLAF-seq
technology. J Appl Genet. 2015;56:365-373.
Xu F., Sun X., Chen Y., et al. Rapid identification of major QTLs associated with rice grain weight and their utilization. PLoS One. 2015;10:e0122206.
Xu X., Lu L., Zhu B., et al. QTL mapping of cucumber fruit flesh thickness by SLAF-seq. Sci Rep. 2015;5:15829.
Xu Y., Huang L., Ji D., et al. Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of
Pyropia haitanensis (Bangiales, Rhodophyta). BMC Plant Biol. 2015;15:228.
Zhang J., Zhang Q., Cheng T., et al. High-density genetic map construction and identification of a locus controlling weeping trait in an ornamental woody plant (Prunus
mume Sieb. et Zucc). DNA Res. 2015;22:183-191.
Zhang Y., Zhang J., Huang L., et al. A high-density genetic map for P genome of Agropyron Gaertn. based on specific-locus amplified fragment sequencing (SLAF-seq).
Planta. 2015;242:1335-1347.

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

15

hyRAD: Hybridization RAD for Degraded DNA
hyRAD37 was developed for use on degraded DNA samples, such as those from museum collections. Museum and preserved
samples offer a rich source of valuable specimens, but their degraded DNA is unable to sustain the double-digestion and sample
fractionation required by ddRAD.38 To address this limitation, hyRAD uses biotinylated probes and streptavidin-covered beads tocapture and enrich the fragments of interest.
The first step in the process is to generate a ddRAD library from high-quality DNA, usually from an extant specimen. The fragments
are size-selected and biotinylated. They can now be used as probes for hybridization capture of shotgun or ddRad libraries.
High quality DNA

Prepare RAD-seq
Adapter removal
library and size-select and labeling

DNA to be captured

Biotinylated
probes

Shotgun libraryHybridize to probes

Streptavidin
pull-downDNA

A schematic overview of hyRAD.

Advantages

Disadvantages•••

Can be used on degraded DNA
Can be used on unsequenced genomes

Coverage not as complete as with high-quality samples

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;.
Holmes M. W., Hammond T. T., Wogan G. O., et al. Natural history collections as windows on evolutionary processes. Mol Ecol. 2016;25:864-881.

References
Suchan T., Pitteloud C., Gerasimova N. S., Kostikova A., Schmid S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing
Genomic Analyses on Collection Specimens. PLoS One. 2016;11:e0151651.
This study describes the hyRAD protocol and demonstrates its performance. The researchers obtained a large set of orthologous loci from fresh and museum samples
from a non–model butterfly species. They discovered a high proportion of SNPs present in all 8 analyzed specimens, including 58-year-old museum samples.
Illumina Technology: MiSeq System, HiSeq System

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

16

37.

Suchan T., Pitteloud C., Gerasimova N. S., et al. Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection
Specimens. PLoS One. 2016;11:e0151651.

38.

Peterson B. K., Weber J. N., Kay E. H., Fisher H. S. and Hoekstra H. E. Double digest RADseq: an inexpensive method for de novo SNP discovery andgenotyping in model and non-model species. PLoS One. 2012;7:e37135.

DNA Sequencing Methods Collection

Rapture: Restriction-Site Associated DNA Capture
Rapture is a massively parallel, targeted DNA sequencing technique that combines RAD-seq and sequence capture to compare
multiple genes of interest among large numbers of samples. The targeted sequencing is based on identifying restriction enzyme
sites specifically near the loci of interest.
gDNA samples are pooled into individual wells in plates and digested with selected restriction enzymes. Biotinylated RAD
adapters with well-specific barcodes are ligated to the sticky ends before pooling all the wells from each plate. The barcoded DNA
fragments are randomly sheared and bound to streptavidin beads. Again, using restriction enzymes, fragments are cleaved fromthe streptavidin beads and used in standard DNA library preparation kits with plate-specific barcode labels. Next, libraries fromboth plates are pooled and hybridized with biotinylated bait-oligos specific to each RAD tag, before a final streptavidin pull-down.
The isolated DNA fragments are sequenced and arranged according to their RAD tags, plate barcodes, and well barcodes.
Plate 1 Plate 2

Restriction sites

Digest withrestriction
enzymeWell 1
Well 2
Add well-specificbarcodesPool wells
and shear

Streptavidin
pull-down

Restriction
enzyme digest

Library prep with Pool Hybridize biotinylated
capture bait
plate barcodes

Streptavidin
pull-downDNA

A schematic overview of Rapture.

Advantages

Disadvantages

•
•

•
•
•
•

Massively parallel, targeted DNA sequencing for SNP identification
Improved number of mapped fragments and locus coveragecompared to RAD-seq

RAD tags needs to be designated prior to the experiment
Additional cost to synthesize baits
Only a small number of loci are interrogated
Biased toward sequences closer to the restriction cut site

Reviews
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;.

References

Ali O. A., O’Rourke S. M., Amish S. J., Meek M. H., Luikart G., et al. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. Genetics.
2016;202:389-400.
This publication describes the original Rapture protocol. To test the method, the researchers analyzed fin clips of 96 rainbow trout individuals. The average coverageat the captured loci was 16x compared, to 0.4x for RAD. In a principal-component analysis, the first principal component separated 2 distinct groups corresponding
to individuals born in Bear Creek and the spring-fed spawning locations (Thousands Springs and Spring Creek). The third component separated individuals fromThousand Springs and Spring Creek. These results represent a remarkably fine scale of spatial analysis.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

39.

Ali O. A., O’Rourke S. M., Amish S. J., et al. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. Genetics. 2016;202:389-400.

An overview of recent DNA-seq publications featuring Illumina technology

17Digenome-seq: Cas9-Digested Whole-Genome Sequencing
Digenome-seq was designed to profile genome-wide Cas9 off-target effects.40 A multiplexed version has also been published.41
It belongs to a family of methods, including HTGTS42 LAM-HTGTS,43 and Guide-seq,44 which are aimed at detecting off-target
effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and other RNA-guided engineered nucleases(RGENs).
This method detects off-target mutations, induced by RGENs in a bulk population of cells, by sequencing in vitro nucleasedigested genomes (digenomes). These digests should produce many DNA fragments with identical 5’ ends, which are verticallyaligned at cleavage sites.
Cas9 target

Deletion
Wild type
InsertTarget site digestion

Sequence

Align and determine
sequence breaks

A schematic overview of Digenome-seq.

Advantages

Disadvantages

•
•

•
•

•

Relies on DNA cleavage rather than binding
Performed in a genomic context and captures sites with a
DNA/RNA bulge
Detects off-target effects with a frequency of 0.1% or lower 45

Requires in vivo cleavage confirmation46
High skill requirement for bioinformatic analysis 46

Reviews
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.
Mol Ther. 2016;24:475-487.
Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics. 2016;43:63-75.

References

Kim D., Kim J., Hur J. K., et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016;34:863-868.
To test whether Cpf1 endonucleases could replace Cas9 to improve the precision of gene editing, the authors used a mismatched CRISPR RNA (crRNA). They foundthat Cpf1 can tolerate single or double mismatches in the 3’-protospacer-adjacent motif (PAM) distal region, but not in the 5’-PAM proximal region. Next, they used
Digenome-seq to perform a genome-wide analysis of cleavage sites for 8 Cpf1 nucleases. Of these, Lachnospiraceae bacterium Cpf1 (LbCpf1) and Acidaminococcus
sp Cpf1 (AsCpf1) showed 6 and 12 cleavage sites in the human genome, respectively, while Cas9 nucleases generally cut over 90 sites. Most of the identified Cpf1
cleavage sites did not produce mutations in cells. The authors also found mismatches in either the 3’-PAM-distal region or in the PAM sequence of 12 off-target sitesthat were validated in vivo. The off-target effects were eliminated by using preassembled recombinant Cpf1 ribonucleoproteins.
Illumina Technology: HiSeq X Ten System, TruSeq DNA Library Prep Kit

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

18

40.

Kim D., Bae S., Park J., et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p
following 243.

41.

Kim D., Kim S., Kim S., Park J. and Kim J. S. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res.
2016;26:406-415.

42.

Chiarle R., Zhang Y., Frock R. L., et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell.
2011;147:107-119.

43.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear
amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

44.

Kim D., Bae S., Park J., et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p
following 243.

45.

Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics. 2016;43:63-75.

46.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

DNA Sequencing Methods Collection

CAP-seq: CXXC Affinity Purification Sequencing
CAP-seq maps methylated CpGs in gDNA.47
In this method, DNA is incubated with CXXC bound to nickel-charged sepharose beads. Next, DNA is eluted from the complex

CXXC

X

CX
XC

XC
CX

Methylated CpG

CX
XC

and sequenced. Deep sequencing provides insights into methylated CpG sites in gDNA.
CXXC

CXXC

Unmethylated CpG

CXXC

CXXC

CXXC

CXXC bound to nickel-charged
sepharose beads

CXXC

Hybridize to sepharose column

Elute unmethylated CpG
enriched fragments

DNA

A schematic overview of CAP-seq.

Advantages

Disadvantages••

Targets CpG islands

None known

Reviews
Vilborg A., Passarelli M. C., Yario T. A., Tycowski K. T. and Steitz J. A. Widespread Inducible Transcription Downstream of Human Genes. Mol Cell. 2015;59:449-461.
Smallwood S. A., Lee H. J., Angermueller C., et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817-820.
Suzuki M. M., Yoshinari A., Obara M., et al. Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells. Epigenetics Chromatin.
2013;6:38.

References
None available yet

Associated Kits
Nextera DNA Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

47.

Illingworth R. S., Gruenewald-Schneider U., Webb S., et al. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet.
2010;6:e1001134.

An overview of recent DNA-seq publications featuring Illumina technology

19

CPT-seq: Contiguity-Preserving Transposition Sequencing
CPT-seq is a method for genome-wide haplotyping based on contiguity-preserving transposition (CPT) and combinatorial
indexing.48
Tn5 transposition is used to modify DNA with adapter and index sequences while preserving contiguity. After DNA dilution andcompartmentalization, the transposase is removed, and the DNA is separated into individually indexed libraries. The libraries
in each compartment are enriched for neighboring genomic elements and are further indexed via PCR. Combinatorial 96-plex
indexing at both the transposition and PCR stage enables the construction of phased synthetic reads from each of the nearly
10,000 virtual compartments.

Divide sample
into 96 reactions

Indexed transposome reactions

EDTA
Pool

Dilute and divide
into 96 reactions

SDS

IndexedPCR primers

PCR

DNA

A schematic overview of CPT-seq.

Advantages

Disadvantages•••

Highly indexed and efficient
FragScaff reported to be highly effective at scaffolding large
genomes from CPT-seq data49• Large effective number of virtual compartments per physicalcompartment could avoid the amplification biases associated withmultiple displacement amplification (MDA)50

Not yet adopted widely by the scientific community

Reviews
Mostovoy Y., Levy-Sakin M., Lam J., et al. A hybrid approach for de novo human genome sequence assembly and phasing. Nat Methods. 2016;13:587-590.
Snyder M. W., Adey A., Kitzman J. O. and Shendure J. Haplotype-resolved genome sequencing: experimental methods and applications. Nat Rev Genet. 2015;16:344-358.

References
Amini S., Pushkarev D., Christiansen L., Kostem E., Royce T., et al. Haplotype-resolved whole-genome sequencing by contiguity-preserving transpositionand combinatorial indexing. Nat Genet. 2014;46:1343-1349.
This publication describes the original CPT-seq method. The authors demonstrate the feasibility of this method by assembling >95% of the heterozygous variants in ahuman genome into long, accurate haplotype blocks (N50 = 1.4–2.3 Mb).
Illumina Technology: HiSeq 2000 System

Associated Kits
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

20

48.

Kuleshov V., Snyder M. P. and Batzoglou S. Genome assembly from synthetic long read clouds. Bioinformatics. 2016;32:i216-i224.

49.

Cao C.-c. and Sun X. Combinatorial pooled sequencing: experiment design and decoding. Quantitative Biology. 2016;4:36-46.

50.

Amini S., Pushkarev D., Christiansen L., et al. Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing.
Nat Genet. 2014;46:1343-1349.

DNA Sequencing Methods CollectionRC-Seq: Retrotransposon Capture Sequencing
RC-seq is a high-throughput protocol to map and study retrotransposon insertions.51
In this method, after gDNA is fractionated, retrotransposon binding sites on DNA hybridize to transposon binding sites on a
microarray. Deep sequencing provides accurate information that can be aligned to a reference sequence to discover novel
retrotransposition events. A single-cell version (scRC-seq) has also been described.52
Retrotransposon
binding sites

Genomic DNA

Sequenced fragment

Read1

Fractionate

DNAfragments

Hybridize Microarray with transposon
binding sites

Read2

Reference sequence

Transposon sites

Known retrotransposon

Align

Novel retrotransposition
eventsA schematic overview of RC-seq.

Advantages

Disadvantages

•
•
•
•

•

Ability to clearly identify and detect novel retrotransposition events
Can specifically study transposon binding sites of interest
High-throughput protocol
PCR validation rate estimated at 98.5%•••

Different types of mobile element insertions (MEIs) require separate
PCR experiments with different primers53
Hybridization errors can lead to sequencing unwanted DNA
fragments
PCR biases can underrepresent GC-rich templates
Similar transposition binding sites can lead to sequence ambiguity
and detection for a transposition event

Reviews
Xing J., Witherspoon D. J. and Jorde L. B. Mobile element biology: new possibilities with high-throughput sequencing. Trends Genet. 2013;29:280-289.

References
Klawitter S., Fuchs N. V., Upton K. R., et al. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.
Nat Commun. 2016;7:10286.
The researchers used RC-seq to map the genomic integration sites of de novo retrotransposon insertions in human induced pluripotent stem cells (hiPSCs). They
wanted to determine if activation of the L1 mobilization machinery produced L1-mediated retrotransposition. They detected 40,608 non–reference retrotransposon
insertions and estimated that each hiPSC carried ~1 de novo L1 insertion.
Illumina Technology: HiSeq 2000 System
Upton K. R., Gerhardt D. J., Jesuadian J. S., Richardson S. R., Sanchez-Luque F. J., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell.
2015;161:228-239.
The authors performed scRC-seq on individual human hippocampal neurons and glia, as well as cortical neurons. They estimated that 13.7 somatic L1 insertions
occurred per hippocampal neuron and carried the sequence hallmarks of target-primed reverse transcription (RT). This result indicates that L1 mosaicism is pervasive atgenomic loci expressed in hippocampal neurons.
Illumina Technology: Unspecified Illumina sequencing system
Solyom S., Ewing A. D., Rahrmann E. P., et al. Extensive somatic L1 retrotransposition in colorectal tumors. Genome Res. 2012;22:2328-2338.
Shukla R., Upton K. R., Munoz-Lopez M., et al. Endogenous retrotransposition activates oncogenic pathways in hepatocellular carcinoma. Cell. 2013;153:101-111.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

51.

Baillie J. K., Barnett M. W., Upton K. R., et al. Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011;479:534-537.

52.

Upton K. R., Gerhardt D. J., Jesuadian J. S., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell. 2015;161:228-239.

53.

Xing J., Witherspoon D. J. and Jorde L. B. Mobile element biology: new possibilities with high-throughput sequencing. Trends Genet. 2013;29:280-289.

An overview of recent DNA-seq publications featuring Illumina technology

21

Tn-Seq: Transposon Sequencing
INSeq: Insertion Sequencing
Tn-seq54 and INSeq55 are nearly identical methods that determine quantitative genetic interactions accurately. INSeq includes apolyacrylamide gel purification step following adapter ligation and PCR, whereas Tn‑seq requires agarose gel purification.56
In this method, a transposon with flanking Mmel digestion sites is transposed into bacteria which, after culturing, can help detect
the frequency of mutations within the transposon. After MmeI digestion and subsequent adapter ligation, PCR amplification andsequencing can provide information about the transposon insertion sites.
Transposon
Inverted MmeI
recognition site

MmeI
recognition site

MmeI

20bp
MmeI

20bp

MmeI digestion

Add adapters

PCR and sequence

Transposon
insertion sites

A schematic overview of Tn-seq.

Advantages

Disadvantages

•
•
•

•
•

Can study mutational frequency of transposons
Can be used to deduce fitness of genes within microorganisms
Robust, reproducible, and sensitive

Limited to bacterial studies
Errors during PCR amplification can lead to inaccurate sequence
reads

Reviews
None available yet.

References

Hooven T. A., Catomeris A. J., Akabas L. H., et al. The essential genome of Streptococcus agalactiae. BMC Genomics. 2016;17:406.
In order to establish the essential genome of group B Streptococcus (GBS), the authors used the Himar1 minitransposon, which inserts at genomic TA dinucleotide
sites. Next, they performed Tn-seq on DNA collected from 3 independent mutant libraries. Each library had at least 135,000 mutants after 24 hour outgrowth in richmedia. From these data, the authors identified 13.5% of genes as essential and 1.2% as critical.
Illumina Technology: HiSeq 4000 System
Le Breton Y., Belew A. T., Valdes K. M., et al. Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes. Sci Rep. 2015;5:9838.
The authors developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools in group A Streptococcus (GAS) by Tn-seq. Highly saturated
transposant libraries carried Krmit insertions at approximately every 25 nucleotides. The authors identified 227 and 241 of those genes in a GAS serotype M1T1
invasive strain 5448 and a GAS nephritogenic serotype M49 strain NZ131, respectively.
Illumina Technology: HiSeq 1500 System

22

54.

van Opijnen T., Bodi K. L. and Camilli A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat Methods.
2009;6:767-772.

55.

Goodman A. L., McNulty N. P., Zhao Y., et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe.
2009;6:279-289.

56.

van Opijnen T. and Camilli A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol. 2013;11:435-442.

DNA Sequencing Methods Collection

Liu F., Wang C., Wu Z., Zhang Q. and Liu P. A zero-inflated Poisson model for insertion tolerance analysis of genes based on Tn-seq data. Bioinformatics.
2016;32:1701-1708.
DeJesus M. A., Ambadipudi C., Baker R., Sassetti C. and Ioerger T. R. TRANSIT--A Software Tool for Himar1 TnSeq Analysis. PLoS Comput Biol. 2015;11:e1004401.
Lee S. A., Gallagher L. A., Thongdee M., et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A.
2015;112:5189-5194.
Meeske A. J., Sham L. T., Kimsey H., et al. MurJ and a novel lipid II flippase are required for cell wall biogenesis in Bacillus subtilis. Proc Natl Acad Sci U S A.
2015;112:6437-6442.
Rubin B. E., Wetmore K. M., Price M. N., et al. The essential gene set of a photosynthetic organism. Proc Natl Acad Sci U S A. 2015;112:E6634-6643.
Turner K. H., Wessel A. K., Palmer G. C., Murray J. L. and Whiteley M. Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum. Proc Natl Acad Sci U S A.
2015;112:4110-4115.
Wu M., McNulty N. P., Rodionov D. A., et al. Genetic determinants of in vivo fitness and diet responsiveness in multiple human gut Bacteroides. Science.
2015;350:aac5992.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

23

TC-Seq: Translocation-Capture Sequencing
TC-Seq was developed to study chromosomal rearrangements and translocations.57
In this method, cells are infected with retrovirus expressing l-Scel sites in cells with and without activation-induced cytidine
deaminase (AICDA or AID) protein. gDNA from cells is sonicated, linker-ligated, purified, and amplified via seminested ligationmediated (LM)-PCR. The linker is cleaved, and the DNA is sequenced. Any AID-dependent chromosomal rearrangement will be
amplified by LM-PCR, while AID-independent translocations will be discarded.
I-SceI site

+AID
-AID

Genomic DNA

Infect I-Sel

A
A
Sonicate, blunt-end
and A-tailA
A
Ligate linkers
Cut I-Scel

Purification

Seminested PCR
Linker cleavage

DNA

A schematic overview of TC-Seq.

Advantages

Disadvantages

•

•
•

•

Can study chromosomal translocations within a given model orenvironment
Random sonication generates unique linker ligation points,
and deep sequencing allows reading through rearrangement
breakpoints

Does not resolve junction structures
Relatively higher cost and lower sensitivity compared with
LAM-HTGTS58• PCR amplification errors• Nonlinear PCR amplification can lead to biases affecting
reproducibility• PCR biases can underrepresent GC-rich templates

Reviews
None available yet

References

Robbiani D. F., Deroubaix S., Feldhahn N., et al. Plasmodium Infection Promotes Genomic Instability and AID-Dependent B Cell Lymphoma. Cell.
2015;162:727-737.
Plasmodium chabaudi (Pc) induces prolonged expansion of germinal centers (GCs). These centers are unique compartments in which B cells undergo rapid clonal
expansion and express AID, a DNA mutator. The authors used TC-Seq to detect the AID-induced damage and found that GC B cells elicited during Pc infection suffer
widespread DNA damage, leading to chromosome translocations. Although infection does not change the overall rate, it modifies lymphomagenesis to favor mature
AID-dependent B cell lymphomas. As a result, malaria infection appears to favor mature B cell cancers by eliciting protracted AID expression in GC B cells.
Illumina Technology: Unspecified HiSeq system
Qian J., Wang Q., Dose M., et al. B cell super-enhancers and regulatory clusters recruit AID tumorigenic activity. Cell. 2014;159:1524-1537.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

24

57.

Klein I. A., Resch W., Jankovic M., et al. Translocation-capture sequencing reveals the extent and nature of chromosomal rearrangements in B lymphocytes. Cell.
2011;147:95-106.

58.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

DNA Sequencing Methods CollectionRep-Seq: Repertoire Sequencing
Ig-seq: DNA Sequencing of Immunoglobulin Genes
MAF: Molecular Amplification fingerprinting
Rep-Seq is a collective term for repertoire sequencing technologies,59 including Ig-seq60 and MAF.61
Ig-seq is a targeted gDNA amplification method performed with primers complementary to the rearranged V-region gene (VDJ
recombinant). Amplification of gDNA is then performed with the appropriate 5’ primers. Although throughput is high, information
regarding which VH and VL chains were paired in the same cell is lost, as cells are lysed in bulk and VH and VL genes areamplified in separate reactions. MAF (illustrated below) was developed to correct the amplification biases in Ig-seq, by using
tagging with unique identifiers (UIDs) before and during the multiplex PCR amplification, as well as a bioinformatics pipeline.
V D JConstant

8N UID
8N UID

Extracted RNA

Reverse transcription

Second-strand synthesis

PCR

PurifyDNA

A schematic overview of Rep-Seq.

Advantages

Disadvantages•• Data analysis is challenging62• In bulk analysis, information regarding which VH and VL chains werepaired in the same cell is lost61

Measures antibody frequencies with up to 99% accuracy

Reviews
Hackl H., Charoentong P., Finotello F. and Trajanoski Z. Computational genomics tools for dissecting tumour-immune cell interactions. Nat Rev Genet. 2016;17:441-458.
Hou X. L., Wang L., Ding Y. L., Xie Q. and Diao H. Y. Current status and recent advances of next generation sequencing techniques in immunological repertoire.
Genes Immun. 2016;17:153-164.
Georgiou G., Ippolito G. C., Beausang J., Busse C. E., Wardemann H., et al. The promise and challenge of high-throughput sequencing of the antibody repertoire.
Nat Biotechnol. 2014;32:158-168.

References
Khan T. A., Friedensohn S., Gorter de Vries A. R., Straszewski J., Ruscheweyh H. J., et al. Accurate and predictive antibody repertoire profiling by molecular
amplification fingerprinting. Sci Adv. 2016;2:e1501371.
This study describes the original MAF method. The authors found that MAF bias correction, when combined with a bioinformatic pipeline, led to measurements ofantibody frequencies with up to 99% accuracy. They also used MAF to correct PCR and sequencing errors, to achieve full-length antibody diversity measurements with
98–100% error correction.
Illumina Technology: MiSeq System

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

59.

Benichou J., Ben-Hamo R., Louzoun Y. and Efroni S. Rep-Seq: uncovering the immunological repertoire through next-generation sequencing. Immunology.
2012;135:183-191.

60.

Georgiou G., Ippolito G. C., Beausang J., Busse C. E., Wardemann H. and Quake S. R. The promise and challenge of high-throughput sequencing of the antibody
repertoire. Nat Biotechnol. 2014;32:158-168.

61.

Khan T. A., Friedensohn S., Gorter de Vries A. R., Straszewski J., Ruscheweyh H. J. and Reddy S. T. Accurate and predictive antibody repertoire profiling by
molecular amplification fingerprinting. Sci Adv. 2016;2:e1501371.

62.

Georgiou G., Ippolito G. C., Beausang J., Busse C. E., Wardemann H. and Quake S. R. The promise and challenge of high-throughput sequencing of the antibody
repertoire. Nat Biotechnol. 2014;32:158-168.

An overview of recent DNA-seq publications featuring Illumina technology25EC-seq: Excision Circle Sequencing
EC-seq allows the capture and analysis of immune locus rearrangements from whole thymic and splenic tissues.
Double-stranded cleavage at recombination signal sequence (RSS) sites, mediated by recombination-activating gene recombinase(RAG), precedes genomic deletion and end-processing by the nonhomologous end-joining (NHEJ) complex. Coding ends arecovalently hair-pinned and reopened prior to ligation. The excision circle junction is then ligated from unprocessed DNA ends withlittle modification and used to prepare a sequencing library.
J1
V1

Lymphocyte-specific recombination activating gene (RAG) recombinase, bound genome

V2

J3

J2

V1

V2 J3

J2

J1

J1

V1

V

J2

Synapsis, cleavage, and coding
end hairpin formation

J4

J3

V3

J4

J4

J5

J5

V3

3

V2

V2

V3

J5

J4

J3

J2

J1

J5

V1

Resolved genomic Liberated excision circle withcoding junction
resolved signal junction

Align read pairs toreference genome

A schematic overview of EC-seq.

Advantages

Disadvantages••

Provides capture and analysis of immune locus rearrangements

Not yet adopted widely by the scientific community

Reviews
None available yet

References

Parkinson N. J., Roddis M., Ferneyhough B., et al. Violation of the 12/23 rule of genomic V(D)J recombination is common in lymphocytes. Genome Res.
2015;25:226-234.
The authors developed EC-seq to study V(D)J recombination in mouse whole thymic and splenic tissues. They found that the 12/23 rule of genomic recombination was
frequently violated under physiological conditions, resulting in unanticipated hybrid recombination. These nonclassical excision circles are liberated during the formationof antigen receptor diversity.
Illumina Technology: HiSeq 2000 System

Associated Kits
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit

26

DNA Sequencing Methods Collection

Bubble-Seq: Libraries of Restriction Fragments that Contain Replication Initiation
Sites (Bubbles)
Bubble-Seq prepares libraries of restriction fragments that contained replication initiation sites (bubbles) in vivo.63
In this method, DNA from origin libraries is biotinylated by a 3’-end tailing reaction with biotin-16-dUTP. The biotinylated DNA issonicated and captured with streptavidin-coated beads. The free DNA fragments are purified and used to prepare a sequencing.

Bubble-containing
fragment

Restriction
digestCast fragments intrapping gel

Run gelRecover bubble-containing plug

DNA extraction

Add sequencing
primersDNA

A schematic overview of Bubble-Seq.

Advantages

Disadvantages••

Simple protocol

Not yet adopted widely by the scientific community

Reviews
None available yet

References
Foulk M. S., Urban J. M., Casella C. and Gerbi S. A. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing
reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res. 2015;25:725-735.
The authors used Bubble-Seq as an orthogonal mapping technique to confirm that G-quadruplex (G4) structures are not general determinants for origin specification,
but they may play a role for a subset.
Illumina Technology: HiSeq System
Mukhopadhyay R., Lajugie J., Fourel N., et al. Allele-specific genome-wide profiling in human primary erythroblasts reveal replication program organization. PLoS Genet.
2014;10:e1004319.

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit

63.

Mesner L. D., Valsakumar V., Cieslik M., Pickin R., Hamlin J. L. and Bekiranov S. Bubble-seq analysis of the human genome reveals distinct chromatin-mediatedmechanisms for regulating early- and late-firing origins. Genome Res. 2013;23:1774-1788.

An overview of recent DNA-seq publications featuring Illumina technology

27

NSCR: Nascent Strand Capture and Release
NSCR isolates and sequences the origin of DNA replication by capturing short nascent strands (SNS), which are single-stranded
RNA-DNA chimeras.64 In short, NSCR captures and purifies RNA-DNA chimeric SNS, and releases the DNA part by cutting thestrand at the end of the initial RNA primer section.
First, gDNA is denatured, size-selected through a sucrose gradient for 400–2000 bp fragments, and 5’-biotinylated. The
biotinylated fragments are isolated by streptavidin pull-down, purifying both SNS and gDNA strands. Next, RNase I is used
to separate the DNA from the initial RNA primer section of the single-stranded RNA-DNA chimera. This process leaves RNA
primers and gDNA on the streptavidin beads. The released nascent DNA strands are amplified and sequenced. This method is an
improvement over widely used SNS purification methods using BrdU and lambda exonuclease (λ-exo).
BiotingDNA

RNA
primer

DNA replication bubble

SNS

Denaturation andsize-selection

gDNA
SNS

5’-biotinylation

Streptavidin pull-down

RNase I
digestion

Isolate andamplify SNS

DNA

A schematic overview of NSCR.

Advantages

Disadvantages

•
•

•

•
•

Maps genome-wide DNA replication origins
Higher discovery of nascent strands than λ-exo–based methods(2231 vs 3922 peaks)
RNase I cleaves with minimal sequence specificity
Can be used for systems that use small RNA primers to produce
RNA-DNA chimeras•

Less than 10% purification yield (1–2 ng of SNS from 1 mg total
DNA)
False reads may arise due to misincorporation of ribonucleotides
into the DNA portion of the SNS

Reviews
None available yet

References
Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. Isolation and sequencing of active origins of DNA replication by nascent strand capture and release(NSCR). J Biol Methods. 2015;2:.
The authors developed NSCR to isolate SNS and identify the locations of DNA replication origins. They performed NSCR on murine cell lines and obtained 3922 SNS
peaks. These peaks showed 79% overlap over the 2231 SNS peaks from a previous experiment using λ-exo–based techniques.
Illumina Technology: HiSeq 2000 System

Associated Kits
TruSeq ChIP Library Prep Kit

64.

28

Kunnev D., Freeland A., Qin M., Wang J. and Pruitt S. C. Isolation and sequencing of active origins of DNA replication by nascent strand capture and release (NSCR).
J Biol Methods. 2015;2:

DNA Sequencing Methods CollectionRepli-Seq: Nascent DNA Replication Strand Sequencing
Repli-Seq maps the sequences of nascent DNA replication strands throughout the whole genome during each of the 6 cellcycle phases.65
This process is achieved by growing cells in media containing BrdU to replace thymidine. The cells are sorted to their current statein cell division using fluorescence-activated cell sorting (FACS). BrdU-labeled DNA strands are immunoprecipitated by anti-BrdU
antibodies on magnetic beads. These immunoprecipitated strands can be prepared for sequencing following the TruSeq DNA
library preparation protocol.
BrdU

Run-on with analog

Sort cells and lyse

Bead coated with
anti-BrdU antibody

Elute
End Repair

PurifyDNA

A schematic overview of Repli-Seq.

Advantages

Disadvantages

•

•

•
•

Maps sequences of newly replicated DNA to the phases of cell
division
Low sample input required (5000 cells) makes it suitable forstudying rare cell populations
Streamlined DNA library preparation step

Limited to cell cultures and other artificial systems, due to therequirement for incubation in the presence of labeled nucleotides

Reviews
Reuter J. A., Spacek D. V. and Snyder M. P. High-Throughput Sequencing Technologies. Mol Cell. 2015;58:586-597.

References

Ohbayashi R., Watanabe S., Ehira S., et al. Diversification of DnaA dependency for DNA replication in cyanobacterial evolution. ISME J. 2015;.
The authors studied the importance of DNA replication initiation factor DnaA, which is highly conserved in prokaryotes. They applied Repli-Seq to identify replication
initiation sites within DnaA disruptants in Synechococcus elongates. The results revealed that deletion of the dnaA gene had no effect in DNA replication or cell growthof the model. The researchers concluded that functional dependency on DnaA was lost in some cyanobacterial lineages during diversification.
Illumina Technology: Genome Analyzer System
Deyle D. R., Hansen R. S., Cornea A. M., et al. A genome-wide map of adeno-associated virus-mediated human gene targeting. Nat Struct Mol Biol. 2014;21:969-975.
Barlow J. H., Faryabi R. B., Callen E., et al. Identification of early replicating fragile sites that contribute to genome instability. Cell. 2013;152:620-632.

Associated Kits
TruSeq Nano DNA Library Prep Kit

65.

Hansen R. S., Thomas S., Sandstrom R., et al. Sequencing newly replicated DNA reveals widespread plasticity in human replication timing. Proc Natl Acad Sci U S A.
2010;107:139-144.

An overview of recent DNA-seq publications featuring Illumina technology

29

NS-Seq: Nascent Strand Sequencing
NS-Seq sequences nascent DNA strands to locate DNA replication origins in the genome. NS-Seq uses λ-exo to digest parental
DNA effectively while leaving the RNA primer–protected nascent strands intact. However, λ-exo inefficiently digests G-quadruplex
structures (G4) and GC-rich motifs; this bias can be normalized by using λ-exo–digested DNA from nonreplicating cells as acontrol.66
In this method, gDNA is enriched, made single-stranded, and 5’-phosphorylated using T4 PNK. Next, the DNA is digested with
λ-exo and purified. The resultant single-stranded nascent strands are converted to double-stranded DNA using random hexamersand fragmented to 100–600 bp. DNA libraries are prepared, using standard kits, and sequenced.

DNA replication origin

Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo)

Digestion

PurifyAmplifyDNA

A schematic overview of NS-seq.

Advantages

Disadvantages••

Locates DNA replication origins by sequencing RNA primer–
protected nascent DNA strands• λ-exo–digested DNA from nonreplicating cells can be used as acontrol to normalize for biases in λ-exo digestion• Replacing K+ with Na+ during λ-exo digestion reduces digestion
inefficiency in G4 regions•

λ-exo does not efficiently digest G4 structures in plasmid and halts
upon GC-rich motifs
Purified samples can be contaminated with GC-rich andG4-protected DNA

Reviews
MacAlpine D. M. ORChestrating the human DNA replication program. Proc Natl Acad Sci U S A. 2016;113:9136-9138.
Song J., Perreault J.-P., Topisirovic I. and Richard S. RNA G-quadruplexes and their potential regulatory roles in translation. Translation. 2016;4:e1244031.

References

Smith O. K., Kim R., Fu H., et al. Distinct epigenetic features of differentiation-regulated replication origins. Epigenetics Chromatin. 2016;9:18.
This study explores the relationship between DNA replication origins and transcriptomic changes, especially in human oncogenic cell lines. The authors performed
NS-Seq on short nascent strands isolated using λ-exo digestion and BrdU substitution. They found that cancer and noncancer cells displayed very similar distributionsof replication origins. Using NS-Seq data, they discovered that cell type–specific replication origins are replicated during late S phase, while shared origins are replicatedthroughout the S phase.
Illumina Technology: HiSeq 2000 System, TruSeq SBS Kit v3

Associated Kits
TruSeq SBS Kit v3

66.

30

Foulk M. S., Urban J. M., Casella C. and Gerbi S. A. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals
phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins. Genome Res. 2015;25:725-735.

DNA Sequencing Methods Collection

DNA BREAK MAPPINGDNA strand breaks are intrinsic to various biological processes, such as transcription and the generation of antigen receptor
diversification in lymphocytes.67 They are key substrates for translocations, deletions, and amplifications associated withvarious cancers.68,69 With the advent of CRISPR/Cas9 gene editing, there is renewed interest in determining cleavage patterns
characteristic of on- and off-target sites.70

Accurate identification of DNA strand breaks are notably used in cancer research and CRISPR-mediated gene editing studies.

Reviews
Kim D., Kim S., Kim S., Park J. and Kim J. S. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res.
2016;26:406-415.
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.
Mol Ther. 2016;24:475-487.
Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediatedhigh-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

67.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

68.

Nussenzweig A. and Nussenzweig M. C. Origin of chromosomal translocations in lymphoid cancer. Cell. 2010;141:27-38.

69.

Alt F. W., Zhang Y., Meng F. L., Guo C. and Schwer B. Mechanisms of programmed DNA lesions and genomic instability in the immune system. Cell. 2013;152:417-429.

70.

Kim D., Kim S., Kim S., Park J. and Kim J. S. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res.
2016;26:406-415.

An overview of recent DNA-seq publications featuring Illumina technology

31

Map DNA Single-Strand Breaks (SSB-Seq)
SSB-Seq71 maps in vivo DNA single-strand breaks (SSBs) on a genome-wide scale.
In this method, gDNA is isolated and subjected to nick-translation by DNA polymerase I in the presence of deoxynucleotide
triphosphates (dNTPs), including digoxigenin-labeled dUTP, and a small amount of dideoxynucleotides. The DNA is sheared, andthe labeled fragments are immunoprecipitated with antidigoxigenin antibodies and purified to prepare a sequencing library.

Genome DNA

DNA Pol I, dU-digoxigenin, and dNTPs

DNA isolation and random shearing

Immunoprecipitate with
anti-digoxigenin antibodyDNA

A schematic overview of SSB-Seq.

Advantages

Disadvantages•••

Simple protocol

Locations of breaks cannot be determined accurately
Not yet adopted widely by the scientific community

Reviews
None available yet

References

Baranello L., Kouzine F., Wojtowicz D., Cui K., Przytycka T. M., et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int J Mol Sci.
2014;15:13111-13122.
This study describes the original SSB-Seq protocol. The authors tested the method in human colon cancer cells and validated the results using the topoisomerase
II (Top2)–poisoning agent, etoposide (ETO). The combination of ETO treatment with break-mapping techniques appears to be a powerful method for elaborating thepattern of Top2 enzymatic activity across the genome.
Illumina Technology: Genome Analyzer System

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit

71.

32

Baranello L., Kouzine F., Wojtowicz D., et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int J Mol Sci. 2014;15:13111-13122.

DNA Sequencing Methods Collection

BLESS: Breaks labeling and Enrichment on Streptavidin and Sequencing
BLESS is a genome-wide approach to map DSBs at nucleotide resolution.72 BLESS is able to detect telomere ends, Sce
endonuclease–induced DSBs, and complex genome-wide DSB landscapes.
In this method, DSBs are ligated in situ to a proximal linker covalently linked to biotin. The gDNA is extracted and fragmented, andthe labeled fragments are captured on streptavidin beads. Next, a distal linker is ligated to the free end of the captured fragments.
The fragments are released by linker digestion with I-SceI and PCR-amplified.
TTTT

TT

TT

TT

DNA isolation andrandom shearing

TTTT

TT

Ligate primer

TTTT

Biotinylated
proximal primer

TT

Capture on streptavidin
beadsDistalprimer

TTTT

TTTT

TTTT

Genome DNA

TT
TT

TTTT

TTTT

TT

TT

TT

TT

TT

Digest with
I-SceI and PCRDNA

A schematic overview of BLESS.

Advantages

Disadvantages•••• High background; only maps unjoined ends73• Susceptible to artifacts associated with cell fixation74

Detects DSBs at nucleotide resolution
Does not depend on proteins that bind to DSBs—a source of bias
Does not depend on single-stranded DNA (ssDNA)—a sourceof bias

Reviews
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.
Mol Ther. 2016;24:475-487.

References

Ran F. A., Cong L., Yan W. X., et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186-191.
The authors applied BLESS to capture a snapshot of Cas9-induced DNA DSBs in cells. They were able to identified off-target sites not previously predicted bysequence similarity to the target or by chromatin immunoprecipitation (ChIP).
Illumina Technology: MiSeq System

Associated Kits
TruSeq Nano DNA LT Library Prep Kit

72.

Crosetto N., Mitra A., Silva M. J., et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013;10:361-365.

73.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

74.

Tsai S. Q., Zheng Z., Nguyen N. T., et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187-197.

An overview of recent DNA-seq publications featuring Illumina technology

33

DSB-Seq: Map DNA Double-Strand Breaks
DSB-Seq maps in vivo DNA DSBs on a genome-wide scale.75
In this method, DNA is biotinylated by a 3’-end tailing reaction with biotin-16-dUTP. The biotinylated DNA is sonicated andcaptured with streptavidin-coated beads. The free DNA fragments are purified and used to prepare a sequencing library.

Genome DNA

TdT and biotinylated-dUTP

DNA isolation and random shearing

Capture on streptavidin beadsDNA

A schematic overview of DSB-Seq.

Advantages

Disadvantages••

Simple protocol

Not yet adopted widely by the scientific community

Reviews
None available yet.

References

Baranello L., Kouzine F., Wojtowicz D., Cui K., Przytycka T. M., et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int J Mol Sci.
2014;15:13111-13122.
This study describes the original DSB-Seq protocol. The authors tested the method in human colon cancer cells and validated the results using the Top2-poisoning
agent, ETO. The combination of ETO treatment with break-mapping techniques appears to be a powerful method for elaborating the pattern of Top2 enzymatic activity
across the genome.
Illumina Technology: Genome Analyzer

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit

75.

34

Baranello L., Kouzine F., Wojtowicz D., et al. DNA break mapping reveals topoisomerase II activity genome-wide. Int J Mol Sci. 2014;15:13111-13122.

DNA Sequencing Methods Collection

Break-seq: Double-Stranded Break Labeling
Break-seq combines DSB labeling with NGS to map chromosome breaks with improved sensitivity and resolution.76
DNA trapped in agarose plugs is end-repaired and labeled with biotin. The agarose is then digested by b-agarase, followed bydilution and fragmentation. The fragmented DNA is purified and end-repaired with unmodified dNTPs, followed by A-tailing. The
fragments are purified on magnetic beads, PCR-amplified, purified, and used to prepare a sequencing library.

5’
3’

Break

3’
5’

DNA trapped inagarose gel

5’3’Index 2

3’
5’

End-repair with dGTP, dCTP,
dTTP, and biotinylated-dATP

Elution andfragmentation

Streptavidin magnetic
bead pull-down

Attachadapters

Index 1

PCR andpurifyDNA

A schematic overview of Break-seq.

Advantages

Disadvantages••

Simple protocol

Not replicated in other laboratories

Reviews
None available yet.

References
Hoffman E. A., McCulley A., Haarer B., Arnak R. and Feng W. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled
conflict between DNA replication and transcription. Genome Res. 2015;25:402-412.
The authors used Break-seq to show that DSBs occur preferentially at genes transcriptionally induced by hydroxyurea (HU) in yeast cells. Notably, different subsets ofthe HU-induced genes produced DSBs in the checkpoint mutant mec1 and MEC1 cells. The authors conclude that replication forks traversed a greater distance in
MEC1 cells than in mec1 cells during recovery from HU.
Illumina Technology: MiSeq System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

76.

Hoffman E. A., McCulley A., Haarer B., Arnak R. and Feng W. Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict
between DNA replication and transcription. Genome Res. 2015;25:402-412.

An overview of recent DNA-seq publications featuring Illumina technology

35

GUIDE-seq: Genome-Wide, Unbiased Identification of DSBs Enabled
by Sequencing
GUIDE-seq77 relies on the integration of double-stranded oligodeoxynucleotides (DSOs) into DSBs. It belongs to a family of
methods—such as HTGTS,78 LAM-HTGTS,79 and Digenome-seq80—that are aimed at detecting off-target effects of CRISPR/Cas9
and other RNA-guided nucleases (RGNs).
RGN-induced DSBs are tagged by integration of blunt-ended DSOs in the genomes of living human cells. The DSO integrationsites are mapped precisely in the genome at the nucleotide level with unbiased amplification and NGS.
dsOligo insert
Genome DNA

UMI

dsOligo tag integrated in live cells

DNA isolation
Random shearing

End repair
Adapter ligation

P5
UMI

Oligo tagIndex 2
Index 1 P7

PCR

dsOligo-specific amplificationDNA

A schematic overview of GUIDE-seq.

Advantages

Disadvantages

•

•

•

Generates global specificity landscapes for RGNs in living
human cells
Targeted sequencing reduces cost

Can miss some targets81

Reviews
Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediatedhigh-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.
Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing.
Mol Ther. 2016;24:475-487.

References

Kleinstiver B. P., Tsai S. Q., Prew M. S., et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol. 2016;.
The authors found that Cpf1 nucleases from Acidaminococcus sp (AsCpf1) and Lachnospiraceae bacterium (LbCpf1) have on-target editing accuracies in human cells
that are comparable to that of Streptococcus pyogenes Cas9. Through GUIDE-seq and targeted deep sequencing analysis performed on both nucleases, they foundno off-target effects for most of the crRNAs used, suggesting that AsCpf1 and LpCpf1 are highly specific in human cells.
Illumina Technology: MiSeq System
Kleinstiver B. P., Pattanayak V., Prew M. S., et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature.
2016;529:490-495.
In this study, the authors created and studied 15 SpCas9 mutants, each with a single, double, triple, or quadruple combination of 4 substitutions (N497A, R661A,
Q695A, and Q926A). Through an assay using a single-guide RNA (sgRNA) targeted to an enhanced green fluorescent protein (EGFP) reporter gene, they identified
the quadruple mutant (referred as SpCas9-HFI) as a high fidelity variant that reduces nonspecific DNA contacts. SpCas9-HFI retained on-target activities with wildtype SpCas9 with > 85% of sgRNAs tested in human cells. With sgRNAs targeted at nonrepetitive sequences, Cas9-HF1 rendered all or nearly all off-target events
undetectable by GUIDE-seq and by targeted deep sequencing. Even for atypical, repetitive target sites, the majority of off-target mutations induced by wild-type
SpCas9 were not detected with SpCas9-HFI.
Illumina Technology: MiSeq System

36

77.

Tsai S. Q., Zheng Z., Nguyen N. T., et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187-197.

78.

Chiarle R., Zhang Y., Frock R. L., et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell.
2011;147:107-119.

79.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

80.

Kim D., Bae S., Park J., et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p
following 243.

81.

Kim D., Kim S., Kim S., Park J. and Kim J. S. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res.
2016;26:406-415.

DNA Sequencing Methods Collection

Yin H., Song C. Q., Dorkin J. R., et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.
Nat Biotechnol. 2016;34:328-333.
In this study, the authors combined lipid nanoparticle-mediated delivery of Cas9 mRNA with adeno-associated viruses (AAV) encoding a sgRNA and a repair templatein a mouse model of human hereditary tyrosinemia. They showed that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting
the causative Fah-splicing mutation. They used GUIDE-seq in cultured liver cells and targeted deep sequencing to check for off-target effects, and found that the in vivo
off-target rate was low for the proposed method.
Illumina Technology: NextSeq 500 System, MiSeq System, Nextera XT DNA Library Prep Kit
Bolukbasi M. F., Gupta A., Oikemus S., et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods. 2015;
12:1150-1156.
To improve the editing precision of Cas9, the authors fused a programmable DNA binding domain (pDBD) to Cas9 and reduced the inherent DNA-binding affinity of
Cas9. They used GUIDE-seq and discovered that the Cas9-pDBD chimera had a significantly improved editing precision, providing a flexible system that can achieve
extremely precise genome-editing results.
Illumina Technology: MiSeq System, TruSeq DNA Library Prep Kit

Friedland A. E., Baral R., Singhal P., et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery
and paired nickase applications. Genome Biol. 2015;16:257.
The authors used GUIDE-seq to compare the specificity of Streptococcus aureus Cas9 (SaCas9) with Streptococcus pyogenes Cas9 (SpCas9), using a guide RNA(gRNA) that has known off-target sites. A substantial number of the SpCas9 off-target sites could be reproduced. By comparison, SaCas9 showed a high number of
on-target reads but only single-digit read counts for comparatively fewer off-target sites.
Illumina Technology: MiSeq System

Kleinstiver B. P., Prew M. S., Tsai S. Q., et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition.
Nat Biotechnol. 2015;33:1293-1298.
The researchers used GUIDE-seq to study the gene-editing specificity of wild-type SaCas9 and a series of mutants with modifications in the protospacer adjacent motif(PAM). One mutant, termed KKH, showed robust genome editing activities and increased the SaCas9 targeting range by 2- to 4-fold. Both wild-type and KKH SaCas9
induced comparable numbers of off-target effects in human cells.
Illumina Technology: MiSeq System

Doench J. G., Fusi N., Sullender M., et al. Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016;34:184-191.
Yin H., Song C. Q., Dorkin J. R., Zhu L. J., Li Y., et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo.
Nat Biotechnol. 2016;34:328-333.
Chari R., Mali P., Moosburner M. and Church G. M. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods.
2015;12:823-826.
Kleinstiver B. P., Prew M. S., Tsai S. Q., et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015;523:481-485.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

37

HTGTS: High-Throughput Genome-Wide Translocation Sequencing
HTGTS was developed to study translocation mechanisms in mammalian cells.82 This approach is particularly suitable for studying
for AID-dependent IgH class-switching (HTGTS-Rep-seq)83 and CRISPR/Cas9 genome modifications.84
In HTGTS-Rep-seq, genomic DNA from B-cell populations is sonicated and linearly amplified with a biotinylated primer thatanneals downstream of a J segment. The biotin-labeled single-stranded DNA products are enriched with streptavidin beads, andthe 3’ ends are ligated to a bridge adaptor containing a 6-nucleotide UMI.
A

A

A

A
A

Fragmentation bysonication

End repair

A

3’ A addition

Adapter ligation
and PCR

Streptavidin magnetic
bead pull-down

NestedPCR

Tagged PCR

PurifyDNA

A schematic overview of HTGTS-Rep-seq.

Advantages

Disadvantages•• Underestimates the frequency of DSBs85• Limited by chromatin accessibility86

Higher efficiency compared to whole-genome sequencing

Reviews
None available yet.

References
Schwer B., Wei P. C., Chang A. N., et al. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor
cells. Proc Natl Acad Sci U S A. 2016;113:2258-2263.
The authors used HTGTS to determine if breakpoint junctions are enriched around transcription start sites (TSSs) that were determined to be active by global run-on
sequencing analyses of primary neural stem/progenitor cells (NSPCs). All actively transcribed genes containing translocation junctions in NSPCs within 2 kb of the TSS
showed a significantly higher transcription rate, on average, than genes at distances greater than 2 kb from the TSS.
Illumina Technology: MiSeq System

Frock R. L., Hu J., Meyers R. M., et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33:179-186.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

38

82.

Chiarle R., Zhang Y., Frock R. L., et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell.
2011;147:107-119.

83.

Lin S. G., Ba Z., Du Z., Zhang Y., Hu J. and Alt F. W. Highly sensitive and unbiased approach for elucidating antibody repertoires. Proc Natl Acad Sci U S A.
2016;113:7846-7851.

84.

Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics. 2016;43:63-75.

85.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

86.

Kim D., Bae S., Park J., et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p
following 243.

DNA Sequencing Methods Collection

LAM-HTGTS: Linear Amplification-Mediated High-Throughput GenomeWide Sequencing
LAM-HTGTS is a method for the genome-wide detection of “prey” DSBs via their translocation to a fixed “bait” DSB in cultured
mammalian cells.87 Bait-prey junctions are cloned directly from isolated gDNA using LAM-PCR and ligated unidirectionally tobridge adapters. Subsequent PCR steps amplify the single-stranded DNA junction library in preparation for sequencing.
NNNN

Fragmentation
by sonication

LAM-PCR withbiotinylated
primerStreptavidin magnetic
bead pull-down

NNNN

Adapter ligation

NestedPCR

Enzymeblocking

Tagged PCR

PurifyDNA

A schematic overview of LAM-HTGTS.

Advantages

Disadvantages••

Sensitive detection of DSBs within chromosomes

Not applicable to limited amounts of material

Reviews
None available yet.

References
Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.
The authors used LAM-HTGTS to study the on-target and off-target activities of the recombination-activating gene (RAG) endonuclease during V(D)J recombination,using endogenous RAG-generated DSBs as bait. Although prior studies detected only a handful of off-target RAG-generated DSBs, the LAM-HTGTS studies identified
thousands of RAG off-target sites, which were restricted tightly within chromosomal loop domains. This result suggests a linear RAG tracking model to explain thegeneration of most RAG off-target events.
Illumina Technology: MiSeq System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

87.

Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplificationmediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871.

An overview of recent DNA-seq publications featuring Illumina technology

39

LOW-LEVEL DNA DETECTION
Single-cell genomics can be used to identify and study circulating tumor cells, cell-free DNA, microbes, and uncultured microbes.
In addition, it can aid preimplantation diagnosis and help us better understand tissue-specific cellular differentiation.88,89 DNA
replication during cell division is not perfect. As a result, progressive generations of cells accumulate unique somatic mutations.
Consequently, each cell in the body has a unique genomic signature, which allows the reconstruction of cell-lineage trees with highprecision.90 These cell-lineage trees can predict the existence of small populations of stem cells. This information is important fordiverse fields, from cancer development91,92 to preimplantation and genetic diagnosis.93,94

Single-cell genomics can help characterize and identify circulating tumor cells as well as microbes.

Reviews
Gawad C., Koh W. and Quake S. R. Single-cell genome sequencing: current state of the science. Nat Rev Genet. 2016;17:175-188.
Huang L., Ma F., Chapman A., Lu S. and Xie X. S. Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.
Annu Rev Genomics Hum Genet. 2015;16:79-102.
Voet T. and Van Loo P. SNES makes sense? Single-cell exome sequencing evolves. Genome Biol. 2015;16:86.
Wang Y. and Navin N. E. Advances and applications of single-cell sequencing technologies. Mol Cell. 2015;58:598-609.

40

88.

Shapiro E., Biezuner T. and Linnarsson S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat Rev Genet. 2013;14:618-630.

89.

Blainey P. C. The future is now: single-cell genomics of bacteria and archaea. FEMS Microbiol Rev. 2013;37:407-427.

90.

Frumkin D., Wasserstrom A., Kaplan S., Feige U. and Shapiro E. Genomic variability within an organism exposes its cell lineage tree. PLoS Comput Biol. 2005;1:e50.

91.

Navin N., Kendall J., Troge J., et al. Tumour evolution inferred by single-cell sequencing. Nature. 2011;472:90-94.

92.

Potter N. E., Ermini L., Papaemmanuil E., et al. Single-cell mutational profiling and clonal phylogeny in cancer. Genome Res. 2013;23:2115-2125.

93.

Van der Aa N., Esteki M. Z., Vermeesch J. R. and Voet T. Preimplantation genetic diagnosis guided by single-cell genomics. Genome Med. 2013;5:71.

94.

Hou Y., Fan W., Yan L., et al. Genome analyses of single human oocytes. Cell. 2013;155:1492-1506.

DNA Sequencing Methods Collection

smMIP: Single-Molecule Molecular Inversion Probes
The smMIP method uses single-molecule tagging and molecular inversion probes to detect and quantify genetic variations
occurring at low frequencies.95 In this method, probes are used to detect targets in gDNA. After the probed targets are copied,
exonuclease digestion leaves the target with a tag, which subsequently undergoes PCR amplification. Sequencing allows for highresolution sequence reads of targets, while greater depth allows for better alignment of every unique molecular tag.
Read1

Degenerate
molecular tag

Genomic DNA

Sample index
Read2

Copy target sequence

Exonuclease

PCR amplification

True variant

Random error
Align fragments from every
unique molecular tag

Corrected
sequence

A schematic overview of smMIPs.

Advantages

Disadvantages

•
•

•
•
•

•

Detects low-frequency targets
Can perform single-cell sequencing or sequencing for samples withlimited starting material
Per-base error of 2.6 x 10-5 in clinical samples96

PCR amplification errors
PCR biases can underrepresent GC-rich templates
Targets smaller than 500 bp are amplified preferentially bypolymerases during PCR

Reviews
Eboreime J., Choi S. K., Yoon S. R., Arnheim N. and Calabrese P. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation
Sequencing. PLoS One. 2016;11:e0158340.

References
Luks V. L., Kamitaki N., Vivero M. P., et al. Lymphatic and other vascular malformative/overgrowth disorders are caused by somatic mutations in PIK3CA.
J Pediatr. 2015;166:1048-1054 e1041-1045.
To test if mutations in somatic phosphatidylinositol-4,5-bisphospate 3-kinase, catalytic subunit alpha gene (PIK3CA) would be found in patients with more common
disorders, including isolated lymphatic malformation (LM) and Klippel-Trenaunay syndrome (KTS), the authors screened a small cohort of patients with these diseases.
In 8 patients, no PIK3CA mutations were detected by whole-exome sequencing, targeted capture sequencing, or droplet digital polymerase chain reaction (ddPCR).
The researchers used smMIPs to screen the entire PIK3CA coding sequence for novel mutations. They identified likely disease-causing PIK3CA mutations in 4 of the 8affected tissue samples, with mutant allele frequencies ranging from 2.8% (8/275) to 37.5% (314/837).
Illumina Technology: MiSeq System

Mirzaa G. M., Conti V., Timms A. E., et al. Characterisation of mutations of the phosphoinositide-3-kinase regulatory subunit, PIK3R2, in perisylvian
polymicrogyria: a next-generation sequencing study. Lancet Neurol. 2015;14:1182-1195.
This study was intended to identify additional genetic causes of bilateral perisylvian polymicrogyria (BPP) and characterize their frequency in the study population. The
researchers used targeted sequencing of the entire PIK3R2 gene by smMIPs on 38 patients with BPP with normal to large head sizes. They found constitutional andmosaic PIK3R2 mutations in 17 additional children.
Illumina Technology: HiSeq 2000 System

95.

Hiatt J. B., Pritchard C. C., Salipante S. J., O’Roak B. J. and Shendure J. Single molecule molecular inversion probes for targeted, high-accuracy detection of lowfrequency variation. Genome Res. 2013;23:843-854.

96.

Eboreime J., Choi S. K., Yoon S. R., Arnheim N. and Calabrese P. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation
Sequencing. PLoS One. 2016;11:e0158340.

An overview of recent DNA-seq publications featuring Illumina technology

41Carlson K. D., Sudmant P. H., Press M. O., et al. MIPSTR: a method for multiplex genotyping of germline and somatic STR variation across many individuals.
Genome Res. 2015;25:750-761.
Carvill G. L., McMahon J. M., Schneider A., et al. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures. Am J Hum Genet.
2015;96:808-815.
Kang H. C., Baek S. T., Song S. and Gleeson J. G. Clinical and Genetic Aspects of the Segmental Overgrowth Spectrum Due to Somatic Mutations in PIK3CA.
J Pediatr. 2015;167:957-962.
Weren R. D., Ligtenberg M. J., Kets C. M., et al. A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis andcolorectal cancer. Nat Genet. 2015;47:668-671.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

42

DNA Sequencing Methods Collection

MIPSTR: Targeted Capture of STR Loci by smMIPs
MIPSTR is a method for multiplex genotyping of germline and somatic short tandem repeat (STR) variation across many
individuals.97 This method is a variation of the smMIP98 approach and uses a novel mapping strategy.
The method uses a smMIP with a common backbone for a PCR primer, sequencing adapters, 12 bp degenerate tag, andtargeting arms with locus-specific and STR-flanking sequences. Capture across genetically diverse individuals identifies germline
STR variation. The use of degenerate tags identifies technical variation, and STR variation across tag-defined read groups isconsidered somatic variation.
Degenerate
molecular tag

Targeted STR

Strain I
Copy target STR

Amplify and sequence

Strain II
Natural variation between individuals

Strain I
Strain I
Somatic variation within an individual

A schematic overview of MIPSTR.

Advantages

Disadvantages••

Capable of distinguishing technical error from somatic
STR mutations

Requires high-quality reference genome

Reviews
Estivill X. Genetic variation and alternative splicing. Nat Biotechnol. 2015;33:357-359.

References
Carlson K. D., Sudmant P. H., Press M. O., Eichler E. E., Shendure J., et al. MIPSTR: a method for multiplex genotyping of germline and somatic STR
variation across many individuals. Genome Res. 2015;25:750-761.
The authors used MIPSTR to determine germline STR genotypes for 102 STR loci across diverse populations of the plant Arabidopsis thaliana. They showed that
putatively functional STRs may be identified by deviation from predicted STR variation and by association with quantitative phenotypes. They also used DNA mixing
experiments, with a mutant deficient in DNA repair, to demonstrate that MIPSTR can detect low-frequency somatic STR variants.
Illumina Technology: MiSeq System

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

97.

Carlson K. D., Sudmant P. H., Press M. O., Eichler E. E., Shendure J. and Queitsch C. MIPSTR: a method for multiplex genotyping of germline and somatic STR
variation across many individuals. Genome Res. 2015;25:750-761.

98.

Hiatt J. B., Pritchard C. C., Salipante S. J., O’Roak B. J. and Shendure J. Single molecule molecular inversion probes for targeted, high-accuracy detection of lowfrequency variation. Genome Res. 2013;23:843-854.

An overview of recent DNA-seq publications featuring Illumina technology

43

MDA: Multiple Displacement Amplification
MDA is a method commonly used for sequencing microbial genomes due to its ability to amplify templates larger than 0.5 Mbp,
but it can also be used to study genomes of other sizes.99
In this method, 3’-blocked random hexamer primers are hybridized to the template, followed by strand-displacement DNA
synthesis with Phi 29 polymerase. The method allows for efficient and rapid DNA amplification. Deep sequencing of the amplified
DNA provides accurate representation of reads, while sequencing depth provides better alignment and consensus for sequences.
Several variations on the original MDA method—such as MIDAS,100 ddMDA,101 SNES,102 and IMS-MDA103—have been developed
to improve the amplification bias and throughput.104

3’ blocked random hexamer primers

Hybridize primers

Phi 29Nascent replication fork

Synthesis

Phi 29Synthesis

S1 nuclease

Amplified DNA

A schematic overview of MDA.

Advantages

Disadvantages•••• Strong amplification bias; genome coverage as low as ~6%105• PCR biases can underrepresent GC-rich templates• Contaminated reagents can impact results106

Templates can be circular DNA (eg, plasmids, bacterial DNA)Can sequence large templates
Can perform single-cell sequencing or sequencing for samples withlimited amounts of starting material

Reviews
Sauvage V. and Eloit M. Viral metagenomics and blood safety. Transfus Clin Biol. 2016;23:28-38.
Zhang X., Marjani S. L., Hu Z., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res. 2016;76:1305-1312.
Hou Y., Wu K., Shi X., et al. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing. Gigascience.
2015;4:37.
Wang Y. and Navin N. E. Advances and applications of single-cell sequencing technologies. Mol Cell. 2015;58:598-609.

References
Newton I. L., Clark M. E., Kent B. N., et al. Comparative Genomics of Two Closely Related Wolbachia with Different Reproductive Effects on Hosts.
Genome Biol Evol. 2016;8:1526-1542.
The authors compared the genomes of 2 closely related Wolbachia pipientis strains (with 0.57% genome-wide synonymous divergence) that differ in their reproductive
effects on hosts. wVitA induces a sperm–egg incompatibility (also known as cytoplasmic incompatibility) in the parasitoid insect Nasonia vitripennis, whereas wUni
causes parthenogenetic development in a different parasitoid, Muscidifurax uniraptor. The authors performed MDA on DNA from both strains. They identified a set ofgenes whose loss or pseudogenization in the wUni lineage implicates them in the phenotypic shift from cytoplasmic incompatibility to induction of parthenogenesis.
Illumina Technology: Genome Analyzer System

99.

Dean F. B., Nelson J. R., Giesler T. L. and Lasken R. S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling
circle amplification. Genome Res. 2001;11:1095-1099.

100. Gole J., Gore A., Richards A., et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol.
2013;31:1126-1132.
101. Rhee M., Light Y. K., Meagher R. J. and Singh A. K. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA
Samples. PLoS One. 2016;11:e0153699.
102. Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
103. Seth-Smith H. M., Harris S. R., Scott P., et al. Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
Nat Protoc. 2013;8:2404-2412.
104. Seth-Smith H. M., Harris S. R., Scott P., et al. Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
Nat Protoc. 2013;8:2404-2412.
105. Navin N., Kendall J., Troge J., et al. Tumour evolution inferred by single-cell sequencing. Nature. 2011;472:90-94.
106. Woyke T., Sczyrba A., Lee J., et al. Decontamination of MDA reagents for single cell whole genome amplification. PLoS One. 2011;6:e26161.

44

DNA Sequencing Methods Collection

Salman V., Berben T., Bowers R. M., et al. Insights into the single cell draft genome of “Candidatus Achromatium palustre”. Stand Genomic Sci. 2016;11:28.
Candidatus Achromatium palustre belong to the group of large sulfur bacteria that can grow to nearly 100 μm in size and store elemental sulfur (S0) intracellularly. As aunique feature, Achromatium spp. can accumulate colloidal calcite (CaCO3) inclusions in great amounts. All Achromatium spp. are unculturable. The authors used MDA
and sequencing to produce a draft genome at 83% completion of a single Candidatus Achromatium palustre cell.
Illumina Technology: MiSeq System
Troell K., Hallstrom B., Divne A. M., et al. Cryptosporidium as a testbed for single cell genome characterization of unicellular eukaryotes. BMC Genomics.
2016;17:471.
To explore the potential use of single-cell genomics methodology for revealing genome-level variation in clinical samples from Cryptosporidium-infected hosts, theauthors sorted individual oocysts for subsequent MDA and full-genome sequencing. The genomics workflow, starting directly from fecal samples, had an 80% successrate with 81% genome coverage.
Illumina Technology: MiSeq System

Ning L., Li Z., Wang G., et al. Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using
hippocampal neurons. Sci Rep. 2015;5:11415.
In this study, the authors compared MDA, multiple annealing and looping–based amplification cycles (MALBAC), and GenomePlex amplification methods in sequencing
of individual hippocampal neurons. They amplified gDNA from individual hippocampal neurons with these methods, followed by sequencing at shallow depth on a
HiSeq 2000 system. Their results showed that single-cell sequencing results from MALBAC and GenomePlex methods were highly reproducible and had high successrates. MALBAC did display significant GC bias, but it was overcome by using bioinformatics tools. Overall, the authors determined that MALBAC and GenomePlex had
better performance in detecting CNVs compared to MDA.
Illumina Technology: HiSeq 2000 System

Brito I. L., Yilmaz S., Huang K., et al. Mobile genes in the human microbiome are structured from global to individual scales. Nature. 2016;535:435-439.
Eloe-Fadrosh E. A., Paez-Espino D., Jarett J., et al. Global metagenomic survey reveals a new bacterial candidate phylum in geothermal springs. Nat Commun.
2016;7:10476.
Hatzenpichler R., Connon S. A., Goudeau D., et al. Visualizing in situ translational activity for identifying and sorting slow-growing archaeal-bacterial consortia.
Proc Natl Acad Sci U S A. 2016;113:E4069-4078.
Moutailler S., Popovici I., Devillers E., Vayssier-Taussat M. and Eloit M. Diversity of viruses in Ixodes ricinus, and characterization of a neurotropic strain of Eyach virus.
New Microbes New Infect. 2016;11:71-81.
Ottolini C. S., Capalbo A., Newnham L., et al. Generation of meiomaps of genome-wide recombination and chromosome segregation in human oocytes. Nat Protoc.
2016;11:1229-1243.
Peterson S. W., Knox N. C., Golding G. R., et al. A Study of the Infant Nasal Microbiome Development over the First Year of Life and in Relation to Their Primary Adult
Caregivers Using cpn60 Universal Target (UT) as a Phylogenetic Marker. PLoS One. 2016;11:e0152493.
Thoendel M., Jeraldo P. R., Greenwood-Quaintance K. E., et al. Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.
J Microbiol Methods. 2016;127:141-145.
Bigdeli S., Dettloff R. O., Frank C. W., Davis R. W. and Crosby L. D. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR andwhole genome amplification. PLoS One. 2015;10:e0117738.
Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
Li N., Wang L., Wang H., et al. The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation Genetic Diagnosis of
Chromosomal Abnormalities. J Genet Genomics. 2015;42:151-159.
Macaulay I. C., Haerty W., Kumar P., et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods. 2015;.
Ottolini C. S., Newnham L. J., Capalbo A., et al. Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection
for maternal recombination rates. Nat Genet. 2015;.
Zhang C. Z., Spektor A., Cornils H., et al. Chromothripsis from DNA damage in micronuclei. Nature. 2015;522:179-184.

Associated Kits
TruSeq Nano DNA Library Prep Kit
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Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

An overview of recent DNA-seq publications featuring Illumina technology

45

MIDAS: Microwell displacement amplification system
IMS-MDA: Immunomagnetic Separation for Targeted Bacterial Enrichment for MDA
ddMDA: Digital Droplet MDA
Several variations on the original MDA method107—such as MIDAS,108 ddMDA,109 SNES,110 and IMS-MDA111—have beendeveloped to improve the amplification bias and throughput.112
Microwell and droplet displacement amplification systems are protocol variations of MDA in which single cells are randomly
distributed into hundreds to thousands of nanoliter wells or droplets, and their genetic material is simultaneously amplified.113
Sequencing individual cells simplifies the genomic diversity and reduces inherent amplification biases among cells. It results inimproved representation and sensitivity in heterogeneous cell populations. In an alternative approach to simplify genomic diversity,
IMS-MDA uses immunomagnetic separation for targeted bacterial enrichment.114

Genome3’ blocked random hexamer primers

Multiple displacement amplification (MDA).
Immunomagnetic separation for targetedbacterial enrichment for MDA (IMS-MDA)
Microwell displacement amplification
system (MIDAS)

Hybridize primers

Phi 29Nascent replication fork

Synthesis

Phi 29Synthesis

S1 nuclease

Amplified DNA

A schematic overview of MIDAS.

Advantages

Disadvantages

•

•

•
•

Templates used for this method can be circular DNA (eg, plasmids,
bacterial DNA)Can sequence large templates
Can perform single-cell sequencing or sequencing for samples withlimited amounts of starting material

PCR biases can underrepresent GC-rich templates

Reviews
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res.
2016;76:1305-1312.
Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810.
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586.
Sun H. J., Chen J., Ni B., Yang X. and Wu Y. Z. Recent advances and current issues in single-cell sequencing of tumors. Cancer Lett. 2015;365:1-10.

107. Dean F. B., Nelson J. R., Giesler T. L. and Lasken R. S. Rapid amplification of plasmid and phage DNA using Phi 29 DNA polymerase and multiply-primed rolling
circle amplification. Genome Res. 2001;11:1095-1099.
108. Gole J., Gore A., Richards A., et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol.
2013;31:1126-1132.
109. Rhee M., Light Y. K., Meagher R. J. and Singh A. K. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited DNA
Samples. PLoS One. 2016;11:e0153699.
110. Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
111. Seth-Smith H. M., Harris S. R., Scott P., et al. Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
Nat Protoc. 2013;8:2404-2412.
112. Seth-Smith H. M., Harris S. R., Scott P., et al. Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
Nat Protoc. 2013;8:2404-2412.
113. Gole J., Gore A., Richards A., et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol.
2013;31:1126-1132.
114. Seth-Smith H. M., Harris S. R., Scott P., et al. Generating whole bacterial genome sequences of low-abundance species from complex samples with IMS-MDA.
Nat Protoc. 2013;8:2404-2412.

46

DNA Sequencing Methods CollectionReferences
Rhee M., Light Y. K., Meagher R. J. and Singh A. K. Digital Droplet Multiple Displacement Amplification (ddMDA) for Whole Genome Sequencing of Limited
DNA Samples. PLoS One. 2016;11:e0153699.
The researchers amplified E. coli genomes by performing tube MDA (20 μl total volume) and ddMDA (42 μl total volume, partitioned into ~300,000 droplets of
150 pl volume), with template DNA concentrations varying from 0.1 pg/μl to 100 pg/μl. They sequenced the same mass (1 ng) of amplicon prepared from each method.
They found that ddMDA had a markedly improved quality of amplification compared to tube MDA at the same concentrations. This effect was most pronounced at low
concentrations of template DNA.
Illumina Technology: MiSeq System, Nextera XT DNA Sample Prep Kit
Sidore A. M., Lan F., Lim S. W. and Abate A. R. Enhanced sequencing coverage with digital droplet multiple displacement amplification. Nucleic Acids Res.
2016;44:e66.
The authors demonstrated the ability to amplify and sequence the genomes of single E. coli cells with 4.7 fg of starting DNA. They obtained sequence coverage
distributions similar to unamplified material.
Illumina Technology: MiSeq System
Lan F., Haliburton J. R., Yuan A. and Abate A. R. Droplet barcoding for massively parallel single-molecule deep sequencing. Nat Commun. 2016;7:11784.
To demonstrate the ability to sequence large DNA molecules, the researchers obtained synthetic read-lengths up to 10 kb from the E. coli genome. Single-molecule
droplet barcoding (SMDB) was able to detect 457 SNPs in 81 haplotypes, while conventional short-read sequencing could only detect 1 SNP and could not
generate haplotypes.
Illumina Technology: MiSeq System

Nishikawa Y., Hosokawa M., Maruyama T., et al. Monodisperse Picoliter Droplets for Low-Bias and Contamination-Free Reactions in Single-Cell Whole
Genome Amplification. PLoS One. 2015;10:e0138733.
Whole-genome amplification (WGA) is a critical component of single-cell sequencing pipelines, and MDA is the most common WGA method in single-cell sequencing.
Despite its widespread use, MDA typically produces uneven genome coverage due to amplification bias and the formation of DNA chimeras. To overcome this problem,the authors developed a droplet MDA method that minimizes these technical artifacts. They used microfluidics to compartmentalize extracted DNA fragments into
67 pl droplets, in which the individual fragments were amplified using MDA. This approach was validated by sequencing the droplet MDA products of E. coli cells, withgenome recovery improving to 89% compared to 59% using traditional MDA.
Illumina Technology: MiSeq System, Nextera XT DNA Library Prep Kit

Bigdeli S., Dettloff R. O., Frank C. W., Davis R. W. and Crosby L. D. A simple method for encapsulating single cells in alginate microspheres allows for direct PCR andwhole genome amplification. PLoS One. 2015;10:e0117738.

Eastburn D. J., Huang Y., Pellegrino M., et al. Microfluidic droplet enrichment for targeted sequencing. Nucleic Acids Res. 2015;43:e86.
Fu Y., Li C., Lu S., et al. Uniform and accurate single-cell sequencing based on emulsion whole-genome amplification. Proc Natl Acad Sci U S A. 2015;112:11923-11928.

Associated Kits
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An overview of recent DNA-seq publications featuring Illumina technology

47

MALBAC: Multiple Annealing and Looping–Based Amplification Cycles
MALBAC is intended to address some of the shortcomings of MDA.115
In this method, MALBAC primers randomly anneal to a DNA template. A polymerase with displacement activity at elevatedtemperatures amplifies the template, generating “semiamplicons.” As the amplification and annealing process is repeated, thesemiamplicons are amplified into full amplicons that have a 3’ end complementary to the 5’ end. As a result, full-amplicon ends
hybridize to form a looped structure, inhibiting further amplification of the looped amplicon, while only the semiamplicons andgDNA undergo amplification. Deep sequencing of the full-amplicon sequences allows for accurate representation of reads,
while sequencing depth provides improved alignment for consensus sequences. This method can also be applied to cDNA fortranscriptome analysis. 116
Hybridize primers

Synthesis

27 bp common sequence
8 random nucleotides

Hybridize primers

Bst DNA
polymerase

Denature

Partial amplicons

Cycles of quasilinear
amplification

Looped full
amplicons

Template

PCR

DNA

Denature

A schematic overview of MALBAC.

Advantages

Disadvantages••• Polymerase is relatively error prone, compared to Phi 29117• Temperature-sensitive protocol• Provides genome coverage up to ~90%,118 but some regions of the
genome are underrepresented consistently119

•
•
•
•

Can sequence large templates
Can perform single-cell sequencing or sequencing for samples withlimited amounts of starting material
Full-amplicon looping inhibits overrepresentation of templates,
reducing PCR bias
Can amplify GC-rich regions
Provides uniform genome coverage
Lower allele dropout rate compared to MDAl

Reviews
Leung M. L., Wang Y., Kim C., et al. Highly multiplexed targeted DNA sequencing from single nuclei. Nat Protoc. 2016;11:214-235.
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res.
2016;76:1305-1312.
Bowers R. M., Clum A., Tice H., et al. Impact of library preparation protocols and template quantity on the metagenomic reconstruction of a mock microbial community.
BMC Genomics. 2015;16:856.
Grun D. and van Oudenaarden A. Design and Analysis of Single-Cell Sequencing Experiments. Cell. 2015;163:799-810.
Hou Y., Wu K., Shi X., Li F., Song L., et al. Comparison of variations detection between whole-genome amplification methods used in single-cell resequencing.
Gigascience. 2015;4:37.
Li N., Wang L., Wang H., Ma M., Wang X., et al. The Performance of Whole Genome Amplification Methods and Next-Generation Sequencing for Pre-Implantation
Genetic Diagnosis of Chromosomal Abnormalities. J Genet Genomics. 2015;42:151-159.
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586.

115. Zong C., Lu S., Chapman A. R. and Xie X. S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science.
2012;338:1622-1626.
116. Briese M., Saal L., Appenzeller S., Moradi M., Baluapuri A. and Sendtner M. Whole transcriptome profiling reveals the RNA content of motor axons.
Nucleic Acids Res. 2016;44:e33.
117. Gole J., Gore A., Richards A., et al. Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol.
2013;31:1126-1132.
118. Lovett M. The applications of single-cell genomics. Hum Mol Genet. 2013;22:R22-26.
119. Lasken R. S. Single-cell sequencing in its prime. Nat Biotechnol. 2013;31:211-212.

48

DNA Sequencing Methods CollectionSnyder M. W., Adey A., Kitzman J. O. and Shendure J. Haplotype-resolved genome sequencing: experimental methods and applications. Nat Rev Genet.
2015;16:344-358.
Sun H. J., Chen J., Ni B., Yang X. and Wu Y. Z. Recent advances and current issues in single-cell sequencing of tumors. Cancer Lett. 2015;365:1-10.
Wang Y. and Navin N. E. Advances and applications of single-cell sequencing technologies. Mol Cell. 2015;58:598-609.

References
Gui B., Yang P., Yao Z., et al. A New Next-Generation Sequencing-Based Assay for Concurrent Preimplantation Genetic Diagnosis of Charcot-Marie-Tooth
Disease Type 1A and Aneuploidy Screening. J Genet Genomics. 2016;43:155-159.
Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A (CMT1A), accountingfor ~70% of CMT1 cases and ~50% of all CMT cases, is transmitted in an autosomal dominant manner. The authors used a MALBAC single-cell WGA method for
amplifying gDNA from a single cell. They showed that it is possible to detect the CMT1A 30 duplication, combined with screening of aneuploidy, in a single test.
Illumina Technology: HiSeq 2500 System

Mehetre G. T., Paranjpe A. S., Dastager S. G. and Dharne M. S. Complete metagenome sequencing based bacterial diversity and functional insights frombasaltic hot spring of Unkeshwar, Maharashtra, India. Genom Data. 2016;7:140-143.
In this study, the authors analyzed the microbial community of the Unkeshwar basaltic hot springs, located in the South East Deccan Continental region of Maharashtra,
India, using whole metagenome shotgun sequencing. They used MALBAC to enrich the low-abundant metagenomic DNA and created a complete representation ofboth abundant and rare members of the microbiome.
Illumina Technology: HiSeq 2500 System

Yan L., Huang L., Xu L., et al. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing
with linkage analyses. Proc Natl Acad Sci U S A. 2015;112:15964-15969.
NGS methods have improved the precision of preimplantation genetic screening and diagnosis. Although the precision has been limited by false-positive and falsenegative SNVs, linkage analysis can overcome this challenge. In this study, the authors developed MARSALA, a method that combines NGS using the HiSeq Systemwith single-cell WGA. The method allows for embryo diagnosis with single-molecule precision and reduces false-positive and false-negative errors significantly. Theirmethod is the first integrated NGS-based preimplantation genetic diagnostic procedure that simultaneously detects disease-causing mutations and chromosome
abnormalities, and performs linkage analyses.
Illumina Technology: HiSeq 2500 System

Briese M., Saal L., Appenzeller S., Moradi M., Baluapuri A., et al. Whole transcriptome profiling reveals the RNA content of motor axons. Nucleic Acids Res.
2016;44:e33.
Huang J., Yan L., Lu S., et al. Validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of blastocysts. Fertil Steril.
2016;105:1532-1536.
Chapman A. R., He Z., Lu S., et al. Single cell transcriptome amplification with MALBAC. PLoS One. 2015;10:e0120889.
Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol.
2015;33:285-289.
Ning L., Li Z., Wang G., Hu W., Hou Q., et al. Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using
hippocampal neurons. Sci Rep. 2015;5:11415.
Yan L., Huang L., Xu L., Huang J., Ma F., et al. Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation
sequencing with linkage analyses. Proc Natl Acad Sci U S A. 2015;112:15964-15969.
Zhang C. Z., Adalsteinsson V. A., Francis J., et al. Calibrating genomic and allelic coverage bias in single-cell sequencing. Nat Commun. 2015;6:6822.

Associated Kits
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An overview of recent DNA-seq publications featuring Illumina technology

49

nuc-seq: Single G2/M Nucleus Sequencing of Cells in S Phase
SNES: Single Nucleus Exome Sequencing
A modified MDA protocol, nuc-seq takes advantage of the fact that a single cell in the G2–M stage of the cell cycle has 4 copies of
the genome. This property allows the cells to be isolated with a cell sorter, and it also significantly increases the genome coverageof single cells.120 SNES is an additional variation that includes targeted selection and sequencing of the exomes.121 Div-Seq is avariation that combines nuc-seq with pulse labeling of proliferating cells by 5-ethynyl-2’-deoxyuridine (EdU).122
Cell 1
Cell 2Cell 3Cell sorting from
G2/M distribution

Lyse cell

NucleusPhi 29Limited amplification

Synthesis

S1 nucleaseDNA

A schematic overview of nuc-seq.

Methods

Advantages

Disadvantages

nuc-seq•

Improves physical coverage performance to more than
90% for single-cell sequencing•

Cannot be applied to cells with low proliferation rates

SNES

•
•

95.94% exome coverage in single cells
Detection efficiencies of 92.37% for SNVs in an isogenic
population•

Limited to exomes

Reviews
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res.
2016;76:1305-1312.
Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586.
Szulwach K. E., Chen P., Wang X., et al. Single-Cell Genetic Analysis Using Automated Microfluidics to Resolve Somatic Mosaicism. PLoS One. 2015;10:e0135007.
Voet T. and Van Loo P. SNES makes sense? Single-cell exome sequencing evolves. Genome Biol. 2015;16:86.
Wang Y. and Navin N. E. Advances and applications of single-cell sequencing technologies. Mol Cell. 2015;58:598-609.

References

Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
Despite the great potential of single-cell sequencing methods to increase the understanding of tissue heterogeneity, current single-cell DNA-Seq methods arechallenged by technical errors and poor physical coverage data. In this study, the authors developed SNES, a single-cell DNA-Seq method that combines flow-sortingof G1/0 or G2/M nuclei, time-limited MDA, exome capture using the TruSeq Exome Enrichment Kit, and sequencing on the HiSeq 2000 system. They validated SNES
by sorting and sequencing single nuclei from a fibroblast cell line. The method generated 96% coverage of individual cells and demonstrated 92% detection efficiency
for SNVs and 85% for indels in single cells.
Illumina Technology: HiSeq 2000 System, TruSeq Exome Enrichment Kit

120. Wang Y., Waters J., Leung M. L., et al. Clonal evolution in breast cancer revealed by single nucleus genome sequencing. Nature. 2014;512:155-160.
121. Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.
122. Habib N., Li Y., Heidenreich M., et al. Div-Seq: A single nucleus RNA-Seq method reveals dynamics of rare adult newborn neurons in the CNS. bioRxiv. 2016;50

DNA Sequencing Methods Collection

Leung M. L., Wang Y., Kim C., Gao R., Jiang J., et al. Highly multiplexed targeted DNA sequencing from single nuclei. Nat Protoc. 2016;11:214-235.
This study describes a refinement to SNES and includes the addition of DNA barcoding to allow multiplexing of 48–96 individual cells into single sequencing reactions.
Compared to SNES,123 this new technique has higher throughput and reduced cost. The authors suggest that it is more suitable for detecting point mutations andindels at base-pair resolution.
Illumina Technology: HiSeq 2000 System

Habib N., Li Y., Heidenreich M., Swiech L., Trombetta J. J., et al. Div-Seq: A single nucleus RNA-Seq method reveals dynamics of rare adult newborn neurons in the
CNS. bioRxiv. 2016;.

Associated Kits
TruSeq Nano DNA Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

123. Leung M. L., Wang Y., Waters J. and Navin N. E. SNES: single nucleus exome sequencing. Genome Biol. 2015;16:55.

An overview of recent DNA-seq publications featuring Illumina technology

51

OS-Seq: Oligonucleotide-Selective Sequencing
OS-Seq124 was developed to improve targeted resequencing by capturing and sequencing gene targets directly on the flow cell.
In this method, target sequences with adapters are used to modify the flow cell primers. Targets in the template are captured onto
the flow cell with the modified primers. Further extension, denaturation, and hybridization provide sequence reads for the target
genes. Deep sequencing provides accurate representation of reads.
Target sequence

Fragment and add single adapters

Sequencing Primers

Single adapter library

Target sequence

Sequence

Adapter sequence

Flow cell

Create target-specific oligos

Extend and
Denature

Extend and
Denature

HybridizeHybridizeExtend and
Denature

Sequence reads
1 and 2A schematic overview of OS-Seq.

Advantages

Disadvantages

•
•
•
•
•
•

•

Can resequence multiple targets at a time
No gel excision or narrow size-purification required
Rapid, single-day protocol
Samples can be multiplexed
Reduced PCR bias due to removal of amplification steps
Avoids loss of material

Primers may interact with similar target sequences, leading tosequence ambiguity

Reviews
None available yet.

References

Akinrinade O., Ollila L., Vattulainen S., et al. Genetics and genotype-phenotype correlations in Finnish patients with dilated cardiomyopathy. Eur Heart J.
2015;36:2327-2337.
The authors used OS-Seq to target the coding regions and splice junctions of 101 genes associated with cardiomyopathies in 145 unrelated Finnish patients withdilated cardiomyopathy (DCM). The diagnostic yield was 35.2% (familial 47.6% and sporadic 25.6%; P = 0.004) when both pathogenic and likely pathogenic variants
are considered as disease-causing. Of these, 53% were titin (TTN) truncations affecting all TTN transcripts.
Illumina Technology: MiSeq System

Vattulainen S., Aho J., Salmenpera P., et al. Accurate genetic diagnosis of Finnish pulmonary arterial hypertension patients using oligonucleotide-selective
sequencing. Mol Genet Genomic Med. 2015;3:354-362.
DNA samples from 21 Finnish patients with pulmonary arterial hypertension (PAH) with BMPR2 and ACVRL1 mutation status had been previously studied using Sanger
sequencing. In this study, the authors used OS-Seq to screen for a panel of 7 PAH genes (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3). The
sequencing panel covered 100% of the targeted base pairs with > 15x sequencing depth. The authors identified pathogenic base substitutions in the BMPR2 gene in
29% of the Finnish PAH cases. Two of the pathogenic variant–positive patients previously had tested negative using Sanger sequencing.
Illumina Technology: MiSeq System

Hopmans E. S., Natsoulis G., Bell J. M., et al. A programmable method for massively parallel targeted sequencing. Nucleic Acids Res. 2014;42:e88.

Associated Kits
TruSeq Nano DNA Library Prep Kit

124. Myllykangas S., Buenrostro J. D., Natsoulis G., Bell J. M. and Ji H. P. Efficient targeted resequencing of human germline and cancer genomes by oligonucleotideselective sequencing. Nat Biotechnol. 2011;29:1024-1027.

52

DNA Sequencing Methods CollectionSafe-SeqS: Safe-Sequencing System is a Unique Molecular Identifier (UMI)
Approach to Detect Rare Variants
Safe-SeqS (or, more commonly, Safe-Seq) is an approach that uses unique molecular identifiers (UMIs) to detect rare variants.125
In the time after the publication of the method in 2011, the use of UMIs has become ubiquitous, particularly in single-cellsequencing approaches, and the name of the method fell into disuse.
Safe-Seq assigns a UMI to each template molecule and amplifies each uniquely tagged template molecule to create UMI families.
The abundance of each UMI can be used to distinguish between rare mutations and technical errors, as well as to correct for PCR
amplification bias.
Mutation

DNA

Shear

Randomly shearedends serve as UMIs

Adapter ligation

Amplify and solid
phase capture

Sequence

Align sequences anddetermine actual ratio

TruemutantA schematic overview of Safe-SeqS.

Advantages

Disadvantages••• Can introduce spurious cross-hybridization• Complex protocol that requires a gel-purification step127• Uses 2 or 4 PCR cycles for barcode addition and does not satisfy
the basic principle of labeling each molecule with a single, uniquebarcode128

Distinguishes between rare mutations and technical errors
Detects 1 mutant template among 5000 to 1,000,000
wild-type templates126• Corrects for PCR amplification bias

Reviews
Gregory M. T., Bertout J. A., Ericson N. G., et al. Targeted single molecule mutation detection with massively parallel sequencing. Nucleic Acids Res. 2016;44:e22.
Wang K., Ma X., Zhang X., et al. Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis. Sci Rep. 2016;6:25310.
Belic J., Koch M., Ulz P., et al. Rapid Identification of Plasma DNA Samples with Increased ctDNA Levels by a Modified FAST-SeqS Approach. Clin Chem.
2015;61:838-849.
Do H. and Dobrovic A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem. 2015;61:64-71.
Heitzer E., Ulz P. and Geigl J. B. Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem. 2015;61:112-123.
Maslov A. Y., Quispe-Tintaya W., Gorbacheva T., White R. R. and Vijg J. High-throughput sequencing in mutation detection: A new generation of genotoxicity tests?
Mutat Res. 2015;776:136-143.
Patel K. M. and Tsui D. W. The translational potential of circulating tumour DNA in oncology. Clin Biochem. 2015;48:957-961.

125. Kinde I., Wu J., Papadopoulos N., Kinzler K. W. and Vogelstein B. Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad
Sci U S A. 2011;108:9530-9535.
126. Kinde I., Bettegowda C., Wang Y., et al. Evaluation of DNA from the Papanicolaou test to detect ovarian and endometrial cancers. Sci Transl Med. 2013;5:167ra164.
127. Stahlberg A., Krzyzanowski P. M., Jackson J. B., Egyud M., Stein L. and Godfrey T. E. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation
detection in liquid biopsies using sequencing. Nucleic Acids Res. 2016;44:e105.
128. Kukita Y., Matoba R., Uchida J., et al. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute
quantitation of mutations in plasma cell-free DNA from cancer patients. DNA Res. 2015;22:269-277.

An overview of recent DNA-seq publications featuring Illumina technology

53

References
Dal Molin M., Zhang M., de Wilde R. F., et al. Very Long-term Survival Following Resection for Pancreatic Cancer Is Not Explained by Commonly MutatedGenes: Results of Whole-Exome Sequencing Analysis. Clin Cancer Res. 2015;21:1944-1950.
The authors sequenced the BRAF, CDKN2A, GNAS, KRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL genes using Safe-SeqS in a panel of 27 additional surgicallyresected ductal adenocarcinomas of the pancreas, obtained from very long-term survivors. KRAS was the most commonly mutated gene, as alterations were found in
27 of 27 (100%) of these validation cancers.
Illumina Technology: Genome AnalyzerIIx System
Eboreime J., Choi S. K., Yoon S. R., Arnheim N. and Calabrese P. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next
Generation Sequencing. PLoS One. 2016;11:e0158340.
The authors used Safe-SeqS to measure ultra-rare de novo mutation frequencies in the human male germline. The results showed that the average backgroundfrequency of some mutation types was at 1.5 x 10-6 per base, which far exceeded the well-documented human genome average frequency per base pair (~10−8). This
result suggested a nonbiological explanation for the data. The authors suggest that most of the base-dependent variation in background frequency is due to a mixtureof deamination and oxidation during the first 2 PCR cycles.
Illumina Technology: HiSeq 2000 system

Wang Y., Springer S., Mulvey C. L., et al. Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas.
Sci Transl Med. 2015;7:293ra104.

Associated Kits
TruSeq Nano DNA Library Prep Kit

54

DNA Sequencing Methods CollectionDuplex-Seq: Duplex Sequencing
Duplex-Seq is a tag-based, error-correction method to improve sequencing accuracy.129
In this method, adapters (with primer sequences and random 12 bp indexes) are ligated onto the template and amplified using
PCR. Deep sequencing provides consensus sequence information from every unique molecular tag. Based on the molecular tags
and sequencing primers, duplex sequences are aligned, determining the true sequence on each DNA strand. The method
is estimated to be > 10,000-fold more accurate than conventional NGS.130 A targeted version of duplex sequencing includes
2 rounds of capture to yield read depth as high as 1,000,000x.131

A mutation occurs
on both strands

P5

α

P7

12 randombase index

Add adaptersβ
12 randombase index

P7

Random error

True variant

P5

Ligate and PCR

Sequence

Consensus

Create single strand
consensus sequence fromevery unique molecular tag

Create duplex sequences
based on molecular tags
and sequencing primers

Rare variant

A schematic overview of Duplex-Seq.

Advantages

Disadvantages••

Can detect a single point mutation among > 107 sequenced
nucleotides132• Very low error rate due to duplex tagging• PCR amplification errors can be detected and removed fromanalysis• No additional library preparation steps after addition of adapters

Complex library construction protocols133

Reviews
Gregory M. T., Bertout J. A., Ericson N. G., Taylor S. D., Mukherjee R., et al. Targeted single molecule mutation detection with massively parallel sequencing.
Nucleic Acids Res. 2016;44:e22.
Do H. and Dobrovic A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem. 2015;61:64-71.
Maslov A. Y., Quispe-Tintaya W., Gorbacheva T., White R. R. and Vijg J. High-throughput sequencing in mutation detection: A new generation of genotoxicity tests?
Mutat Res. 2015;776:136-143.

129. Schmitt M. W., Kennedy S. R., Salk J. J., Fox E. J., Hiatt J. B. and Loeb L. A. Detection of ultra-rare mutations by next-generation sequencing.
Proc Natl Acad Sci U S A. 2012;109:14508-14513.
130. Ahn E. H., Hirohata K., Kohrn B. F., Fox E. J., Chang C. C. and Loeb L. A. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex
Sequencing. PLoS One. 2015;10:e0136216.
131. Schmitt M. W., Fox E. J., Prindle M. J., et al. Sequencing small genomic targets with high efficiency and extreme accuracy. Nat Methods. 2015;12:423-425.
132.

Schmitt M. W., Kennedy S. R., Salk J. J., Fox E. J., Hiatt J. B. and Loeb L. A. Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci
U S A. 2012;109:14508-14513, Schmitt M. W., Fox E. J., Prindle M. J., et al. Sequencing small genomic targets with high efficiency and extreme accuracy.
Nat Methods. 2015;12:423-425.

133. Stahlberg A., Krzyzanowski P. M., Jackson J. B., Egyud M., Stein L. and Godfrey T. E. Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation
detection in liquid biopsies using sequencing. Nucleic Acids Res. 2016;44:e105.

An overview of recent DNA-seq publications featuring Illumina technology

55

References
Krimmel J. D., Schmitt M. W., Harrell M. I., et al. Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations
in noncancerous tissues. Proc Natl Acad Sci U S A. 2016;113:6005-6010.
The authors used Duplex-Seq to analyze TP53 mutations in 17 peritoneal fluid samples from women with high-grade serous ovarian carcinomas (HGSOCs) and20 samples from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC, at a frequency
as low as 1 mutant per 24,736 normal genomes.
Illumina Technology: HiSeq 2500 System

Ahn E. H., Hirohata K., Kohrn B. F., Fox E. J., Chang C. C., et al. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex
Sequencing. PLoS One. 2015;10:e0136216.
mtDNA is vulnerable to damage mediated by reactive oxygen species (ROS), and it is more prone to accumulating mutations than nuclear DNA. This study presents
an ultra-deep mutation analysis for the whole mtDNA genome in human breast normal epithelial cells (non–stem cells vs stem cells) using Duplex-Seq. The results
indicated that stochastic rare mutation frequency was lower in stem cells than in the corresponding non–stem cells. The authors conclude that the mitochondrial
genome is maintained with greater fidelity in stem cells than in non–stem cells.
Illumina Technology: HiSeq 2500 System

Schmitt M. W., Fox E. J., Prindle M. J., Reid-Bayliss K. S., True L. D., et al. Sequencing small genomic targets with high efficiency and extreme accuracy.
Nat Methods. 2015;12:423-425.
To detect rare mutations, the researchers adapted the Duplex-Seq protocol to include 2 rounds of capture, essentially creating a single-gene targeted duplex
sequencing method. The double-capture approach resulted in extremely high depth and uniformity of coverage up to 1,000,000x depth, with average and minimum
depths of 830,000x and 250,000x, respectively. This approach yielded an average of more than 1000 unique DNA molecules sampled at every nucleotide position
within the ABL1 gene target.
The authors used their protocol to sequence the ABL1 gene from an individual with chronic myeloid leukemia who relapsed after treatment with the targeted therapy,
imatinib. This individual did not show any ABL1 mutations with conventional high-throughput sequencing. In contrast, Duplex-Seq revealed a single mutation with amutant fraction of 1%. This mutation, E279K, confers imatinib resistance.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq Nano DNA Library Prep Kit

56

DNA Sequencing Methods Collection

DR-Seq: DNA-mRNA Sequencing
DR-Seq studies the genomic and transcriptomic relationship in single cells.134 Nucleic acid amplification prior to physicalseparation reduces sample loss and the risk of contamination. DR-Seq involves multiple amplification steps, including a quasilinear
amplification technique similar to MALBAC.
First, mRNA from lysed single-cells is reverse-transcribed using poly(dT) primers with Ad-1x adapters, producing single-stranded
cDNA. The Ad-1x adapter sequence contains cell-identifying barcodes, 5’ Illumina adaptors, and a T7 promoter. Next, bothgDNA and sscDNA are simultaneously amplified via quasilinear WGA with Ad-2 primers. These primers are similar to MALBAC
adapters, containing an 8 nt random sequence for random priming followed by a constant 27 nt tag at the 5’ end. The productsof this amplification step are split into 2 batches. One half is prepared for genome sequencing, in which gDNA is PCR-amplified
and liberated from the Ad-2 adapters before DNA library preparation and sequencing. The other half is used for transcriptome
sequencing, where double-stranded cDNA is synthesized and RNA is amplified by in vitro transcription. The resulting amplified
RNA is produced only from cDNA fragments flanked with Ad-1x and Ad-2, suppressing amplification of the gDNA fragments. The
RNA library is prepared for sequencing following the Illumina small-RNA protocol. Sequencing gDNA and mRNA from the same
cell preserves information between the genome and its expression levels.
RNA

AA(A)nAAAAAAA
TTTTTTTTTT

DNA
Single Lyse cell
cell

RT with barcoded primer

Ad-2
primerQuasilinear
amplification

SplitsamplesSecond strand
synthesis
PCR and remove
adapters

cDNA amplification
gDNA amplification
Sequence

A schematic overview of DR-Seq.

Advantages

Disadvantages

•
•

•
•
•

•
•

Interrogates genomic and transcriptomic behavior from a single cell
Amplification prior to separation reduces sample loss andcontamination
Length-based identifier used to remove duplicate reads
Quasilinear amplification reduces PCR bias

Manual single-cell isolation prevents high-throughput adaptation
Quasilinear amplification is temperature-sensitive
RNA reads are 3’-end-biased135

Reviews
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res.
2016;76:1305-1312.
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586.

References

Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol.
2015;33:285-289.
In this study, the authors describe the original DR-Seq method. They amplified gDNA and cDNA from mouse embryonic stem cells (ESCs), subsequently divided thenucleic acids for further amplification and library construction, and sequenced both libraries using a HiSeq 2500 system. They demonstrated that genes with high cellto-cell variability in transcript numbers have low CNVs, and vice versa.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq Small RNA Library Prep Kit
TruSeq Nano DNA Library Prep Kit

134. Dey S. S., Kester L., Spanjaard B., Bienko M. and van Oudenaarden A. Integrated genome and transcriptome sequencing of the same cell. Nat Biotechnol. 2015;135. Macaulay I. C., Haerty W., Kumar P., et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods. 2015;

An overview of recent DNA-seq publications featuring Illumina technology

57

G&T-Seq: Genome and Transcriptome Sequencing
G&T-seq can separate and sequence gDNA and full-length mRNA from single cells.136
In this method, single cells are isolated and lysed. RNA is captured using biotinylated oligo(dT) capture primers and separated fromDNA using streptavidin-coated magnetic beads. Smart-seq2 is used to amplify captured RNA on the bead, while MDA is used
to amplify DNA. After sequencing, integrating the DNA and RNA sequences provides insights into the gene expression profiles ofsingle cells.
RNA
DNA
Cell
suspension

Isolate single
cell

Lyse cellAA(A)nAAAAAAA
TTTTTTTTTTAAAAAAA
TTTTTTTTTT

Streptavidin magnetic beadwith mRNA capture primer

Separate the DNA and the RNA

On-bead transcriptome
amplification with Smart-seq2
Whole genome amplification
with MDA
Sequence

Align RNA andgenomeA schematic overview of G&T-seq.

Advantages

Disadvantages

•
•

•

•

Compatible with any WGA method
No 3’-end bias in sequence reads because full-length transcripts
are captured
Because DNA and RNA are physically separated and amplified
independently, there is no need to mask coding sequences during
analysis•

Physical separation of DNA and RNA can increase risk of sample
loss or contamination
Increased handling time

Reviews
Zhang X., Marjani S. L., Hu Z., Weissman S. M., Pan X., et al. Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects. Cancer Res.
2016;76:1305-1312.
Saadatpour A., Lai S., Guo G. and Yuan G. C. Single-Cell Analysis in Cancer Genomics. Trends Genet. 2015;31:576-586.

References

Macaulay I. C., Haerty W., Kumar P., et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods. 2015;12:519-522.
The authors performed G&T-seq-enabled transcriptome analysis using a modified Smart-Seq2 protocol,137,138 and they automated the method on a robotic liquidhandling platform. The authors sequenced human cancer cells, reversine-treated mouse embryo blastomeres, and neurons derived from induced pluripotent stem cells(iPSCs). Notably, G&T-seq analysis of aneuploid blastomeres demonstrated that chromosomal gains/losses led to increases/losses in chromosome-wide relative gene
expression, during a single cell division.
Illumina Technology: MiSeq System, HiSeq 2500 System, HiSeq X Ten System, Nextera XT DNA Library Prep Kit

Associated Kits
TruSeq RNA Library Prep Kit v2
TruSeq Nano DNA Library Prep Kit

136. Macaulay I. C., Haerty W., Kumar P., et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods. 2015;
137. Picelli S., Bjorklund A. K., Faridani O. R., Sagasser S., Winberg G. and Sandberg R. Smart-seq2 for sensitive full-length transcriptome profiling in single cells.
Nat Methods. 2013;10:1096-1098.
138. Picelli S., Faridani O. R., Bjorklund A. K., Winberg G., Sagasser S. and Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc.
2014;9:171-181.

58

DNA Sequencing Methods Collection

scM&T-Seq: Single-Cell Methylome and Transcriptome Sequencing
scM&T-seq allows parallel analysis of both epigenetic and gene expression patterns from single cells using Smart-seq2 and scBSseq.139 scM&T-Seq is based on G&T-seq, but instead of using MDA for DNA sequencing, it uses scBS-Seq to determine DNA
methylation patterns.
Single cells are isolated and individually lysed. The mRNAs are captured with streptavidin-coupled mRNA capture primers toseparate them physically from the DNA strands. Smart-seq2 uses RT with template switching and tagmentation to generate cDNA
libraries from the mRNA. DNA libraries are prepared via scBS-seq, which involves bisulfite conversion of DNA strands to identify
methylated cytosines. Both libraries are ready for sequencing.
RNA
DNA
Cell
suspension

Isolate single
cell

Lyse cellAA(A)nAAAAAAA
TTTTTTTTTTAAAAAAA
TTTTTTTTTT

Streptavidin magnetic beadwith mRNA capture primer

Separate the DNA and the RNA

On-bead transcriptome
amplification with Smart-seq2
Whole genome bisulfite
sequencing with scBS-seq
Sequence

Align RNA andmethylome

A schematic overview of scM&T-seq.

Advantages

Disadvantages•• Smart-seq2 is not strand-specific and applicable to only poly(A)+ RNA• Does not distinguish between 5-methylcytosine (5mC) and
5-hydroxymethylcytosine (5hmC)•

Investigates links between epigenetic and transcriptional
heterogeneity in single cells
Because DNA and RNA are physically separated and amplified
independently, there is no need to mask coding sequences
during analysis

Reviews
Clark S. J., Lee H. J., Smallwood S. A., Kelsey G. and Reik W. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.
Genome Biol. 2016;17:72.
Wen L. and Tang F. Single-cell sequencing in stem cell biology. Genome Biol. 2016;17:71.

References
Angermueller C., Clark S. J., Lee H. J., Macaulay I. C., Teng M. J., et al. Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity.
Nat Methods. 2016;13:229-232.
The authors used the G&T-seq protocol to purify single-cell DNA that was then subjected to single-cell bisulfite sequencing (scBS-seq).140 They performed single-cellmethylome and transcriptome sequencing (scM&T-seq) on 61 mouse ESCs. They found that gene expression levels of many pluripotency factors were negatively
associated with DNA methylation. These data demonstrate that epigenetic heterogeneity is an important mechanism of fluctuating pluripotency in ESCs. They also
demonstrate that scM&T-seq can reveal the relationship between transcriptional and DNA-methylation heterogeneity in single cells.
Illumina Technology: HiSeq 2000 System, Nextera XT DNA Library Prep Kit

Associated Kits
Nextera XT DNA Library Preparation Kit

139. Angermueller C., Clark S. J., Lee H. J., et al. Parallel single-cell sequencing links transcriptional and epigenetic heterogeneity. Nat Methods. 2016;13:229-232.
140. Smallwood S. A., Lee H. J., Angermueller C., et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817-820.

An overview of recent DNA-seq publications featuring Illumina technology

59

scTrio-seq: Single-Cell Triple Omics Sequencing
scTrio-seq can analyze genomic CNVs, the DNA methylome, and the transcriptome of an individual mammalian cell
simultaneously.141 This approach is an extension of previous methods, such as scMT-seq.142
A single cell is lysed with a mild lysis buffer and the lysis product centrifuged. The supernatant is transferred to a new tube
for transcriptome sequencing analyses, while the pellet (containing the nucleus) is bisulfite-converted for genome andepigenome sequencing.
RNA
SupernatantDNA
Nucleus
Cell
Isolatesuspension single
cell

Lyse andcentrifuge

AA(A)nAdd carrier RNA

AA(A)nT T (T)nHybridize oligo

AA(A)ncDNA synthesis

Add poly(A) with TDT

Add sequencing adapters

PCR and sequence

PCR and sequence

Methylated DNA
MspI
digestion

Methylated Methylated
regions
adapter

End repair
and ligation

Bisulfite
conversion

Convertedfragments

PCR and sequence

Align sequences

A schematic overview of scTrio-seq.

Advantages

Disadvantages

•

•
•

•

Accurately analyzes the mechanism by which the transcriptome,
genome, and DNA methylome regulate each other
CNVs can be identified reliably using single-cell reducedrepresentation bisulfite sequencing (scRRBS) data

Lower transcriptome coverage than scMT-seq141
Results in 3’-biased transcriptome

Reviews
None available yet.

References
Hou Y., Guo H., Cao C., Li X., Hu B., et al. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular
carcinomas. Cell Res. 2016;26:304-319.
The authors used scTrio-seq to analyze 25 single cells derived from a human hepatocellular carcinoma (HCC) tissue sample. They found 2 subpopulations distinct in
DNA copy numbers, DNA methylation, and RNA expression levels. By comparing the differences between the 2 HCC subpopulations, the researchers discovered thatthe minor subpopulation I, which harbored more copy-gain CNVs, expressed more invasive cell markers and was more likely to evade immune surveillance.
Illumina Technology: HiSeq 2000 System, HiSeq 2500 System

Associated Kits
TruSeq Nano DNA Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

141. Hou Y., Guo H., Cao C., et al. Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas. Cell Res.
2016;26:304-319.
142. Hu Y., Huang K., An Q., et al. Simultaneous profiling of transcriptome and DNA methylome from a single cell. Genome Biol. 2016;17:88.

60

DNA Sequencing Methods Collection

scBS-Seq: Single-Cell Bisulfite Sequencing
scBS-seq is a version of the well-established bisulfite sequencing (BS-seq) and post-bisulfite adapter tagging (PBAT) protocols,
modified to detect methylated cytosines in gDNA from single cells.143
In this method, after single cells are isolated, gDNA is treated with sodium bisulfite, which fragments the DNA. The converted DNA
then undergoes random priming several times and is PCR-amplified for sequencing. Deep sequencing provides single-nucleotide
resolution mapping of methylated cytosines.
Random primer 1

IsolatedLysesingle cell

Methylated DNA

Bisulfite
conversion

Random primer 2

AdapterFirst randompriming

Repeat
4 times

ExtendExo I andpurifyAdapter
PCR
Second randompriming

Align fragments from every Sequence
unique molecular tag

A schematic overview of scBS-seq.

Advantages

Disadvantages••

Measure DNA methylation of up to 48% (10.1 M) CpG sites within
a single cell

DNA degradation after bisulfite step144

Reviews
Pisanic T. R., 2nd, Athamanolap P. and Wang T. H. Defining, distinguishing and detecting the contribution of heterogeneous methylation to cancer heterogeneity.
Semin Cell Dev Biol. 2016;.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.
Greenleaf W. J. Assaying the epigenome in limited numbers of cells. Methods. 2015;72:51-56.
Liang J., Cai W. and Sun Z. Single-cell sequencing technologies: current and future. J Genet Genomics. 2014;41:513-528.
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.

References
Smallwood S. A., Lee H. J., Angermueller C., Krueger F., Saadeh H., et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic
heterogeneity. Nat Methods. 2014;11:817-820.
The authors tested the performance of scBS-seq on ovulated metaphase II mouse oocytes. They obtained methylation scores on an average of 3.7 million CpG
dinucleotides, corresponding to 17.7% of all CpGs.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

143. Smallwood S. A., Lee H. J., Angermueller C., et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817-820.
144. Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

An overview of recent DNA-seq publications featuring Illumina technology

61

scAba-seq: Detect 5hmC Marks in Single Cells With AbaSI NucleasescAba-seq is a single-cell, genome-wide, and strand-specific 5hmC sequencing technology.145
5hmC marks in DNA from individual cells are glucosylated with T4 phage b-glucosyltransferase (T4-bGT), and the DNA is digested
with the restriction endonuclease AbaSI. The digested DNA is ligated to an adapter containing a cell-specific barcode, an Illumina
5’ adapter, and a T7 promoter. The ligated DNA from different cells is pooled and amplified using in vitro transcription mediated by
T7 RNA polymerase.
Adapter with cell-specific barcode
Illumina 5’ adapter
T7 promoter

Hydroxy-methylated DNA

T4-βGT

Glucosylated 5-hmC

AbaSI

PrimerLigatePool

T7 amplificationDNA

A schematic overview of scAba-seq.

Advantages

Disadvantages•• Ambiguity can exist in 13% of the cleaved molecules146• Sequence bias in AbaSI activity147

Determines strand-specific 5hmC marks in individual cells

Reviews
None available yet.

References
Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability
and enables lineage reconstruction. Nat Biotechnol. 2016;34:852-856.
In this study, the authors describe the development of the scAba-seq method. They applied it in 2- and 4-cell mouse embryos to reconstruct their lineage. Their
analysis of individual chromosomes in the sister cells of the 2-cell embryos showed a strong anticorrelation between the bias of the chromosomes from sister cell 1
and sister cell 2. This observation is consistent with each sister cell receiving an old strand, containing a high level of 5hmC marks, and a new strand containing fewer
5hmC marks.
Illumina Technology: NextSeq 500 System

Associated Kits
TruSeq Nano DNA Library Prep Kit

145. Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability andenables lineage reconstruction. Nat Biotechnol. 2016;34:852-856.
146. Serandour A. A., Avner S., Mahe E. A., et al. Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies
evolutionarily unconserved CpGs as TET targets. Genome Biol. 2016;17:56.

62

DNA Sequencing Methods Collection

Single-cell RC-Seq: Single-Cell Retrotransposon Capture Sequencing
Single-cell RC-seq uses sequence capture to enrich DNA for the junctions between retrotransposon termini and adjacent genomic
regions, followed by paired-end sequencing.148
In this method, nuclei are purified first by fluorescence-activated cell sorting (FACS) and then picked with a micromanipulator. The
DNA is extracted from nuclei and subjected to linear WGA, followed by exponential PCR in 2 separate reactions for each nucleus,
using different enzymes. The products are combined and analyzed via WGS to assess genome coverage. The libraries preparedare enriched by hybridization to locked nucleic acid (LNA) probes and sequenced.147
Retrotransposon
binding site

Genomic DNA

Cellsuspension

FACS
isolation

Pick
nuclei

Nucleus

Whole-genome
amplification

Create sequencing
library

Sequence capture

Enriched library

A schematic overview of single-cell RC-seq.

Advantages

Disadvantages••

Detects somatic mutations in single cells

Low throughput due to manual single-nucleus isolation

Reviews
None available yet.

References

Upton K. R., Gerhardt D. J., Jesuadian J. S., Richardson S. R., Sanchez-Luque F. J., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell.
2015;161:228-239.
The authors carried out single-cell RC-seq on individual human hippocampal neurons and glia, as well as cortical neurons. They found that an estimated 13.7
somatic long interspersed nuclear elements (LINE-1 or L1) insertions occurred per hippocampal neuron and carried the sequence hallmarks of target-primed reverse
transcription. Hippocampal neuron L1 insertions were specifically enriched in transcribed neuronal stem cell enhancers and hippocampus genes, which may indicatethat they are functionally relevant.
Illumina Technology: MiSeq System

Associated Kits
TruSeq Nano DNA Library Prep Kit
Nextera DNA Library Prep Kit

147. Sun Z., Dai N., Borgaro J. G., et al. A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol Cell.
2015;57:750-761.
148. Upton K. R., Gerhardt D. J., Jesuadian J. S., et al. Ubiquitous L1 mosaicism in hippocampal neurons. Cell. 2015;161:228-239.

An overview of recent DNA-seq publications featuring Illumina technology

63scATAC-Seq (Cell index variation): Single-Cell Assay for TransposaseAccessible Chromatin
This version of scATAC-seq uses combinatorial cellular indexing to measure chromatin accessibility in thousands of single cells
per assay. This method avoids the need for compartmentalization of individual cells, which makes the system scalable to analyze
thousands of cells at a time.149
In this method, nuclei are isolated and molecularly tagged in bulk with barcoded Tn5 transposases in wells. Next, the nuclei arepooled and a limited number redistributed into a second set of wells. A second barcode is introduced during the PCR amplification
step, and the fragments are pooled for library preparation and sequencing.

Cell suspension

Isolate nuclei

SplitsampleBarcode each wellwith Tn5 transposase

Pool anddiluteSplit sample

PCR-barcode
every well

Pool for library
prep

DNA

A schematic overview of scATAC-seq: combinatorial indexing version.

Advantages

Disadvantages••

High throughput and scalable

None reported

Reviews
Liu S. and Trapnell C. Single-cell transcriptome sequencing: recent advances and remaining challenges. F1000Res. 2016;5:.
Lu F., Liu Y., Inoue A., et al. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development. Cell. 2016;165:1375-1388.

References
Corces M. R., Buenrostro J. D., Wu B., et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution.
Nat Genet. 2016;48:1193-1203.
This study used an improved scATAC-seq protocol, named Fast-ATAC, to create a comprehensive map of chromatin accessibility and transcriptional landscapes of
13 human primary blood cell types in a total of 137 samples. The authors found that single acute myeloid leukemia (AML) cells exhibited distinctive mixed regulome
profiles, corresponding to disparate developmental stages, possibly caused by HOX factors. This result could provide insights into hematopoietic development
and disease.
Illumina Technology: HiSeq 2000 System, HiSeq 2500 System, NextSeq 500 System

Cusanovich D. A., Daza R., Adey A., Pliner H. A., Christiansen L., et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellularindexing. Science. 2015;348:910-914.
The researchers applied combinatorial cellular indexing with scATAC-seq to measure chromatin accessibility in thousands of single cells per assay and reported
chromatin accessibility maps for > 15,000 single cells. They predicted that their combinatorial cellular indexing scheme could be scaled feasibly to collect data from
~17,280 cells per experiment by using 384-by-384 barcoding and sorting 100 nuclei per well.
Illumina Technology: MiSeq System

Associated Kits
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

149. Cusanovich D. A., Daza R., Adey A., et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015;348:910-914.

64

DNA Sequencing Methods Collection

scATAC-Seq (Microfluidics variation): Single-Cell Assay for TransposaseAccessible Chromatin
This version of scATAC-seq is integrated into a programmable microfluidics platform.150 The method is similar to scATAC-seq(combinatorial indexing version), which relies on indexing to identify single cells.151
In this protocol the cells are captured in a fluidics device, lysed, and treated with Tn5 transposases. The ends of the Tn5 fragments
are extended, and the fragments are PCR-amplified with dual-index primers, pooled, purified, and sequenced.
Microfluidics device

Cellsuspension

Isolatesingle cell

Lyse and introduce Insert in regions of open chromatin
Tn5 transposase

Fragmented and primed

Amplify withcell-specificbarcodesPool libraries
from all cellsDNA

A schematic overview of scATAC-seq: microfluidics version.

Advantages

Disadvantages••

Maps the accessible genome of individual cells

Requires dedicated fluidics

Reviews
Liu S. and Trapnell C. Single-cell transcriptome sequencing: recent advances and remaining challenges. F1000Res. 2016;5:.
Lu F., Liu Y., Inoue A., Suzuki T., Zhao K., et al. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development. Cell. 2016;165:1375-1388.

References
Buenrostro J. D., Wu B., Litzenburger U. M., Ruff D., Gonzales M. L., et al. Single-cell chromatin accessibility reveals principles of regulatory variation.
Nature. 2015;523:486-490.
Using scATAC-seq (microfluidics version), the researchers investigated single-cell epigenomic heterogeneity and were able to link cis- and trans-effectors to variability inaccessibility profiles within individual epigenomes. They also identified trans-factors associated with increased accessibility variance. Other trans-factors, such as CTCF,
appeared to buffer variability, perhaps by providing a stable anchor for chromatin accessibility or an insulator function that dampens potential fluctuations.
Illumina Technology: MiSeq System, HiSeq 2500 System, NextSeq 500 System

Associated Kits
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

150. Buenrostro J. D., Wu B., Litzenburger U. M., et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 2015;523:486-490.
151. Cusanovich D. A., Daza R., Adey A., et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015;348:910-914.

An overview of recent DNA-seq publications featuring Illumina technology

65

Drop-ChIP or scChIP-seq: Droplet-Based Single-Cell Chromatin
Immunoprecipitation Sequencing
Drop-ChIP or scChIP-seq analyzes the chromatin states of single cells by using microfluidics, unique molecular barcodes,
and NGS.152
In this method, single cells are isolated into droplets containing lysis buffer and micrococcal nuclease (MNase), and then fused toa droplet carrying distinct oligonucleotides. These oligonucleotides contain the sequences for cell-specific barcodes, sequencing
adapters, and restriction sites. In addition, DNA ligase is fused with the droplet to complete the tagging process. Next, carrier
chromatin is introduced into the pooled droplets before chromatin immunoprecipitation. Library preparation is carried out
according to standard ChIP-Seq procedures before sequencing.
Droplet withunique oligos
Cellsuspension

Load single cells into dropletswith lysis buffer and MNase

Fuse droplets

Pool all droplets

Chromatin immunoprecipitation

Sequence

Barcoded sequencesfrom single cells

A schematic overview of Drop-ChIP.

Advantages

Disadvantages

•

•

•

Analyzes chromatin states from single cells in a highly
parallel manner
Unique molecular barcoding reduces the risk posed byunspecific antibodies

Data from each cell are sparse

Reviews
Clark S. J., Lee H. J., Smallwood S. A., Kelsey G. and Reik W. Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.
Genome Biol. 2016;17:72.
Wen L. and Tang F. Single-cell sequencing in stem cell biology. Genome Biol. 2016;17:71.
Shin J., Ming G. L. and Song H. Decoding neural transcriptomes and epigenomes via high-throughput sequencing. Nat Neurosci. 2014;17:1463-1475.

References
Rotem A., Ram O., Shoresh N., Sperling R. A., Goren A., et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.
Nat Biotechnol. 2015;.
The authors demonstrated the utility of scChIP-seq by assaying thousands of individual cells. They used the data to deconvolute a mixture of ESCs, fibroblasts,
and hematopoietic progenitors into high-quality chromatin state maps for each cell type. By assaying thousands of ESCs, they were able to identify a spectrum of
subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq ChIP Library Prep Kit

152. Rotem A., Ram O., Shoresh N., et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015;66

DNA Sequencing Methods CollectionSMDB: Single-Molecule Droplet Barcoding
SMDB is a method to leverage short-read sequencing to obtain long and accurate reads.153
Using a microfluidics system, the method isolates, amplifies, fragments, and barcodes single DNA molecules in aqueous picoliter
droplets. This approach allows the full-length molecules to be sequenced with multi-fold coverage, using short-read sequencing.

DNA templates

Single-template
encapsulation

Template amplification

Template fragmentation

Barcode every droplet

Pool for library
prep

DNA

A schematic overview of SMDB.

Advantages

Disadvantages••

Limit of detection scales with the number of molecules sequenced andcan be orders of magnitude more sensitive than conventional NGS

Requires specialized hardware

Reviews
None available yet.

References

Lan F., Haliburton J. R., Yuan A. and Abate A. R. Droplet barcoding for massively parallel single-molecule deep sequencing. Nat Commun. 2016;7:11784.
To demonstrate the ability of SMDB to detect rare SNPs confidently, the authors generated a population of DNA templates via 35 cycles of PCR with a bacterial plasmid
extracted from a culture grown from a single colony. In a population such as this, every sequence shares significant homology, but rare variants exist. The authors
identified 457 high-confidence SNPs in ~10% of templates, whereas ~90% of the templates contained no SNPs compared to the reference. A conventional analysis
without barcodes identified only 1 SNP.
Illumina Technology: MiSeq System

Associated Kits
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

153. Lan F., Haliburton J. R., Yuan A. and Abate A. R. Droplet barcoding for massively parallel single-molecule deep sequencing. Nat Commun. 2016;7:11784.

An overview of recent DNA-seq publications featuring Illumina technology

67

EPIGENETICS
DNA methylation and hydroxymethylation are involved in development, X-chromosome inactivation, cell differentiation, tissuespecific gene expression, plant epigenetic variation, imprinting, and cancers and other diseases.154-157 Methylation usually occurs at
the 5’ position of cytosines, and it plays a crucial role in gene regulation and chromatin remodeling.

The active agouti gene in mice codes for yellow coat color. When pregnant mice with the active agouti gene are fed a diet rich in methyl donors, the offspring areborn with the agouti gene turned off. This effect has been used as an epigenetic biosensor for nutritional and environmental alterations of the fetal epigenome.159

Most cytosine methylation occurs on cytosines located near guanines, called CpG sites. These CpG sites are often locatedupstream of promoters, or within the gene body. CpG islands are defined as regions that are greater than 500 bp in length withgreater than 55% GC and an expected/observed CpG ratio of > 0.65.
While cytosine methylation (5mC) is known as a silencing mark that represses genes, cytosine hydroxymethylation (5hmC)
is shown to be an activating mark that promotes gene expression and is a proposed intermediate in the DNA demethylation
pathway.1,4,6 Similar to 5mC, 5hmC is involved during development, cell differentiation, and cancers and other diseases.160
5mC and/or 5hmC can be a diagnostic tool to help identify the effects of nutrition, carcinogens,161 and environmental factors in
relation to diseases. The impact of these modifications on gene regulation depends on their locations within the genome. It istherefore important to determine the exact position of the modified bases.

154. Smith Z. D. and Meissner A. DNA methylation: roles in mammalian development. Nat Rev Genet. 2013;14:204-220.
155. Jullien P. E. and Berger F. DNA methylation reprogramming during plant sexual reproduction? Trends Genet. 2010;26:394-399.
156. Schmitz R. J., He Y., Valdes-Lopez O., et al. Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population. Genome Res.
2013;23:1663-1674.
157. Koh K. P. and Rao A. DNA methylation and methylcytosine oxidation in cell fate decisions. Curr Opin Cell Biol. 2013;25:152-161.
158. Dolinoy D. C., Weidman J. R., Waterland R. A. and Jirtle R. L. Maternal genistein alters coat color and protects Avy mouse offspring from obesity by modifying
the fetal epigenome. Environ Health Perspect. 2006;114:567-572.
159. Dolinoy D. C. The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome. Nutr Rev. 2008;66
Suppl 1:S7-11, Dolinoy D. C. and Faulk C. Introduction: The use of animals models to advance epigenetic science. ILAR J. 2012;53:227-231.
160. Pfeifer G. P., Kadam S. and Jin S. G. 5-hydroxymethylcytosine and its potential roles in development and cancer. Epigenetics Chromatin. 2013;6:10.
161. Thomson J. P., Lempiainen H., Hackett J. A., et al. Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome. Genome Biol.
2012;13:R93.

68

DNA Sequencing Methods CollectionSequencing Reads Created by Various Bisulfite Conversion Methods
Base

Sequence

BS Sequence

oxBS Sequence

TAB Sequence

RRBS Sequence

C

C

T

T

T

T

5mC

C

C

C

T

C

5hmC

C

C

T

C

C

BS, bisulfite sequencing; oxBS, oxidative bisulfite sequencing; TAB, TET-assisted bisulfite sequencing; RRBS, reduced-representation bisulfite sequencing.

Reviews
Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights from advances ingenomic technologies. Neurosci Lett. 2016;625:47-55.
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

References
Bak M., Boonen S. E., Dahl C., et al. Genome-wide DNA methylation analysis of transient neonatal diabetes type 1 patients with mutations in ZFP57. BMC Med Genet.
2016;17:29.
Prezza N., Vezzi F., Kaller M. and Policriti A. Fast, accurate, and lightweight analysis of BS-treated reads with ERNE 2. BMC Bioinformatics. 2016;17 Suppl 4:69.

An overview of recent DNA-seq publications featuring Illumina technology

69

BS-Seq, Bisulfite-seq, and WGBS: Whole-Genome Bisulfite Sequencing (WGBS)
BS-Seq/Bisulfite-seq or WGBS is a well-established protocol to detect methylated cytosines in gDNA.162
In this method, gDNA is treated with sodium bisulfite and then sequenced, providing single-base resolution of methylated
cytosines in the genome. Upon bisulfite treatment, unmethylated cytosines are deaminated to uracils which, upon sequencing, areconverted to thymidines. Simultaneously, methylated cytosines resist deamination and are read as cytosines. The location of themethylated cytosines can then be determined by comparing treated and untreated sequences. Bisulfite treatment of DNA converts
unmethylated cytosines to thymidines, leading to reduced sequence complexity. Very accurate deep sequencing serves to mitigate
this loss of complexity.
The TruSeq DNA Methylation Kit (previously known as the EpiGnome Methyl-Seq Kit) uses a unique library construction methodthat incorporates bisulfite conversion as the first step.
C

T

T

C

G

T

C

G

T

C

G

Bisulfite

T

U
PCR

Methylated DNA

Shear DNA

Bisulfite conversion

T

T

DNA

A schematic overview of BS-Seq.

T

C

T

C

G

T

C

G

T

C

G

Bisulfite

T

U
PCR

Methylated DNA

Bisulfite conversion

T

T

Converted single-strandedfragments

Random priming
DNA synthesis

3’ tagging

PCR

DNA

A schematic overview of the TruSeq DNA Methylation Kit procedure.

Advantages

Disadvantages

BS-Seq or WGBS
• CpG and non-CpG methylation throughout the genome is coveredat single-base resolution• Covers 5mCs in dense and less dense repeat regions•••

TruSeq DNA Methylation Kit• Pre-library bisulfite conversion• Low input gDNA (50 ng)• Provides uniform CpG, CHG, and CHH coverage• No fragmentation and no methylated adapters• Retains sample diversity

•
•
•
•

Bisulfite converts unmethylated cytosines to thymidines, reducing
sequence complexity, which can make it difficult to create alignments
SNPs where a cytosine is converted to thymidine will be missed uponbisulfite conversion
Bisulfite conversion does not distinguish between 5mC and 5hmC
Bisulfite converts unmethylated cytosines to thymidines,
reducing sequence complexity, which can make it difficult tocreate alignments
SNPs where a cytosine is converted to thymidine will be missed
upon bisulfite conversion
Bisulfite conversion does not distinguish between 5mC and 5hmC
Higher duplicate percentage

Reviews
Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights from advances ingenomic technologies. Neurosci Lett. 2016;625:47-55.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

162. Feil R., Charlton J., Bird A. P., Walter J. and Reik W. Methylation analysis on individual chromosomes: improved protocol for bisulphite genomic sequencing.
Nucleic Acids Res. 1994;22:695-696.

70

DNA Sequencing Methods CollectionReferences
Derks M. F., Schachtschneider K. M., Madsen O., et al. Gene and transposable element methylation in great tit (Parus major) brain and blood.
BMC Genomics. 2016;17:332.
The authors used WGBS of whole blood and brain tissue in the great tit (Parus major) to correlate gene features, CpG islands (CGIs), TEs, and their functional roles.
They found that 6877 (~21%) of the CGIs were differentially methylated between blood and brain, of which 1186 and 2055 were annotated to promoter and intragenic
regions, respectively. The dominant sequence motifs for brain non-CpG methylation were similar to those found in mammals, suggesting that a conserved non-CpG
regulatory mechanism was already present in a common ancestor.
Illumina Technology: HiSeq 2000 System, TruSeq Stranded RNA Sample Preparation Kit

Lu Y. C., Feng S. J., Zhang J. J., et al. Genome-wide identification of DNA methylation provides insights into the association of gene expression in rice
exposed to pesticide atrazine. Sci Rep. 2016;6:18985.
The authors used BS-seq to identify the pattern of cytosine methylation in atrazine (ATR)-exposed rice and detected altered DNA methylation patterns in the genome.
They profiled the transcriptome of ATR-exposed rice to reveal the link between DNA methylation and gene expression.
Illumina Technology: Genome AnalyzerIIx System, HiSeq 2000 System
Wang X., Werren J. H. and Clark A. G. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA
Methylation in Nasonia. PLoS Biol. 2016;14:e1002500.
In this study, the authors quantified allele-specific expression and methylation genome-wide in the parasitic wasp species Nasonia vitripennis and Nasonia giraulti, as
well as their reciprocal F1 hybrids. They found no parent-of-origin effect in allelic expression for > 8000 covered genes, suggesting a lack of genomic imprinting in adult
Nasonia. All 178 differentially methylated genes were also differentially methylated between the 2 alleles in F1 hybrid offspring, recapitulating the parental methylation
status with nearly 100% fidelity. The authors suggest that this effect may be due to the presence of strong cis-elements driving the target of gene-body methylation.
Illumina Technology: HiSeq 2000 System, TruSeq RNA Sample Prep Kit v2
Zhang Y., Zhang D., Li Q., et al. Nucleation of DNA repair factors by FOXA1 links DNA demethylation to transcriptional pioneering. Nat Genet. 2016;48:1003-1013.
The researchers performed WGBS in human mammary carcinoma MCF-7 cells and obtained methylomes at single-base resolution in 2 replicates, with a combined
total of 886 million mapped reads (100 bp paired-end reads). Genome-wide DNA methylomes demonstrated that the FOXA1 DNA repair complex was functionallylinked to DNA demethylation in a lineage-specific fashion. Depletion of FOXA1 resulted in localized reestablishment of methylation in a large portion of FOXA1bound regions.
Illumina Technology: HiSeq 2500/4000 System

Bogdanovic O., Smits A. H., de la Calle Mustienes E., et al. Active DNA demethylation at enhancers during the vertebrate phylotypic period. Nat Genet. 2016;48:417-426.
Groth M., Moissiard G., Wirtz M., et al. MTHFD1 controls DNA methylation in Arabidopsis. Nat Commun. 2016;7:11640.
Jeong Y. H., Lu H., Park C. H., et al. Stochastic anomaly of methylome but persistent SRY hypermethylation in disorder of sex development in canine somatic cell
nuclear transfer. Sci Rep. 2016;6:31088.
Kaaij L. J., Mokry M., Zhou M., et al. Enhancers reside in a unique epigenetic environment during early zebrafish development. Genome Biol. 2016;17:146.
Qu W., Tsukahara T., Nakamura R., et al. Assessing Cell-to-Cell DNA Methylation Variability on Individual Long Reads. Sci Rep. 2016;6:21317.
Rehan S. M., Glastad K. M., Lawson S. P. and Hunt B. G. The Genome and Methylome of a Subsocial Small Carpenter Bee, Ceratina calcarata. Genome Biol Evol.
2016;8:1401-1410.
Thienpont B., Steinbacher J., Zhao H., et al. Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature. 2016;537:63-68.
Wallner S., Schroder C., Leitao E., et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics Chromatin. 2016;9:33.
Yang Y., Zhou R., Mu Y., et al. Genome-wide analysis of DNA methylation in obese, lean, and miniature pig breeds. Sci Rep. 2016;6:30160.
Zhang X., Su J., Jeong M., et al. DNMT3A and TET2 compete and cooperate to repress lineage-specific transcription factors in hematopoietic stem cells. Nat Genet.
2016;48:1014-1023.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

An overview of recent DNA-seq publications featuring Illumina technology

71

PBAT: Post-bisulfite Adapter Tagging
In PBAT, bisulfite treatment precedes adaptor tagging to avoid the bisulfite-induced fragmentation of adaptor-tagged
template DNAs.163
Bisulfite treatment is followed by adapter tagging and 2 rounds of random-primer extension. This procedure generates a
substantial number of unamplified reads from subnanogram quantities of DNA.164
Streptavidin

Random primer 2

BiotinAdapter

Methylated DNA

Bisulfite
conversion

AdapterFirst randompriming

Second randompriming

Capture first strand on Streptavidin-coated magnetic beads

Generate second strand

Elution

DNA with adapters

A schematic overview of PBAT.

Advantages

Disadvantages••

Requires only 100 ng of DNA for amplification-free WGBS of
mammalian genomes••

Bisulfite converts unmethylated cytosines to thymidines, reducing
sequence complexity, which can make it difficult to create alignments
SNPs where a cytosine is converted to thymidine will be missed uponbisulfite conversion
Bisulfite conversion does not distinguish between 5mC and 5hmC

Reviews
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

References

Guo F., Yan L., Guo H., et al. The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells. Cell. 2015;161:1437-1452.
Human primordial germ cells (PGCs) show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes,
with a subset of them displaying developmental stage–specific features. The authors used PBAT to analyze the DNA methylome of human PGCs. Approximately
10–11 weeks after gestation, the PGCs were nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female
PGCs, respectively. The study paves the way toward deciphering the complex epigenetic reprogramming of the germline, with the aim of restoring totipotency infertilized oocytes.
Illumina Technology: HiSeq 2500 System
Sakashita A., Kawabata Y., Jincho Y., et al. Sex Specification and Heterogeneity of Primordial Germ Cells in Mice. PLoS One. 2015;10:e0144836.
The authors used PBAT to determine the impact of demethylation on mouse PGC development. They found that, except for the retrotransposon regions, PGCs were
uniformly hypomethylated at embryonic day 13.5.
Illumina Technology: MiSeq System

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays163. Miura F., Enomoto Y., Dairiki R. and Ito T. Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging. Nucleic Acids Res. 2012;40:e136.
164. Smallwood S. A., Lee H. J., Angermueller C., et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods. 2014;11:817-820.

72

DNA Sequencing Methods Collection

BSPP: Bisulfite Sequencing With Padlock Probes
BSPP is a targeted method that isolates selected locations for methylation profiling.165,166
Padlock probes are ~100 nt DNA fragments designed to hybridize to genomic DNA targets in a horseshoe manner. After the gap
between the 2 hybridized, locus-specific arms of a padlock probe is filled and ligated, the circular strand of DNA can be amplified
and sequenced.165
MmeI AluI MmeIH1P1 P2 H2CpG island Bisulfite
conversion

Bisufite-converted
DNA

Padlock
probe

Extensionand ligation

Hybridize

Exonuclease
digestion

PCR

End repair
and adapter
ligation

DNA with
adapters

A schematic overview of BSPP.

Advantages

Disadvantages••

Requires only 100 ng of DNA for amplification-free WGBS of
mammalian genomes

Fails to capture areas with lower CpG densities167

Reviews
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References
None available yet.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

165. Deng J., Shoemaker R., Xie B., et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat Biotechnol.
2009;27:353-360.
166. Ball M. P., Li J. B., Gao Y., et al. Targeted and genome-scale strategies reveal gene-body methylation signatures in human cells. Nat Biotechnol. 2009;27:361-368.
167. Tanaka T., Reilly B., Diep D., Zhang K. and Bejar R. Cytosine Methylation Patterns As Biomarkers in MDS. Blood. 2015;126:5226-5226.

An overview of recent DNA-seq publications featuring Illumina technology

73

RRBS-Seq: Reduced-Representation Bisulfite Sequencing
RRBS uses one or multiple restriction enzymes on the genomic DNA to produce sequence-specific fragmentation.168
The fragmented genomic DNA is treated with bisulfite and sequenced. This is the method of choice to study specific regionsof interest. It is particularly effective where methylation levels are high, such as in promoters and repeat regions.

Methylated
DNA

MspI
digestion

Methylated
regions

Methylated
adapter

Bisulfite
conversion

End repair
and ligation

Converted fragments

PCR

DNA

A schematic overview of RRBS.

Advantages

Disadvantages

•

•

•

Provides genome-wide coverage of CpGs in islands at single-baseresolution
Covers CpG methylation in dense regions•••

Restriction enzymes cut at specific sites, providing biased sequence
selection
Method measures 10-15% of all CpGs in the genome
Cannot distinguish between 5mC and 5hmC
Does not cover non-CpG areas, genome-wide CpGs, and CpGs inareas without the enzyme restriction site

Reviews
Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights from advances ingenomic technologies. Neurosci Lett. 2016;625:47-55.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

References

Auclair G., Borgel J., Sanz L. A., et al. EHMT2 directs DNA methylation for efficient gene silencing in mouse embryos. Genome Res. 2016;26:192-202.
The authors used RRBS in a model of knockout mice to explore the role of the lysine methyltransferase, EHMT2, in DNA methylation during mouse embryogenesis.
Their results showed that DNA methylation was instrumental in EHMT2-mediated gene silencing during embryogenesis. This result implies that EHMT2 is a critical factor
that facilitates repressive DNA methylation at specific genomic loci during mammalian development.
Illumina Technology: HiSeq 2500 System

Day S. E., Coletta R. L., Kim J. Y., et al. Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes.
Clin Epigenetics. 2016;8:77.
The authors studied the role of skeletal muscle DNA methylation, in combination with transcriptomic changes in obesity. They obtained skeletal muscle biopsies
basally from lean (n = 12; body mass index (BMI) = 23.4 ± 0.7 kg/m2) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m2) study participants. They found 13,130 differentiallymethylated cytosines (DMCs) (uncorrected P < 0.05) that were altered in the promoter and 5’- and 3’-untranslated regions (UTRs) in the obese vs lean analysis. They
also observed a negative relationship between gene expression and DNA methylation.
Illumina Technology: HiSeq 2000 System

Heller G., Topakian T., Altenberger C., et al. Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic
myeloid leukemia. Leukemia. 2016;30:1861-1868.
The authors used RRBS and RNA-Seq to analyze samples from chronic myeloid leukemia (CML) patients at chronic phase (CP), accelerated phase (AP), and blastcrisis (BC) for differences in CpG site methylation and expression. They demonstrated that CpG site methylation increases dramatically during the progression from CPCML to AP-CML/BC-CML, and they identified genes that are transcriptionally regulated by methylation in BC-CML samples. Some of these genes are involved in thepathogenesis of various malignancies.
Illumina Technology: HiSeq 2000 System

168. Meissner A., Gnirke A., Bell G. W., Ramsahoye B., Lander E. S. and Jaenisch R. Reduced representation bisulfite sequencing for comparative high-resolution DNA
methylation analysis. Nucleic Acids Res. 2005;33:5868-5877.

74

DNA Sequencing Methods Collection

Baheti S., Kanwar R., Goelzenleuchter M., et al. Targeted alignment and end repair elimination increase alignment and methylation measure accuracy for reduced
representation bisulfite sequencing data. BMC Genomics. 2016;17:149.
Flinders C., Lam L., Rubbi L., et al. Epigenetic changes mediated by polycomb repressive complex 2 and E2a are associated with drug resistance in a mouse model oflymphoma. Genome Med. 2016;8:54.
Orlanski S., Labi V., Reizel Y., et al. Tissue-specific DNA demethylation is required for proper B-cell differentiation and function. Proc Natl Acad Sci U S A.
2016;113:5018-5023.
Page A., Paoli P., Moran Salvador E., et al. Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation landscape. J Hepatol.
2016;64:661-673.
Pegoraro M., Bafna A., Davies N. J., Shuker D. M. and Tauber E. DNA methylation changes induced by long and short photoperiods in Nasonia. Genome Res.
2016;26:203-210.
Yuan X. L., Gao N., Xing Y., et al. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing. Sci Rep.
2016;6:22138.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

An overview of recent DNA-seq publications featuring Illumina technology

75

BSAS: Bisulfite Amplicon Sequencing
BSAS169 is a targeted BS-Seq method that uses PCR enrichment of targeted regions and transposome-mediated library
construction for rapid generation of sequencing libraries, from low (1 ng) sample input.
Genomic DNA is bisulfite-converted and subjected to PCR, using primers specific for bisulfite-converted DNA. The amplicons
are subjected to Nextera XT library preparation, including dual indexing. The final libraries consist of a random insert of bisulfiteconverted amplified DNA, capture probes, and specific index sequences. These libraries are multiplexed and sequenced.

Methylated DNA

Bisulfite
conversion

Bisufite-converted DNA

PCR with primers specific forbisulfite converted DNA

Amplicons

Transposome withadapter

TagmentationDNA

A schematic overview of BSAS.

Advantages

Disadvantages

•

•
•

•

Can be applied to any genomic region from any DNA source,
including tissue and cell culture.
Rapid and highly quantitative

Does not cover the whole genome
Genome and target must be known

Reviews
None available yet.

References
Sun L., Wang J., Yin X., et al. Identification of a 5-Methylcytosine Site that may Regulate C/EBPbeta Binding and Determine Tissue-Specific Expression of
the BPI Gene in Piglets. Sci Rep. 2016;6:28506.
Bactericidal/permeability-increasing protein (BPI) plays an important role in innate immune defense in mammals. The authors used BSAS to quantify CpG islands in
the BPI gene promoter, in 11 different tissue types from weaned Yorkshire piglets. They confirmed that methylation of the mC-15 residue could inhibit the ability of
C/EBPb binding to the BPI promoter and affect gene expression. They suggest that this mechanism plays a role in the tissue-specificity of BPI gene expression in
weaned piglets.
Illumina Technology: MiSeq System
Ou X., Thakali K. M., Shankar K., Andres A. and Badger T. M. Maternal adiposity negatively influences infant brain white matter development. Obesity (Silver Spring).
2015;23:1047-1054.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

169. Masser D. R., Berg A. S. and Freeman W. M. Focused, high accuracy 5-methylcytosine quantitation with base resolution by benchtop next-generation sequencing.
Epigenetics Chromatin. 2013;6:33.

76

DNA Sequencing Methods Collection

MRE-Seq and Methyl-Seq: Methylation-Sensitive Restriction Enzyme Sequencing
MSRE/MRE-seq and Methyl-seq are protocols that use methylation-sensitive restriction enzymes (MSREs) on genomic
DNA to study DNA methylation.170,171 MRE-seq enriches unmethylated DNA and can cover 1.7 million CpG sites across thehuman genome.172
In this method, gDNA is first separately digested with different MSREs. The library is prepared from size-selected restriction
fragments and sequenced. Deep sequencing allows for accurate detection of methylation sites in the genome.

Methylated sites
in the genome

Split sample

C
C

C
C

G
G

G
G

C
C

C
C

G
G

G
G

MspI
HpaII
Restriction
enzyme digest

C
C

C
C

C
C

C
C

G
G
G

G
G

G
G G

Sequence

Align sequences anddetermine undigested sites

Identified
methylation site

A schematic overview of MSRE.

Advantages

Disadvantages••

Allows the estimation of relative DNA methylation levels

Relatively low coverage of the genome, because CpG-containing
recognition sites are limited

Reviews
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References
Lee H. J., Lowdon R. F., Maricque B., et al. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos. Nat
Commun. 2015;6:6315.
To understand how DNA methylation changes during zebrafish embryogenesis, the researchers generated high-resolution DNA methylome maps of 6 developmental
stages. For each developmental stage, they constructed 2 sequencing libraries using methylated DNA immunoprecipitation sequencing (MeDIP-seq) and MRE-seq.
Overall, the average genic DNA methylation levels of each developmental stage were almost identical. The authors observed a negative correlation in the methylationlevel and the proximity to the TSS. The DNA methylation level increased in the 3’-UTR and stayed high in exons, which resembled the DNA methylation pattern overgenic regions in human ESCs.
Illumina Technology: HiSeq System
Chen K., Chen Z., Wu D., et al. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes. Nat Genet.
2015;.
Elliott G., Hong C., Xing X., et al. Intermediate DNA methylation is a conserved signature of genome regulation. Nat Commun. 2015;6:6363.
Gascard P., Bilenky M., Sigaroudinia M., et al. Epigenetic and transcriptional determinants of the human breast. Nat Commun. 2015;6:6351.
Lee H. J., Lowdon R. F., Maricque B., Zhang B., Stevens M., et al. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos.
Nat Commun. 2015;6:6315.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Infinium HumanMethylation450 BeadChip Kit

170. Maunakea A. K., Nagarajan R. P., Bilenky M., et al. Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature. 2010;466:253-257.
171. Brunner A. L., Johnson D. S., Kim S. W., et al. Distinct DNA methylation patterns characterize differentiated human embryonic stem cells and developing human fetalliver. Genome Res. 2009;19:1044-1056.
172. Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

An overview of recent DNA-seq publications featuring Illumina technology

77

EpiRADseq: Double-Digest Restriction Site–Associated DNA Marker Generation
with a Methylation-Sensitive Restriction Enzyme
EpiRADseq is similar to the widely used double-digest RAD-seq method (ddRADseq), except that it uses an MSRE.173
DNA samples are digested with PstI and HpaII followed by purification. Double-stranded sequencing adapters with uniquebarcodes are ligated to each sample. Size-selected libraries are then amplified with indexed primers.
Restriction sites

Restriction
digestion

Add barcoded adapters

Second restriction digest withmethylation-sensitive HpaII

Add P2
adapter

AmplifyDNA

A schematic overview of EpiRADseq.

Advantages

Disadvantages••

Does not need a reference genome

Measures a subset of methylation sites

Reviews
None available yet.

References
Schield D. R., Walsh M. R., Card D. C., Andrew A. L., Adams R. H., et al. EpiRADseq: scalable analysis of genomewide patterns of methylation using nextgeneration sequencing. Methods in Ecology and Evolution. 2016;7:60-69.
The researchers tested the utility of EpiRADseq by identifying shifts in the epigenome of clonal water fleas (Daphnia ambigua) in response to exposure to fish predator
cues. These cues are known to induce transgenerational changes in life-history traits. In response to predator cues, the authors found significant transgenerational
shifts in the methylation states of the sampled loci within treatment groups.
Illumina Technology: MiSeq System

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

173. Schield D. R., Walsh M. R., Card D. C., et al. EpiRADseq: scalable analysis of genomewide patterns of methylation using next-generation sequencing.
Methods in Ecology and Evolution. 2016;7:60-69.

78

DNA Sequencing Methods Collection

T-WGBS: Tagmentation-Based Whole-Genome Bisulfite Sequencing
T-WGBS uses the Tn5 transposome and bisulfite conversion to study 5mC.174
In this method, DNA is incubated with Tn5 transposome containing methylated primers, which fragments the DNA and ligates
adapters. The tagged DNA first undergoes oligonucleotide displacement, followed by methylated oligonucleotide replacement and
gap repair, ensuring the addition of methylated adapters to the tagmented DNA. Next, the DNA is treated with sodium bisulfite,
PCR-amplified, and sequenced. Deep sequencing provides single-base resolution of 5mC in the genome.
Displaced oligo

Methylated DNA

Transposome withmethylated adapter

Tagmentation

Oligo withmethylated
adapter

Displace oligo
Hybridize methylated
adapter and gap repair

Bisulfite
conversion

PCR

DNA

A schematic overview of T-WGBS.

Advantages

Disadvantages

•
•
•

•

Can sequence samples with very limited starting material (~20 ng)
Fast protocol with few steps
Elimination of multiple steps minimizes loss of DNA••

Bisulfite converts unmethylated cytosines to thymidines, reducing
sequence complexity, which can make it difficult to create alignments
SNPs where a cytosine is converted to thymidine will be missed uponbisulfite conversion
Bisulfite conversion does not distinguish between 5mC and 5hmC

Reviews
Tang J., Fang F., Miller D. F., et al. Global DNA methylation profiling technologies and the ovarian cancer methylome. Methods Mol Biol. 2015;1238:653-675.
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.
Weichenhan D. and Plass C. The evolving epigenome. Hum Mol Genet. 2013;22:R1-6.

References
Wang Q., Gu L., Adey A., Radlwimmer B., Wang W., et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc. 2013;8:2022-2032.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

174. Wang Q., Gu L., Adey A., et al. Tagmentation-based whole-genome bisulfite sequencing. Nat Protoc. 2013;8:2022-2032.

An overview of recent DNA-seq publications featuring Illumina technology

79

JBP1-seq: J-Binding Protein 1 Sequencing
JBP1-seq is a method for genome-wide profiling of 5hmC. It relies on the strong affinity of the J-binding protein 1 (JBP1) for
glucosylated 5hmC. The method uses a recombinant JBP1 protein with an additional His tag and Avi tag that is biotinylated andsubsequently conjugated to streptavidin-coated magnetic beads.175
The JBP1-seq workflow involves 4 main steps: (1) gDNA is tagmented to fragment it and add adapter sequences, (2) 5hmC sites
are glucosylated by T4-bGT, (3) JBP1-magnetic beads are used to enrich for DNA fragments containing b-glu- 5hmC, and finally(4) enriched library is amplified to add P5 and P7 adapters and barcodes for sequencing.

Hydroxy-methylated DNA

Transposomes

Tagmentation

T4-βGT

Glucosylated 5-hmC

JBP1-magnetic beadpull-down

PCR

DNA

A schematic overview of JBP1-seq.

Advantages

Disadvantages•••

Streamlined protocol

Regions that contain high levels of 5hmC can be overrepresented
Optimal resolution is approximately 50 bp

Reviews
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.

References

Cui L., Chung T. H., Tan D., Sun X. and Jia X. Y. JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC. Genomics. 2014;104:368-375.
This paper provides a detailed description of the protocol.
Illumina Technology: HiSeq System, Nextera DNA Sample Prep Kit

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

175. Cui L., Chung T. H., Tan D., Sun X. and Jia X. Y. JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC. Genomics. 2014;104:368-375.

80

DNA Sequencing Methods CollectionAba-seq: AbaSI Coupled with Sequencing
Aba-seq is a method for high-resolution mapping of the 5hmC methylome that provides sensitive detection of 5hmC at lowoccupancy regions.176
The method relies on the unique property of the restriction enzyme AbaSI to recognize glucosylated 5hmC with high specificity,
compared to 5mC and C. A single-cell variation, scAba-seq, is also available.177 A related method uses PvuRts1I to overcome thesequence bias in AbaSI.178

Hydroxy-methylated DNA

T4-βGT

Glucosylated 5-hmC

AbaSI

Biotinylated
primers

LigateFragment

Streptavidin magnetic
bead pull-downDNA

A schematic overview of Aba-seq.

Advantages

Disadvantages

•

•

•
•

Covers CpG and non-CpG methylation throughout the genome atsingle-base resolution
Covers 5mC in dense and less dense repeat regions
Clearly differentiates between 5mC and 5hmC

Ambiguity can exist in 13% of the cleaved molecules in the
Aba-seq assay179• Sequence bias in AbaSI activity180

Reviews
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.

References
Sun Z., Terragni J., Borgaro J. G., Liu Y., Yu L., et al. High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem
cells. Cell Rep. 2013;3:567-576.
The authors used Aba-seq to generate a high-resolution hydroxymethylome map of mouse E14 ESCs. The results indicated that 5hmC was enriched at poisedenhancers with monomethylated histone H3 lysine 4 (H3K4me1), but not at active enhancers with acetylated histone H3 lysine 27 (H3K27Ac). The authors also
observed that the mitochondrial genome contained non-CG hydroxymethylated sites.
Illumina Technology: HiSeq System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

176. Sun Z., Terragni J., Borgaro J. G., et al. High-resolution enzymatic mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cells. Cell Rep.
2013;3:567-576.
177. Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability andenables lineage reconstruction. Nat Biotechnol. 2016;34:852-856.
178. Sun Z., Dai N., Borgaro J. G., et al. A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol Cell.
2015;57:750-761.
179. Serandour A. A., Avner S., Mahe E. A., et al. Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease digestion identifies
evolutionarily unconserved CpGs as TET targets. Genome Biol. 2016;17:56.
180. Sun Z., Dai N., Borgaro J. G., et al. A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol Cell.
2015;57:750-761.

An overview of recent DNA-seq publications featuring Illumina technology

81

TAmC-Seq: Tet-Assisted 5-Methylcytosine Sequencing
TAmC-Seq selectively tags 5mC with an azide residue that can be further labeled with biotin for affinity purification.181 The method
relies on the properties of the Ten-eleven translocation (Tet) family of iron(II)/a-ketoglutarate-dependent dioxygenases that are
involved in the demethylation of 5mC.
In this method, the endogenous 5hmC in genomic DNA is protected by glucosylation with normal glucose. The 5mC residues
are converted to 5hmC via mTet1-catalyzed oxidation. The 5hmC is then labeled via b-GT-mediated glucosylation with a modified
glucose moiety (6-N3-glucose) to generate 6-N3-b-glucosyl-5-hydroxymethylcytosine (N3-5gmC). This molecule can be labeled
further, using click chemistry, for detection, affinity purification, and sequencing.
5mC
C

T

5hmC
C

5mC

βGT

C G

C

T

g5hmC
C

C G

Glucosylation

5cmC

TET
C

T

Oxidation

C

N3

g5hmC
C

C G

C

C G

βGT-catalyzed
UDP-6-N3 glucosylation

Biotin
g5hmC

N3S-S

g5hmC
T

C

Biotinylation

T

C

C G

Streptavidin pull-down
and DTT cleavage

A schematic overview of TAmC-Seq.

Advantages

Disadvantages••

Captures a larger fraction of methylated CpGs with lessdensity bias

Not yet adopted widely by the scientific community

Reviews
None available yet.

References
Zhang L., Szulwach K. E., Hon G. C., Song C. X., Park B., et al. Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection andsequencing. Nat Commun. 2013;4:1517.
The authors used TAmC-Seq to screen for 5mC in several mouse and human cell lines. The results showed that TAmC-Seq provided a wider coverage of CpGcontaining genomic regions. Further, it captured a larger fraction of methylated CpGs with less density bias than other affinity-enrichment methods.
Illumina Technology: HiSeq System, cBot System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

181. Zhang L., Szulwach K. E., Hon G. C., et al. Tet-mediated covalent labelling of 5-methylcytosine for its genome-wide detection and sequencing. Nat Commun.
2013;4:1517.

82

DNA Sequencing Methods CollectionDNAfC-Seal: A 5-Formylcytosine-Selective Chemical LabelingIn demethylation pathways, the TET family of proteins oxidize 5mC to 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine(5caC) in a stepwise fashion. Conversely, in vivo, 5fmC residues can be converted to cytosine by base-excision repair.
In fC-Seal, 5hmC residues in the genome are blocked with regular glucose using bGT, and 5fC residues are reduced to 5hmC by
NaBH4. The newly generated 5hmC residues are modified, enriched, and sequenced.182
CC mC
5m 5h 5fc 5ca
C

C

C

C

Blocked 5hmC residue

C

C

C

C

βGT-catalyzed
glucosylation

CC

5hmC residue
C

NaBH4

C

C

C

Convert 5fc to
5hmc

N3S-S

N3

C

C

C

C

C

βGT-catalyzed
UDP-6-N3 glucosylation

CC

Biotinylation

C

C

C

BiotinC

Streptavidin pull-down
and DTT cleavage

DNA

A schematic overview of fC-Seal.

Advantages

Disadvantages••

Does not require antibodies

Cannot map 5fC and 5caC at single-nucleotide resolution anddetermine their relative abundance183

Reviews
Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.

ReferencesSong C. X., Szulwach K. E., Dai Q., Fu Y., Mao S. Q., et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell.
2013;153:678-691.
In this study, genome-wide mapping of 5fC in mouse ESCs revealed that 5fC preferentially occurs at poised enhancers, among other gene regulatory elements.
The researchers resolved 5fC at single-base resolution by hydroxylamine-based protection from bisulfite-mediated deamination, thereby confirming sites of
5fC accumulation.
Illumina Technology: HiSeq System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

182. Song C. X., Szulwach K. E., Dai Q., et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell. 2013;153:678-691.
183. Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.

An overview of recent DNA-seq publications featuring Illumina technology

83

fC-CET: 5fC Chemical Labeling and C-to-T Transition During PCR
fC-CET is a bisulfite-free method for whole-genome analysis of 5fC. The method is based on selective chemical labeling of 5fC
and subsequent C-to-T transition during PCR.184
In this method, gDNA is labeled sequentially with an azido derivative of 1,3-indandione (AI) and then conjugated to biotin using
click chemistry. The gDNA is enriched by streptavidin pull-down and ligated to adapters, followed by PCR and sequencing. C-to-T
transitions during PCR amplification are searched specifically to define 5fC sites in the genome.
CC mC
5m 5h 5fc 5ca
C

C

C

C

C

N 3S-S

N3
C

C

C

C

Azido 1,3-indandione
(AI)

C

C

C

C

C

BiotinN 3SH

CDBCO-S-S-PEG3-biotin

C

C

C

C

Pull-down,
NaOH and DTTC

C

Adapter ligation
and PCR

C

C

T

C

PurifyDNA

A schematic overview of fC-CET.

Advantages

Disadvantages

•
•
•

•

Results are in agreement with fC-Seal
Sequences with a single 5fC are enriched ~100-fold
No noticeable DNA degradation; ideal for analyses of precious
DNA samples

Provides only relative enrichment of cytosines marked by 5fC185

Reviews
None available yet.

References

Xia B., Han D., Lu X., Sun Z., Zhou A., et al. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods. 2015;12:1047-1050.
Using fC-CET, the researchers generated a base-resolution map of 5fC in the whole genome of wild-type mouse ESCs. With a requirement of positive hits in both
replicates, they identified 32,685 and 139,027 high-confidence 5fC sites in Tdgfl/fl and Tdg−/− mouse ESCs, respectively.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

184. Xia B., Han D., Lu X., et al. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods. 2015;12:1047-1050.
185. Wu H., Wu X. and Zhang Y. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc. 2016;11:1081-1100.

84

DNA Sequencing Methods Collection

CAB-Seq: Chemical Modification–Assisted Bisulfite Sequencing
CAB-Seq can detect 5caC with single-base resolution in DNA.186 It is based on the observation that chemically modified 5caC cansurvive bisulfite treatment without deamination.
CC mC
5m 5h 5fc 5cao-acylisourea
CC mC
5m 5h 5fc 5ca

CC mC
5m 5h 5fc 5ca
C

C

C

C

C

C

C

C

C

C

1-ethyl-3-[3-dimethylamino-propyl]-carbodiimide hydrochloride (EDC) chemistry

Nu

S-S

Linker

C mC
5m 5h 5fc 5caC S-S
C

C

C

C

BiotinControl

C

C

C

C

C

T

C

C

T

T

T

C

C

T

C

5caC SCCC

Streptavidin pull-down
and DTT cleavage

C

C

C

Bisulfite treatment
PCR amplification

Sequence

A schematic overview of CAB-Seq.

Advantages

Disadvantages•••

Single-base resolution

Relatively low protection rate of 5caC deamination (50–60%)187
Requires the sequencing of a nontreated DNA control, and the callingof the modified cytosines is based on a subtractive analysis; requires
high sequencing depth to reduce the false-discovery rate

Reviews
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.

References
Lu X., Song C. X., Szulwach K., Wang Z., Weidenbacher P., et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in
DNA. J Am Chem Soc. 2013;135:9315-9317.

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

186. Lu X., Song C. X., Szulwach K., et al. Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA. J Am Chem Soc.
2013;135:9315-9317.
187. Wu H., Wu X., Shen L. and Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol.
2014;32:1231-1240.

An overview of recent DNA-seq publications featuring Illumina technology

85

oxBS-Seq: Oxidative Bisulfite Sequencing
oxBS-Seq differentiates between 5mC and 5hmC.188
In this method, 5hmC is oxidized to 5fC with a selective chemical agent, while 5mC remains unchanged. Sodium bisulfite
treatment of 5fC results in its deamination to uracil which, upon sequencing, is read as a thymidine. Deep sequencing of oxBStreated DNA and sequence comparison of treated vs untreated libraries can identify 5hmC locations at single-base resolution.

CC mC
5m 5h 5fc 5ca
C

C

C

C

C

C
C mC
5m 5h 5fc 5ca

Control
KRuO

4

C

C

C

C

C

Bisulfite treatment
PCR amplification

T

C

C

T

T

C

C

Bisulfite treatment
PCR amplification

T

C

T

T

T

5fc
C

C

C

DNA

A schematic overview of oxBS-Seq.

Advantages

Disadvantages

•

•
•

•
•

Covers CpG and non-CpG methylation throughout the genome atsingle-base resolution
Covers 5mC in dense and less dense repeat regions
Clearly differentiates between 5mC and 5hmC

Harsh oxidation conditions lead to a substantial loss of DNA (99.5%)
Must be combined with BS-Seq in order to distinguish and quantify C,
5mC, and 5hmC

Reviews
Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights from advances ingenomic technologies. Neurosci Lett. 2016;625:47-55.
Marx V. Genetics: profiling DNA methylation and beyond. Nat Methods. 2016;13:119-122.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.
Meldi K. M. and Figueroa M. E. Cytosine modifications in myeloid malignancies. Pharmacol Ther. 2015;152:42-53.
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References
Hadad N., Masser D. R., Logan S., et al. Absence of genomic hypomethylation or regulation of cytosine-modifying enzymes with aging in male and female
mice. Epigenetics Chromatin. 2016;9:30.
The authors used oxBS-Seq to quantify 5mC and 5hmC genome-wide in the hippocampus of young and old male and female mice. They showed that specific levels of
5mC and 5hmC differ with aging and by sex, depending on the chromosomal, promoter, and CG context.
Illumina Technology: NextSeq System
Feng J., Shao N., Szulwach K. E., et al. Role of Tet1 and 5-hydroxymethylcytosine in cocaine action. Nat Neurosci. 2015;18:536-544.
The researchers identified 5hmC induction in putative enhancers and coding regions of genes that have pivotal roles in drug addiction. Such induction of 5hmC, which
occurred similarly following TET1 knockdown alone, correlated with increased expression of these genes as well as with their alternative splicing in response to cocaine
administration. In addition, 5hmC alterations at certain loci persisted for at least 1 month after cocaine exposure. The researchers used oxBS-Seq to confirm inductionof 5hmC at each of these genes in an independent cohort of animals.
Illumina Technology: HiSeq 2000 System

188. Booth M. J., Branco M. R., Ficz G., et al. Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science.
2012;336:934-937.

86

DNA Sequencing Methods Collection

Aijo T., Huang Y., Mannerstrom H., et al. A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.
Genome Biol. 2016;17:49.
Page A., Paoli P., Moran Salvador E., White S., French J., et al. Hepatic stellate cell transdifferentiation involves genome-wide remodeling of the DNA methylation
landscape. J Hepatol. 2016;64:661-673.
Wallner S., Schroder C., Leitao E., Berulava T., Haak C., et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics Chromatin. 2016;9:33.

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit
TruSeq Nano DNA Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

87

redBS-Seq: Reduced Bisulfite Sequencing
caMAB-seq: 5-Carboxylcytosine Methylase-Assisted Bisulfite Sequencing
redBS-Seq relies on the chemical reduction of 5fC to 5hmC by NaBH4. The 5hmC is detected in the same manner as 5mC.189
In caMAB-seq, 5fC is first reduced by NaBH4 to 5hmC. Owing to the combination of NaBH4 reduction with M.SssI treatment,
5caC is sequenced as thymidine after bisulfite conversion, whereas 5mC, 5hmC, and 5fC are read as cytosine.190

CC mC
5m 5h 5fc 5ca
C

C

C

C

C

C
C mC
5m 5h 5fc 5ca

Control
NaBH

4

C

C

C

C

C

mC

5h
C

C

C

C

C

Bisulfite treatment
PCR amplification

T

C

C

T

T

Bisulfite treatment
PCR amplification

T

C

C

C

T

DNA

A schematic overview of redBS-Seq.

Advantages

Disadvantages••

Base-resolution approach to map 5fC and 5caC

Requires a high sequencing depth to reduce the false-discovery rate191

Reviews
Iatrou A., Kenis G., Rutten B. P., Lunnon K. and van den Hove D. L. Epigenetic dysregulation of brainstem nuclei in the pathogenesis of Alzheimer’s disease: looking in
the correct place at the right time? Cell Mol Life Sci. 2017;74:509-523.
Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.

References
Van Haute L., Dietmann S., Kremer L., et al. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying mutations in
NSUN3. Nat Commun. 2016;7:12039.
The authors used whole-exome sequencing to identify loss-of-function mutations in the gene for a 5mC methyltransferase, NSUN3, in a patient presenting withcombined mitochondrial respiratory chain complex deficiency. They showed that NSun3 is required for deposition of 5mC at the anticodon loop in the mitochondrially
encoded transfer RNA methionine (mt-tRNAMet). They also showed that 5mC deficiency in mt-tRNAMet results in the lack of 5fC at the same tRNA position. The
authors used RedBS-Seq to detect 5fC in mitochondrial RNA from wt/wt and mut/mut samples.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

189. Booth M. J., Marsico G., Bachman M., Beraldi D. and Balasubramanian S. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem.
2014;6:435-440.
190. Wu H., Wu X. and Zhang Y. Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq. Nat Protoc. 2016;11:1081-1100.
191. Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.

88

DNA Sequencing Methods Collection

fCAB-seq: 5-Formylcytosine Chemical Modification–Assisted Bisulfite Sequencing
fCAB-Seq is a method for EtONH2-modified bisulfite sequencing for base-resolution detection of 5fC in gDNA.192 It iscomplementary to fC-Seal, which was developed as a highly selective chemical labeling approach for the affinity purification andgenome-wide profiling of 5fC.191
CC mC
5m 5h 5fc 5ca
C

C

C

C

C

C
C mC
5m 5h 5fc 5ca

ControlO-ethylhydroxylamine (EtONH2)

C

C

C

C

C

Blocked
C

C

C

C

C

Bisulfite treatment
PCR amplification

T

C

C

T

T

Bisulfite treatment
PCR amplification

T

C

C

C

T

DNA

A schematic overview of fCAB-Seq.

Advantages

Disadvantages••

Single-base resolution

Requires the sequencing of a matched nontreated DNA control 193

Reviews
Balasubramanian S. Chemical biology on the genome. Bioorg Med Chem. 2014;22:4356-4370.
Booth M. J., Marsico G., Bachman M., Beraldi D. and Balasubramanian S. Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution. Nat Chem.
2014;6:435-440.
Rivera C. M. and Ren B. Mapping human epigenomes. Cell. 2013;155:39-55.

References

Wang L., Zhang J., Duan J., et al. Programming and inheritance of parental DNA methylomes in mammals. Cell. 2014;157:979-991.
The authors used fCAB-Seq to generate a base-resolution 5fC map of 2-cell mouse embryos. The distribution of CpGs at different genomic elements showed that thehighly formylated elements are also the demethylated elements.
Illumina Technology: HiSeq 2000 System

Van Haute L., Dietmann S., Kremer L., Hussain S., Pearce S. F., et al. Deficient methylation and formylation of mt-tRNA(Met) wobble cytosine in a patient carrying
mutations in NSUN3. Nat Commun. 2016;7:12039.
Xia B., Han D., Lu X., Sun Z., Zhou A., et al. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods. 2015;12:1047-1050.
Becker D., Lutsik P., Ebert P., et al. BiQ Analyzer HiMod: an interactive software tool for high-throughput locus-specific analysis of 5-methylcytosine and its oxidized
derivatives. Nucleic Acids Res. 2014;42:W501-507.

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

192. Song C. X., Szulwach K. E., Dai Q., et al. Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell. 2013;153:678-691.
193. Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.

An overview of recent DNA-seq publications featuring Illumina technology89

MAB-seq: M.SssI Methylase-Assisted Bisulfite Sequencing
MAB-seq allows simultaneous and quantitative mapping of both 5fC and 5caC at single-base resolution.194 This method iscomplementary to caMAB-seq, a method for direct 5caC mapping.193
In this method, gDNA is treated with the bacterial CpG methyltransferase M.SssI, which methylates CpG dinucleotides. Next,
bisulfite conversion of methylase-treated DNA leads to deamination of only 5fC and 5caC; originally unmodified CpGs areprotected as 5mCpG. After sequencing, 5fC and 5caC are read as thymidine, while 5mC is read as cytosine.

CC mC
5m 5h 5fc 5ca
C

C

C

C

C

C
C mC
5m 5h 5fc 5ca

ControlC

M.SssI

C

C

C

C

C

Bisulfite treatment
PCR amplification

T

C

C

T

T

C

C

C

Bisulfite treatment
PCR amplification

C

C

C

T

T

C
5m
C

DNA

A schematic overview of MAB-seq.

Advantages

Disadvantages••

Can provide a quantitative measurement of the abundance of
5fC/5caC within CpG dyads

Unable to distinguish 5fC/5caC from unmodified C within a non-CpG
context

Reviews
Meldi K. M. and Figueroa M. E. Cytosine modifications in myeloid malignancies. Pharmacol Ther. 2015;152:42-53.

References
Aijo T., Huang Y., Mannerstrom H., Chavez L., Tsagaratou A., et al. A probabilistic generative model for quantification of DNA modifications enables analysis ofdemethylation pathways. Genome Biol. 2016;17:49.
Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.
Neri F., Incarnato D., Krepelova A., et al. Single-Base Resolution Analysis of 5-Formyl and 5-Carboxyl Cytosine Reveals Promoter DNA Methylation Dynamics. Cell Rep.
2015;10:674-683.

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

194. Wu H., Wu X., Shen L. and Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol.
2014;32:1231-1240.

90

DNA Sequencing Methods Collection

RRMAB-seq: Reduced-Representation M.SssI Methylase-Assisted
Bisulfite Sequencing
RRMAB-seq is based on the pretreatment of gDNA with the enzyme MspI, which recognizes and cuts the CCGG consensus toenrich CpG-rich regions, like gene promoters.195 This method is an adapted version of MAB-seq.196
CC mC
5m 5h 5fc 5ca
C

Digestwith MspI

Add methylated
adapters

C

C

C

C

C
C mC
5m 5h 5fc 5ca

ControlC

M.SssI

C

C

C

C

C

Bisulfite treatment
PCR amplification

T

C

C

T

T

C

C

C

Bisulfite treatment
PCR amplification

C

C

C

T

T

C
5m
C

DNA

A schematic overview of RRMAB-seq.

Advantages

Disadvantages

•

•

•

Increased coverage of CpG-rich regions reduces sequencing costs
and increases read depth
Reduced number of chemical or enzymatic DNA treatments
required before bisulfite treatment

Unable to distinguish 5fC/5caC from unmodified C within a non-CpG
context195

Reviews
None available yet.

References
Neri F., Incarnato D., Krepelova A., Parlato C. and Oliviero S. Methylation-assisted bisulfite sequencing to simultaneously map 5fC and 5caC on a genome-wide scale
for DNA demethylation analysis. Nat Protoc. 2016;11:1191-1205.

Associated Kits
TruSeq DNA Methylation Kit
TruSeq DNA PCR-Free Library Prep Kit

195. Neri F., Incarnato D., Krepelova A., et al. Single-Base Resolution Analysis of 5-Formyl and 5-Carboxyl Cytosine Reveals Promoter DNA Methylation Dynamics.
Cell Rep. 2015;10:674-683.
196. Wu H., Wu X., Shen L. and Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol.
2014;32:1231-1240.

An overview of recent DNA-seq publications featuring Illumina technology91TAB-Seq: Tet-Assisted Bisulfite Sequencing
TAB-Seq is a novel method that uses bisulfite conversion and Tet proteins to study 5hmC.197
In this method, 5hmC is first protected selectively with a glucose moiety, followed by subsequent oxidation of 5mC to 5caC by Tet
proteins. Next, the oxidized gDNA is treated with bisulfite; 5hmC remains unchanged and is read as a cytosine, while 5mC andunmethylated cytosines are deaminated to uracil and read as thymidines upon sequencing. Deep sequencing of TAB-treated DNA
compared with untreated DNA provides accurate representation of 5hmC localization in the genome.
CC mC
5m 5h 5fc 5ca
C

C

C

C

mC
5g

βGT

C

C

C

Glucosylation

CC

C mC C C
5ca 5g 5ca 5ca

TET
C

C

C

C

C

Bisulfite treatment
PCR amplification

C

T

T

C

T

T

DNA

Oxidation

A schematic overview of TAB-Seq.

Advantages

Disadvantages

•

•

•
•

Covers CpG and non-CpG hydroxymethylation throughout the
genome at single-base resolution
Covers 5hmC in dense and less dense repeat regions
Clearly differentiates between 5hmC and 5mC, specifically
identifying 5hmC

Harsh oxidation can lead to substantial loss of DNA5

Reviews
Aijo T., Huang Y., Mannerstrom H., Chavez L., Tsagaratou A., et al. A probabilistic generative model for quantification of DNA modifications enables analysis ofdemethylation pathways. Genome Biol. 2016;17:49.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References
Bogdanovic O., Smits A. H., de la Calle Mustienes E., Tena J. J., Ford E., et al. Active DNA demethylation at enhancers during the vertebrate phylotypic
period. Nat Genet. 2016;48:417-426.
The authors generated single base–resolution maps of 5hmC in zebrafish, Xenopus, and mouse embryos using TAB-Seq. The 5hmC signal in zebrafish, Xenopus, andmouse coincided with developmental demethylation taking place in both early and late differentially methylated regions.
Illumina Technology: HiSeq 1500 System
Chen K., Zhang J., Guo Z., et al. Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer. Cell Res. 2016;26:103-118.
The authors used TAB-Seq to show no global loss of 5mC in kidney tumors compared with matched normal tissues. However, they observed global loss of 5hmC in
virtually all kidney tumor tissues. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model.
Illumina Technology: HiSeq 2000 System

Greco C. M., Kunderfranco P., Rubino M., et al. DNA hydroxymethylation controls cardiomyocyte gene expression in development and hypertrophy.
Nat Commun. 2016;7:12418.
The authors studied the hydroxymethylome in embryonic, neonatal, adult, and hypertrophic mouse cardiomyocytes. They found that DNA hydroxymethylation
was highly dynamic during cardiac development and disease. TAB-Seq analysis showed strong hydroxymethylation of LINE-1, accompanied CG demethylation,in hypertrophic cardiomyocytes. The authors suggest that, in hypertrophic heart, 5hmC may play a role in rendering chromatin structure more permissive in
LINE-1 regions.
Illumina Technology: HiSeq 2000 System

197. Yu M., Hon G. C., Szulwach K. E., et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell. 2012;149:1368-1380.

92

DNA Sequencing Methods Collection

Yu P., Ji L., Lee K. J., et al. Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine. PLoS One. 2016;11:e0154949.
The authors used TAB-Seq analysis to demonstrate that 5hmC levels were remarkably dynamic in gene bodies of various classes of visceral adipose tissue (VAT)
nuclei. These levels dropped 3.8-fold from the highest quintile of expressed genes to the lowest. Their results suggest that VAT-derived adipocytes are more active in
remodeling chromatin than non-adipocytes.
Illumina Technology: NextSeq 500 System

Mooijman D., Dey S. S., Boisset J. C., Crosetto N. and van Oudenaarden A. Single-cell 5hmC sequencing reveals chromosome-wide cell-to-cell variability and enables
lineage reconstruction. Nat Biotechnol. 2016;34:852-856.
Serandour A. A., Avner S., Mahe E. A., Madigou T., Guibert S., et al. Single-CpG resolution mapping of 5-hydroxymethylcytosine by chemical labeling and exonuclease
digestion identifies evolutionarily unconserved CpGs as TET targets. Genome Biol. 2016;17:56.
Thienpont B., Steinbacher J., Zhao H., D’Anna F., Kuchnio A., et al. Tumour hypoxia causes DNA hypermethylation by reducing TET activity. Nature. 2016;537:63-68.
Guo F., Yan L., Guo H., Li L., Hu B., et al. The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells. Cell. 2015;161:1437-1452.
Li Q., Suzuki M., Wendt J., et al. Post-conversion targeted capture of modified cytosines in mammalian and plant genomes. Nucleic Acids Res. 2015;43:e81.
Xia B., Han D., Lu X., Sun Z., Zhou A., et al. Bisulfite-free, base-resolution analysis of 5-formylcytosine at the genome scale. Nat Methods. 2015;12:1047-1050.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

An overview of recent DNA-seq publications featuring Illumina technology

93

MIRA: Methylated-CpG Island Recovery Assay (MIRA)MIRA uses the high affinity of a methylated-CpG-binding protein complex (MBD2B and MBD3L1) to enrich regions withmethylated CpG dinucleotides.198 This approach can be applied to both array-based DNA analysis and NGS, which are sometimes
distinguished as MIRA-chip199 and MIRA-seq.200
In this procedure, the fragmented gDNA is incubated with purified GST-MBD2B and His-MBD3L1 proteins. The high-affinity
MBD2B/MBD3L1 complex binds to the methylated gDNA templates. The methylated gDNA fragments are captured on
glutathione-coated magnetic beads. The enriched methylated DNA fraction is amplified, labeled, and analyzed by NGS.
C

C G C

C G C G C G CC G C G

C

5m

5m

C G C

C G C

Fractionate

C G C GC G C G

MBD2B/MBD3L1
protein complex

Isolate on glutathione-coatedbeads

DNA purification
PCR amplificationDNA

A schematic overview of MIRA.

Advantages

Disadvantages

•
•
•

•
•

High specificity and sensitivity, requiring as little as 1 ng input gDNA
Does not require DNA denaturation
Independent of restriction sites

Resolution limited to 100 bases
Requires a minimum of 2 methylated CpGs in the captured
fragment201

Reviews
None available yet.

References
Benjamin A. L., Green B. B., Crooker B. A., McKay S. D. and Kerr D. E. Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS
challenge occurs without major differences in the methylome. BMC Genomics. 2016;17:258.
The authors established fibroblast cultures from dairy (Holstein) and beef (Angus) cattle and challenged them with lipopolysaccharide (LPS) to examine breed-dependent
differences in the innate immune response. Based on RNA-Seq, the Holstein breed displayed a more robust response to LPS than the Angus breed. MIRA-seq showedsimilar patterns of genome-wide DNA methylation between breeds, with overall hypomethylation of gene promoters. However, the authors discovered 49 regions ofdifferential methylation between Holstein and Angus fibroblasts.
Illumina Technology: HiSeq 1000 System, cBot System
Jin S. G., Xiong W., Wu X., Yang L. and Pfeifer G. P. The DNA methylation landscape of human melanoma. Genomics. 2015;106:322-330.
The authors used MIRA-seq to characterize the DNA methylome of normal melanocytes and 27 metastatic melanoma samples. They found 179 tumor-specificmethylation peaks present in all (27/27) melanomas and suggest that these peaks may act as potential disease biomarkers.
Illumina Technology: HiSeq System

Yoneda A., Henson W. R., Goldner N. K., et al. Comparative transcriptomics elucidates adaptive phenol tolerance and utilization in lipid-accumulating Rhodococcus
opacus PD630. Nucleic Acids Res. 2016;44:2240-2254.

Associated Kits
TruSeq DNA Methylation Kit
Infinium HumanMethylation450 Arrays

198. Rauch T. A. and Pfeifer G. P. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA). Methods. 2010;52:213-217.
199. Jung M., Jin S. G., Zhang X., et al. Longitudinal epigenetic and gene expression profiles analyzed by three-component analysis reveal down-regulation of genes
involved in protein translation in human aging. Nucleic Acids Res. 2015;43:e100.
200. Benjamin A. L., Green B. B., Crooker B. A., McKay S. D. and Kerr D. E. Differential responsiveness of Holstein and Angus dermal fibroblasts to LPS challenge occurs
without major differences in the methylome. BMC Genomics. 2016;17:258.
201. Rauch T. A. and Pfeifer G. P. Methods for Assessing Genome-wide DNA Methylation. 2011;135-147.

94

DNA Sequencing Methods Collection

MeDIP-Seq: Methylated DNA Immunoprecipitation
DIP-seq: DNA Immunoprecipitation Followed by High-Throughput Sequencing
MeDIP-seq is used to study 5mC modification.202 It is based on MeDIP, originally developed as an approach for immunocapturing
methylated DNA followed by microarray analysis.203 A variation on this method detects 5hmC to provide a more complete view ofcytosine modifications (see hMeDIP-seq).204
In this method, anti-5mC antibodies are used to isolate methylated DNA from fragmented gDNA via immunoprecipitation.
The isolated DNA fragments are purified and used to prepare a sequencing library. Deep sequencing provides greater genome
coverage, representing the majority of immunoprecipitated methylated DNA.

Methylated DNA

Extract DNA

Fractionate
Denature

Immunoprecipitate

DNA purificationDNA

A schematic overview of MeDIP-seq.

Advantages

Disadvantages

•
•
•

•

•

Covers CpG and non-CpG 5mC throughout the genome
Covers 5mC in dense and less dense repeat regions
Antibody-based selection is independent of sequence and does
not enrich for 5hmC due to antibody specificity
Can be a cost-effective approach when single-base resolution is
not desired205••

Base-pair resolution is lower (~150 bp), as opposed to single-baseresolution with other methods
Antibody specificity and selectivity must be tested to avoid
nonspecific interaction
Biased toward hypermethylated regions204

Reviews
Sharma G., Sowpati D. T., Singh P., et al. Genome-wide non-CpG methylation of the host genome during M. tuberculosis infection. Sci Rep. 2016;6:25006.
Soto J., Rodriguez-Antolin C., Vallespin E., de Castro Carpeno J. and Ibanez de Caceres I. The impact of next-generation sequencing on the DNA methylation-basedtranslational cancer research. Transl Res. 2016;169:1-18 e11.
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

202. Weber M., Davies J. J., Wittig D., et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed
human cells. Nat Genet. 2005;37:853-862.
203. Weber M., Davies J. J., Wittig D., et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed
human cells. Nat Genet. 2005;37:853-862.
204. Xu Y., Wu F., Tan L., et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol Cell. 2011;42:451-464.
205. Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

An overview of recent DNA-seq publications featuring Illumina technology

95

References
Staunstrup N. H., Starnawska A., Nyegaard M., et al. Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots.
Clin Epigenetics. 2016;8:81.
There has been an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. In
this study, the authors demonstrated the feasibility of using MeDIP-seq to interrogate the methylome from a single 3.2 mm DBS punch (60 ng DNA) from filter cards
archived for up to 16 years. The authors conclude that the enrichment profile, sequence quality, and distribution of reads across genetic regions were comparable
among samples archived 16 years, 4 years, and a freshly prepared control.
Illumina Technology: HiSeq 2000 System

Tang A., Huang Y., Li Z., et al. Analysis of a four generation family reveals the widespread sequence-dependent maintenance of allelic DNA methylation insomatic and germ cells. Sci Rep. 2016;6:19260.
To evaluate transgenerational DNA methylation patterns in humans, the authors used MeDIP-seq to analyze the DNA methylomes of somatic and germ cells in a
4-generation family. They found that the allelic methylation patterns for the vast majority of cis-regulated loci were shared between the somatic and germ cells from thesame individual.
Illumina Technology: Genome AnalyzerIIx System
Han B., Li W., Chen Z., et al. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure. PLoS One. 2016;11:e0160358.
The authors performed MeDIP-seq to reveal changes in the genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) under coldpressure. They discovered that 21% of the methylation peaks were significantly altered after cold treatment. Approximately 8% of the altered DNA methylation peaks
were located in promoter regions, while the majority of them were located in noncoding regions. The methylation of multiple genes involved in biological processes(such as development and the immune response) were significantly affected, suggesting that these processes are responsive to cold stress through the regulation of
DNA methylation.
Illumina Technology: HiSeq 2000 System
Halder R., Hennion M., Vidal R. O., et al. DNA methylation changes in plasticity genes accompany the formation and maintenance of memory. Nat Neurosci.
2016;19:102-110.
To examine the epigenetic processes underlying short- and long-term memory, the authors studied chromatin modification changes in 2 distinct mouse brain regions,
2 cell types, and 3 time points before and after contextual learning. Using ChIP-Seq and MeDIP-seq, they found that histone modifications predominantly changed
during memory acquisition and correlated surprisingly little with changes in gene expression. The data provide evidence for a molecular framework of memoryacquisition and maintenance, wherein DNA methylation could alter the expression and splicing of genes involved in functional plasticity and synaptic wiring.
Illumina Technology: HiSeq 2000 System

Chowdhury B., Seetharam A., Wang Z., et al. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated
Microgravity in Human Lymphoblastoid Cells. PLoS One. 2016;11:e0147514.
Lucas E. S., Dyer N. P., Murakami K., et al. Loss of Endometrial Plasticity in Recurrent Pregnancy Loss. Stem Cells. 2016;34:346-356.
Pheiffer C., Erasmus R. T., Kengne A. P. and Matsha T. E. Differential DNA methylation of microRNAs within promoters, intergenic and intragenic regions of type 2
diabetic, pre-diabetic and non-diabetic individuals. Clin Biochem. 2016;49:433-438.
Su Y., Fan Z., Wu X., et al. Genome-wide DNA methylation profile of developing deciduous tooth germ in miniature pigs. BMC Genomics. 2016;17:134.
Sun L. X., Wang Y. Y., Zhao Y., et al. Global DNA Methylation Changes in Nile Tilapia Gonads during High Temperature-Induced Masculinization. PLoS One.
2016;11:e0158483.
Yang Y., Zhou R., Mu Y., Hou X., Tang Z., et al. Genome-wide analysis of DNA methylation in obese, lean, and miniature pig breeds. Sci Rep. 2016;6:30160.
Zeng Y., Yao B., Shin J., et al. Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression. Mol Cell. 2016;61:153-160.

Associated Kits
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment Kit

96

DNA Sequencing Methods Collection

hMeDIP-seq: Hydroxymethylated DNA Immunoprecipitation and Sequencing
hMeDIP-Seq is used to study 5hmC modifications.206 It is a slight variation of MeDIP-seq, which is based on the original MeDIP
method described by Weber et al.207 Although this method is technically almost identical to MeDIP-seq, it is treated as a separate
method due to the substantially different biological insight it provides. For a comprehensive insight into epigenetic changes, both
5mC and 5hmC modifications should be measured. 208
Methylated DNA is isolated from genomic DNA via immunoprecipitation. Anti-5hmC antibodies are incubated with fragmented
gDNA and precipitated, followed by DNA purification and preparation of a sequencing library. Deep sequencing provides greater
genome coverage, representing the majority of immunoprecipitated hydroxymethylated DNA.

Hydroxymethylated
DNA

Extract DNA

Fractionate
Denature

Immunoprecipitate

DNA purificationDNA

A schematic overview of hMeDIP-seq.

Advantages

Disadvantages•••

Covers 5hmC in dense and less dense repeat regions
Antibody-based selection is independent of sequence and does
not enrich for 5mC, due to antibody specificity••

Base-pair resolution is lower (~150 bp), as opposed to single-baseresolution with other methods
Antibody specificity and selectivity must be tested to avoid
nonspecific interaction
Biased toward hypermethylated regions

Reviews
Kamdar S. N., Ho L. T., Kron K. J., Isserlin R., van der Kwast T., et al. Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates
distinct biological pathways in prostate carcinogenesis. Clin Epigenetics. 2016;8:32.
Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights from advances ingenomic technologies. Neurosci Lett. 2016;625:47-55.
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References
Chowdhury B., Seetharam A., Wang Z., Liu Y., Lossie A. C., et al. A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene
Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells. PLoS One. 2016;11:e0147514.
Although microgravity has a wide variety of physiological effects, its influence on the epigenome has not been characterized. In this study, the authors used MeDIPseq, hMeDIP-seq, and RNA-Seq to examine the effects of induced changes on DNA methylation (5mC), hydroxymethylation (5hmC), and gene expression in cultured
human lymphoblastoid cells subjected to simulated microgravity. They found that simulated microgravity induced alterations in the methylomes: ~60% of differentiallymethylated regions (DMRs) were hypomethylated and ~92% of differentially hydroxymethylated regions (DHMRs) were hyperhydroxymethylated. These inducedchanges occurred both in promoter regions and gene bodies.
Illumina Technology: HiSeq 2000 System

206. Xu Y., Wu F., Tan L., et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol Cell. 2011;42:451-464.
207. Weber M., Davies J. J., Wittig D., et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed
human cells. Nat Genet. 2005;37:853-862.
208. Kamdar S. N., Ho L. T., Kron K. J., et al. Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways inprostate carcinogenesis. Clin Epigenetics. 2016;8:32.

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97

Devall M., Roubroeks J., Mill J., Weedon M. and Lunnon K. Epigenetic regulation of mitochondrial function in neurodegenerative disease: New insights fromadvances in genomic technologies. Neurosci Lett. 2016;625:47-55.
Changes in mtDNA methylation have been implicated in a number of neurodegenerative diseases. The researchers used MeDIP-seq, hMeDIP-seq, BS-Seq, ChIP-Seq
and RNA-Seq to determine the structure and function of Dnmt3a and Dnmt3b adult stem cells. They found the genes differ in their mechanisms of enhancer regulation:
Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes
DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function.
Illumina Technology: HiSeq 2000 System

Kamdar S. N., Ho L. T., Kron K. J., Isserlin R., van der Kwast T., et al. Dynamic interplay between locus-specific DNA methylation and hydroxymethylation
regulates distinct biological pathways in prostate carcinogenesis. Clin Epigenetics. 2016;8:32.
The authors studied functional pathways and genes regulated by epigenetic modifications in prostate cancer. They used hMeDIP-seq on cancer-derived cells andfound disruption of hydroxymethylation distribution, with global loss and highly specific gain in promoter and CpG island regions. They observed locus-specific retentionof hydroxymethylation marks in specific intronic and intergenic regions. The authors suggest that these modifications may play a novel role in the regulation of gene
expression in critical functional pathways, such as BARD1 signaling and steroid hormone receptor signaling, in cancer.
Illumina Technology: HiSeq 2000 System, HiSeq 2500 System

Bogdanovic O., Smits A. H., de la Calle Mustienes E., Tena J. J., Ford E., et al. Active DNA demethylation at enhancers during the vertebrate phylotypic period.
Nat Genet. 2016;48:417-426.
Uribe-Lewis S., Stark R., Carroll T., et al. 5-hydroxymethylcytosine marks promoters in colon that resist DNA hypermethylation in cancer. Genome Biol. 2015;16:69.
Zhu L., Lv R., Kong L., et al. Genome-Wide Mapping of 5mC and 5hmC Identified Differentially Modified Genomic Regions in Late-Onset Severe Preeclampsia: A Pilot
Study. PLoS One. 2015;10:e0134119.

Associated Kits
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

98

DNA Sequencing Methods Collection

MBDCap-seq: Methyl-CpG Binding Domain (MBD)–Based Capture and Sequencing
MethylCap-Seq: Capture of Methylated DNA Using the Methyl-CpG Binding
Domain MBD domain of MeCP2
MiGS: Methyl-CpG Binding Domain–Isolated Genome Sequencing
MBDCap209,210 and MethylCap211,212 enrich methylated DNA by affinity purification. These methods are particularly suitable toinvestigate regional blocks of hypermethylation. The term MiGS is easily confused with the common term MIGS (minimum
information about a genome sequence), and is rarely used.
In these methods, gDNA is first sonicated and incubated with tagged MBD proteins that can bind methylated cytosines. Next, theprotein-DNA complex is precipitated with antibody-conjugated beads that are specific to the protein tag. The DNA is purified andused to prepare a sequencing library. Deep sequencing provides greater genome coverage, representing the majority of MBDbound methylated DNA.

MBD

Methylated
DNA

Extract DNA

Fractionate

BiotinStreptavidin

Capture biotinylated MBD on
Streptavidin coated magnetic beads

Elute with increasing
salt concentrationDNA
purificationDNA

A schematic overview of MBDCap-seq.

Advantages

Disadvantages•••

Interrogates regional blocks of hypermethylation
Compared to HM450K arrays, MBDCap-seq regional coverage ofshores (77% vs 28%), enhancers (12% vs 2%), and insulators(11% vs 1%) is much greater213• MBDCap-Seq from formalin-fixed paraffin-embedded (FFPE)
tissues provides equivalent methylation data to fresh-frozen DNA212• MDB proteins can discriminate between 5mC and 5hmC214•

Base-pair resolution is lower (~150 bp), as opposed to single-baseresolution with other methods
Biased toward hypermethylated regions

Reviews
Soto J., Rodriguez-Antolin C., Vallespin E., de Castro Carpeno J. and Ibanez de Caceres I. The impact of next-generation sequencing on the DNA methylation-basedtranslational cancer research. Transl Res. 2016;169:1-18 e11.
Tang J., Fang F., Miller D. F., Pilrose J. M., Matei D., et al. Global DNA methylation profiling technologies and the ovarian cancer methylome. Methods Mol Biol.
2015;1238:653-675.
Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

209. Rauch T. A., Zhong X., Wu X., et al. High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer. Proc Natl Acad Sci U S A. 2008;105:252-257.
210. Rauch T. A. and Pfeifer G. P. The MIRA method for DNA methylation analysis. Methods Mol Biol. 2009;507:65-75.
211. Bock C., Tomazou E. M., Brinkman A. B., et al. Quantitative comparison of genome-wide DNA methylation mapping technologies. Nat Biotechnol. 2010;28:1106-1114.
212. Brinkman A. B., Simmer F., Ma K., Kaan A., Zhu J. and Stunnenberg H. G. Whole-genome DNA methylation profiling using MethylCap-seq. Methods. 2010;52:232-236.
213. Stirzaker C., Zotenko E., Song J. Z., et al. Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value.
Nat Commun. 2015;6:5899.
214. Yong W. S., Hsu F. M. and Chen P. Y. Profiling genome-wide DNA methylation. Epigenetics Chromatin. 2016;9:26.

An overview of recent DNA-seq publications featuring Illumina technology

99References
Boers A., Wang R., van Leeuwen R. W., et al. Discovery of new methylation markers to improve screening for cervical intraepithelial neoplasia grade 2/3.
Clin Epigenetics. 2016;8:29.
The authors used MethylCap-seq to identify new methylation markers for the detection of high-grade cervical intraepithelial neoplasia (CIN): CIN2/3, or CIN2 andhigher (CIN2+). They found that methylation levels of 8/9 genes were significantly higher in carcinoma compared to normal scrapings of cervical tissue. For all 8 genes,
methylation levels increased with the severity of the underlying histological lesion in scrapings from patients referred with an abnormal Pap smear.
Illumina Technology: Genome Analyzer II System

Stirzaker C., Zotenko E., Song J. Z., Qu W., Nair S. S., et al. Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters withprognostic value. Nat Commun. 2015;6:5899.
The authors used MBDCap-seq to analyze samples of FFPE tissues from triple-negative breast cancer (TNBC) and matched normal tissues. They identified regional
methylation profiles specific to TNBC, which they validated using methylation data extracted from The Cancer Genome Atlas (TCGA) breast cancer cohort. They also
reported the first potential TNBC-specific methylation signature of survival and suggest that it can have prognostic value in larger cohorts.
Illumina Technology: Genome AnalyzerIIx System
Wang Y., Jadhav R. R., Liu J., et al. Roles of Distal and Genic Methylation in the Development of Prostate Tumorigenesis Revealed by Genome-wide DNA
Methylation Analysis. Sci Rep. 2016;6:22051.
The authors used MBDCap-seq analysis on prostate cancer tissue specimens classified into low, high, and very high risk groups, based on Gleason score. They also
analyzed normal and tumor-adjacent tissue samples. The results, correlated with RNA-Seq data from TCGA, suggest that DNA methylation in distal and genic regions
plays critical roles in contributing to prostate tumorigenesis. It may act either positively or negatively with TSS to alter gene regulation in tumors.
Illumina Technology: Genome AnalyzerII System
Clausen M. J., Melchers L. J., Mastik M. F., et al. Identification and validation of WISP1 as an epigenetic regulator of metastasis in oral squamous cell carcinoma.
Genes Chromosomes Cancer. 2016;55:45-59.
Zhang X. L., Wu J., Wang J., et al. Integrative epigenomic analysis reveals unique epigenetic signatures involved in unipotency of mouse female germline stem cells.
Genome Biol. 2016;17:162.
Locke W. J., Zotenko E., Stirzaker C., et al. Coordinated epigenetic remodelling of transcriptional networks occurs during early breast carcinogenesis. Clin Epigenetics.
2015;7:52.

Associated Kits
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment

100

DNA Sequencing Methods Collection

BisChIP-seq: Bisulfite-Treated Chromatin-Immunoprecipitated DNA
ChIP-BS-seq: ChIP of Bisulfite-treated chromatin-Bisulfite Sequencing
ChIP-BMS: Chromatin Immunoprecipitation with Bisulfite Methylation
Sequencing Assay
BisChIP-seq, ChIP-BS-seq, and ChIP-BMS all refer to essentially the same method.215 It is a direct, quantitative approach toassess DNA methylation patterns associated with chromatin modifications or chromatin-associated factors.216
The ChIP-capturing step is used to obtain a restricted representation of the genome occupied by the epigenetic feature of interest.
The captured DNA fragments are subjected to end-repair, adapter ligation using methylated adapters, bisulfite conversion, PCR
amplification, and NGS.
T

C

T

C

G

T

U

T

C

G

T

T

C

G

BisulfiteT

DNA-protein complex withmethylated histones andmethylated DNA

Sonicate

Immunoprecipitate

Purify DNA
and shear

PCR

Bisulfite conversion

DNA

A schematic overview of BisChIP-seq.

Advantages

Disadvantages

•

•

•

Genome-wide coverage of 5mC in dense CpG areas andrepeat regions
MBD proteins do not interact with 5hmC••

Does not cover genome-wide CpGs and non-CpG methylation;
misses areas with less dense 5mC
Base-pair resolution is lower (~150 bp), as opposed to single-baseresolution with other methods
Protein-based selection is biased toward hypermethylated regions

Reviews
Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References

Jin J., Lian T., Gu C., et al. The effects of cytosine methylation on general transcription factors. Sci Rep. 2016;6:29119.
The authors used BisChIP-seq to study the effect of non-CpG methylation on DNA/protein interactions in 3 human transcription factors. They found that the
glucocorticoid receptor could recognize highly methylated sites within chromatin in cells. They conclude that non-CpG methylation of DNA can provide a mechanism forregulating gene expression by directly affecting the binding of transcription factors.
Illumina Technology: HiSeq 2000 System

215. Plongthongkum N., Diep D. H. and Zhang K. Advances in the profiling of DNA modifications: cytosine methylation and beyond. Nat Rev Genet. 2014;15:647-661.
216. Brinkman A. B., Gu H., Bartels S. J., et al. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation crosstalk. Genome Res. 2012;22:1128-1138.

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101

Varshney D., Vavrova-Anderson J., Oler A. J., et al. SINE transcription by RNA polymerase III is suppressed by histone methylation but not by DNA
methylation. Nat Commun. 2015;6:6569.
The authors studied the methylation of short interspersed nuclear elements (SINEs), such as Alu, to examine the effects on retrotransposition of these elements.
They used ChIP-BS-seq to assess the methylation status of Alu SINEs bound to RNA polymerase III (RNAPIII) and found high levels of CpG methylation. This DNA
methylation did not deter the binding of RNAPIII to Alu SINEs, and loss of DNA methylation had little effect on accessibility of SINEs to the transcriptional machinery.
The authors conclude that methylation of histones, rather than DNA, plays a dominant role in suppressing SINE transcription.
Illumina Technology: HiSeq System

Associated Kits
Infinium HumanMethylation450 Arrays
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit
Nextera Rapid Capture Exome/Custom Enrichment

102

DNA Sequencing Methods Collection

DNA-PROTEIN INTERACTIONS
Chromatin remodeling is a dynamic process driven by factors that change DNA-protein interactions. These epigenetic factors
can involve protein modifications, such as histone methylation, acetylation, phosphorylation, and ubiquitination.217 Histone
modifications affect gene activation by recruiting regulatory factors and maintaining an open or closed chromatin state. Epigenetic
factors play roles in tissue development,218 embryogenesis, cell fate, immune response, and diseases such as cancer.219
Bacterial pathogens can elicit transcriptional repression of immune genes by chromatin remodeling.220 The study of protein-DNA
interactions has also demonstrated that chromatin remodeling can respond to external factors, such as excessive alcohol-seeking
behaviors,221 cigarette smoking,222 and clinical drugs.

Cigarette smoking disrupts DNA-protein interactions, leading to the development of cancers or pulmonary diseases.

Reviews

Chaitankar V., Karakulah G., Ratnapriya R., et al. Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research.
Prog Retin Eye Res. 2016;55:1-31.
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;.
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

217. Rivera C. M. and Ren B. Mapping human epigenomes. Cell. 2013;155:39-55.
218. Pinello L., Xu J., Orkin S. H. and Yuan G. C. Analysis of chromatin-state plasticity identifies cell-type-specific regulators of H3K27me3 patterns.
Proc Natl Acad Sci U S A. 2014;111:E344-353.
219. Yeh H. H., Young D., Gelovani J. G., et al. Histone deacetylase class II and acetylated core histone immunohistochemistry in human brains with Huntington’s disease.
Brain Res. 2013;1504:16-24.
220. Kahrstrom C. T. Epigenetics: Legionella makes its mark on histones. Nat Rev Genet. 2013;14:370.
221. Warnault V., Darcq E., Levine A., Barak S. and Ron D. Chromatin remodeling--a novel strategy to control excessive alcohol drinking. Transl Psychiatry. 2013;3:e231.
222. Marwick J. A., Kirkham P. A., Stevenson C. S., et al. Cigarette smoke alters chromatin remodeling and induces proinflammatory genes in rat lungs.
Am J Respir Cell Mol Biol. 2004;31:633-642.

An overview of recent DNA-seq publications featuring Illumina technology

103

ChIP-Seq: Chromatin Immunoprecipitation Sequencing
HT-ChIP: High-Throughput ChIP
ChIP-exo: Exonuclease trimmed ChIP
Mint-ChIP: Multiplexed ChIP
ChIP-Seq is a well-established method to map specific protein-binding sites.223 It has given rise to a vast number of derivatives,
such as AHT-ChIP-Seq,224 BisChIP-Seq,225 CAST-ChIP,226 ChIP-BMS,227 ChIP-BS-seq,228 ChIPmentation,229 Drop-ChIP,230
Mint-ChIP,231 PAT–ChIP,232 reChIP-seq,233 scChIP-seq,234 and X-ChIP.235 Sequential ChIP-seq (reChIP) can also show theassociation of different proteins on the chromatin.236
In this method, DNA-protein complexes are crosslinked in vivo. Next, samples are fragmented and treated with an exonuclease totrim unbound oligonucleotides. Protein-specific antibodies are used to immunoprecipitate the DNA-protein complex. The DNA isextracted, purified, and sequenced, giving high-resolution sequences of the protein-binding sites.

DNA-protein complex

Crosslink proteins and DNA

Sample fragmentation

Exonuclease digestion

ImmunoprecipitateDNA
extractionDNA

A schematic overview of ChIP-Seq.

Advantages

Disadvantages

•
•
•

•

Base-pair resolution of protein-binding sites
Can map specific regulatory factors or proteins
Exonuclease use eliminates contamination by unbound DNA237•

Nonspecific antibodies can dilute the pool of DNA-protein
complexes of interest
Target protein must be known and be able to raise an antibody

Reviews
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

223. Solomon M. J., Larsen P. L. and Varshavsky A. Mapping protein-DNA interactions in vivo with formaldehyde: evidence that histone H4 is retained on a highly
transcribed gene. Cell. 1988;53:937-947.
224. Aldridge S., Watt S., Quail M. A., et al. AHT-ChIP-seq: a completely automated robotic protocol for high-throughput chromatin immunoprecipitation. Genome Biol.
2013;14:R124.
225. Statham A. L., Robinson M. D., Song J. Z., Coolen M. W., Stirzaker C. and Clark S. J. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq)
directly informs methylation status of histone-modified DNA. Genome Res. 2012;22:1120-1127.
226. Schauer T., Schwalie P. C., Handley A., Margulies C. E., Flicek P. and Ladurner A. G. CAST-ChIP maps cell-type-specific chromatin states in the Drosophila central
nervous system. Cell Rep. 2013;5:271-282.
227. Li Y. and Tollefsbol T. O. Combined chromatin immunoprecipitation and bisulfite methylation sequencing analysis. Methods Mol Biol. 2011;791:239-251.
228. Brinkman A. B., Gu H., Bartels S. J., et al. Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation crosstalk. Genome Res. 2012;22:1128-1138.
229. Schmidl C., Rendeiro A. F., Sheffield N. C. and Bock C. ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nat Methods.
2015;12:963-965.
230. Rotem A., Ram O., Shoresh N., et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015;33:1165-1172.
231. van Galen P., Viny A. D., Ram O., et al. A Multiplexed System for Quantitative Comparisons of Chromatin Landscapes. Mol Cell. 2016;61:170-180.
232. Fanelli M., Amatori S., Barozzi I. and Minucci S. Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue.
Nat Protoc. 2011;6:1905-1919.
233. Truax A. D. and Greer S. F. ChIP and Re-ChIP assays: investigating interactions between regulatory proteins, histone modifications, and the DNA sequences to which
they bind. Methods Mol Biol. 2012;809:175-188.
234. Rotem A., Ram O., Shoresh N., et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nat Biotechnol. 2015;33:1165-1172.
235. Skene P. J., Hernandez A. E., Groudine M. and Henikoff S. The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin
remodeler Chd1. Elife. 2014;3:e02042.
236. Elsasser S. J., Noh K. M., Diaz N., Allis C. D. and Banaszynski L. A. Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells.
Nature. 2015;522:240-244.
237. Zentner G. E. and Henikoff S. Surveying the epigenomic landscape, one base at a time. Genome Biol. 2012;13:250.

104

DNA Sequencing Methods CollectionReferences

Rinaldi L., Datta D., Serrat J., et al. Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis. Cell Stem Cell.
2016;19:491-501.
The endogenous function of DNA methyltransferases Dnmt3a and Dnmt3b in adult stem cells are unknown. In this study, the authors used ChIP-Seq in humanepidermal stem cells to show that Dnmt3a and Dnmt3b bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce
their associated enhancer RNAs. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function.
Illumina Technology: HiSeq 2000 System

Woolnough J. L., Atwood B. L., Liu Z., Zhao R. and Giles K. E. The Regulation of rRNA Gene Transcription during Directed Differentiation of Human
Embryonic Stem Cells. PLoS One. 2016;11:e0157276.
To compare the timing of the downregulation of rRNA synthesis with the binding of the RNA polymerase I transcription factor, UBTF, the authors performed ChIP-Seq
on untreated H9 ESCs. They compared the binding to H9 ESCs after 6 hours of treatment with the TGBF-β family member, ACTIVIN A. The overall binding of UBTF to
the rRNA gene was reduced roughly 50% after a 6 hour ACTIVIN A treatment. This result was consistent with the reduction in total rRNA synthesis measured by 32P
metabolic labeling.
Illumina Technology: HiSeq System

Schmidt S. F., Madsen J. G., Frafjord K. O., et al. Integrative Genomics Outlines a Biphasic Glucose Response and a ChREBP-RORgamma Axis Regulating Proliferation
in beta Cells. Cell Rep. 2016;16:2359-2372.
Nelson D. M., Jaber-Hijazi F., Cole J. J., et al. Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescenceand tumor suppression through preservation of genetic and epigenetic stability. Genome Biol. 2016;17:158.
Wu T. P., Wang T., Seetin M. G., et al. DNA methylation on N(6)-adenine in mammalian embryonic stem cells. Nature. 2016;532:329-333.
Zwart W., Flach K. D., Rudraraju B., et al. SRC3 Phosphorylation at Serine 543 Is a Positive Independent Prognostic Factor in ER-Positive Breast Cancer.
Clin Cancer Res. 2016;22:479-491.
Yasuma K., Yasunaga J., Takemoto K., et al. HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM
Domain (TIGIT). PLoS Pathog. 2016;12:e1005372.
De S., Mitra A., Cheng Y., Pfeifer K. and Kassis J. A. Formation of a Polycomb-Domain in the Absence of Strong Polycomb Response Elements. PLoS Genet.
2016;12:e1006200.
Bevington S. L., Cauchy P., Piper J., et al. Inducible chromatin priming is associated with the establishment of immunological memory in T cells. EMBO J. 2016;35:515-535.
Hay D., Hughes J. R., Babbs C., et al. Genetic dissection of the alpha-globin super-enhancer in vivo. Nat Genet. 2016;48:895-903.
Weiner A., Hsieh T. H., Appleboim A., et al. High-resolution chromatin dynamics during a yeast stress response. Mol Cell. 2015;58:371-386.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

105

DNaseI Seq or DNase-Seq: DNase l Hypersensitive Sites Sequencing
DNase I footprinting was first published in 1978238 and predates both Sanger sequencing and NGS. The first published use with
NGS was published by Boyle et al. 2008239 and later optimized for sequencing.240 A high-sensitivity protocol is also available(scDNase-seq).241
In this method, DNA-protein complexes are treated with DNase l, followed by DNA extraction and sequencing. Sequences boundby regulatory proteins are protected from DNase l digestion. Deep sequencing provides accurate representation of the locationof regulatory proteins in the genome. In a variation on this approach, the DNA-protein complexes are stabilized by formaldehyde
crosslinking before DNase I digestion. The crosslinking is reversed before DNA purification. In an alternative modification, called
GeF-seq, both the crosslinking and the DNase I digestion are carried out in vivo, within permeabilized cells.242

Active chromatin

DNase I digestion

Isolate trimmed complexes

DNA extractionDNA

A schematic overview of DNase-seq.

Advantages

Disadvantages• Can detect “open” chromatin243• No prior knowledge of the sequence or binding protein is required• Compared to formaldehyde-assisted isolation of regulatory
elements and sequencing (FAIRE-seq), has greater sensitivity
at promoters244•

DNase l is sequence-specific and hypersensitive sites might notaccount for the entire genome245• DNA loss through the multiple purification steps limits sensitivity246• Integration of DNase I with ChIP data is necessary to identify anddifferentiate similar protein-binding sites

Reviews
Chaitankar V., Karakulah G., Ratnapriya R., Giuste F. O., Brooks M. J., et al. Next generation sequencing technology and genomewide data analysis: Perspectives forretinal research. Prog Retin Eye Res. 2016;55:1-31.
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

References
Qiu Z., Li R., Zhang S., et al. Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit
Development. Mol Plant. 2016;9:1168-1182.
The authors studied the development and ripening of tomato fruit, events that are controlled precisely by the accessibility of regulatory elements to transcription
factors. They used DNase-seq to generate stage-specific, genome-wide, high-resolution DNase I hypersensitive site maps from fruit tissues of the tomato cultivar“Moneymaker.” They combined the data with gene expression data sets at 20 days after anthesis and break stage, to characterize chromatin accessibility andexpression changes to fruit developmental cues.
Illumina Technology: HiSeq 2500 System

238. Galas D. J. and Schmitz A. DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 1978;5:3157-3170.
239. Boyle A. P., Davis S., Shulha H. P., et al. High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008;132:311-322.
240. Anderson S. Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucleic Acids Res. 1981;9:3015-3027.
241. Jin W., Tang Q., Wan M., et al. Genome-wide detection of DNase I hypersensitive sites in single cells and FFPE tissue samples. Nature. 2015;528:142-146.
242. Chumsakul O., Nakamura K., Kurata T., et al. High-resolution mapping of in vivo genomic transcription factor binding sites using in situ DNase I footprinting and ChIPseq. DNA Res. 2013;20:325-338.
243. Zentner G. E. and Henikoff S. Surveying the epigenomic landscape, one base at a time. Genome Biol. 2012;13:250.
244. Kumar V., Muratani M., Rayan N. A., et al. Uniform, optimal signal processing of mapped deep-sequencing data. Nat Biotechnol. 2013;31:615-622.
245. Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.
246. Lu F., Liu Y., Inoue A., Suzuki T., Zhao K. and Zhang Y. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development. Cell.
2016;165:1375-1388.

106

DNA Sequencing Methods Collection

Frank C. L., Manandhar D., Gordan R. and Crawford G. E. HDAC inhibitors cause site-specific chromatin remodeling at PU.1-bound enhancers in K562 cells.
Epigenetics Chromatin. 2016;9:15.
The authors used DNase-seq and RNA-Seq to examined changes in response of K562 cells to sub-lethal treatment with of histone deacetylase inhibitors (HDACi).
As cell proliferation slowed, chromatin accessibility increased or decreased in several thousand gene regulatory elements. These changes coincided with nearby gene
expression changes and likely represent enhancer element activation or deactivation events.
Illumina Technology: HiSeq 2000 System
Lu F., Liu Y., Inoue A., Suzuki T., Zhao K., et al. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development. Cell.
2016;165:1375-1388.
The authors used DNase-seq to generate DNase I hypersensitive site maps of mouse preimplantation embryos from 1-cell to the morula stage. Their data revealed the
dynamics of chromatin accessibility during mouse preimplantation development. They identified key transcription factors involved in the establishment of the regulatory
landscape early in mammalian life.
Illumina Technology: HiSeq 2500 System
Badal S. S., Wang Y., Long J., et al. miR-93 regulates Msk2-mediated chromatin remodelling in diabetic nephropathy. Nat Commun. 2016;7:12076.
The authors used DNase I digestion to assess miR-93–modulated DNase1 hypersensitivity changes in podocytes cultured in high-glucose (HG) conditions. A numberof HG-related DNase I hypersensitive sites were reversed upon miR-93 overexpression. Some of these hypersensitive sites were located at the TSS of several genes
critical in the development of diabetic nephropathy.
Illumina Technology: HiSeq 2000 System

Adar S., Hu J., Lieb J. D. and Sancar A. Genome-wide kinetics of DNA excision repair in relation to chromatin state and mutagenesis. Proc Natl Acad Sci U S A.
2016;113:E2124-2133.
Bevington S. L., Cauchy P., Piper J., Bertrand E., Lalli N., et al. Inducible chromatin priming is associated with the establishment of immunological memory in T cells.
EMBO J. 2016;35:515-535.
Browne J. A., Yang R., Eggener S. E., Leir S. H. and Harris A. HNF1 regulates critical processes in the human epididymis epithelium. Mol Cell Endocrinol. 2016;425:94-102.
Chaitankar V., Karakulah G., Ratnapriya R., Giuste F. O., Brooks M. J., et al. Next generation sequencing technology and genomewide data analysis: Perspectives forretinal research. Prog Retin Eye Res. 2016;55:1-31.
Corces M. R., Buenrostro J. D., Wu B., Greenside P. G., Chan S. M., et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis andleukemia evolution. Nat Genet. 2016;48:1193-1203.
Georgakilas G., Vlachos I. S., Zagganas K., et al. DIANA-miRGen v3.0: accurate characterization of microRNA promoters and their regulators. Nucleic Acids Res.
2016;44:D190-195.
Lensing S. V., Marsico G., Hansel-Hertsch R., et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat Methods. 2016;13:855-857.
Metser G., Shin H. Y., Wang C., et al. An autoregulatory enhancer controls mammary-specific STAT5 functions. Nucleic Acids Res. 2016;44:1052-1063.
Schmidt S. F., Madsen J. G., Frafjord K. O., Poulsen L., Salo S., et al. Integrative Genomics Outlines a Biphasic Glucose Response and a ChREBP-RORgamma Axis
Regulating Proliferation in beta Cells. Cell Rep. 2016;16:2359-2372.
Shin H. Y., Willi M., Yoo K. H., et al. Hierarchy within the mammary STAT5-driven Wap super-enhancer. Nat Genet. 2016;48:904-911.
Thompson B., Varticovski L., Baek S. and Hager G. L. Genome-Wide Chromatin Landscape Transitions Identify Novel Pathways in Early Commitment to Osteoblast
Differentiation. PLoS One. 2016;11:e0148619.
Yang R., Kerschner J. L., Gosalia N., et al. Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus.
Nucleic Acids Res. 2016;44:3082-3094.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

107

DNase I SIM: DNase I Simplified in-Nucleus Method for plants
This method is a simplified DNase I protocol specifically intended for plants. It contains an additional step of nuclei purification in
Percoll gradients prior to DNase I digestion, in order to remove cellular debris and starch granules more efficiently. A DNA endpolishing step, using T4 DNA polymerase, is performed directly in the nuclei following DNase I digestion. These additional steps
obviate the need for gel purification and its attendant loss of material.
SupernatantNucleus

Nuclei
Cells

Lyse andcentrifuge

Sort
nuclei

Nucleus

DNase I digestion

Terminate DNase I
digestion

Polish ends

DNA
extractionDNA

A schematic overview of DNase I SIM.

Advantages

Disadvantages••

Does not require gel purification

Optimized for plants only

Reviews
None available yet.

References
Cumbie J. S., Filichkin S. A. and Megraw M. Improved DNase-seq protocol facilitates high resolution mapping of DNase I hypersensitive sites in roots in Arabidopsis
thaliana. Plant Methods. 2015;11:42.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

108

DNA Sequencing Methods CollectionMNase-Seq: Micrococcal Nuclease Sequencing
MAINE-Seq: Micrococcal Nuclease–Assisted Isolation of Nucleosomes
Nucleo-Seq: Isolated Nucleosome Sequencing
Nuc-seq: Isolated Nucleosome Sequencing
Micrococcal nuclease (MNase) is derived from Staphylococcus aureus, and its first use to determine chromatin structure dates
back to 1975, when the method was called, variously, staphylococcal nuclease or micrococcal nuclease digestion of nuclei
or chromatin.247,248 With the advent of NGS, MNase digestion249 became more popular and the term MNase-Seq was coined
finally.250 The terms MNase-assisted isolation of nucleosomes sequencing (MAINE-seq),251,252 Nucleo-Seq, 254 and Nuc-seq are
not commonly used. MNase, fused to the protein of interest, has been also been used for calcium-dependent cleavage to study
specific genomic loci in vivo (ChEC-seq).255
In MNase-Seq, gDNA is treated with MNase. Sequences bound by chromatin proteins are protected from MNase digestion.
Next, the DNA from the DNA-protein complexes is extracted and used to prepare a sequencing library. Deep sequencing provides
accurate representation of the location of regulatory DNA-binding proteins in the genome. 256

Open chromatin

MNase digestion

Isolate trimmed complexes

DNA extractionDNA

A schematic overview of MNase-Seq.

Advantages

Disadvantages••• MNase sites might not account for the entire genome• AT-dependent sequence bias259• Integration of MNase with ChIP data is necessary to identify anddifferentiate similar protein-binding sites

Can map nucleosomes and other DNA-binding proteins
Can footprint subnucleosomal particles protecting as little
as ~25 bp258• Identifies location of various regulatory proteins in the genome• Covers a broad range of regulatory sites

Reviews
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

247. Sollner-Webb B. and Felsenfeld G. A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease. Biochemistry. 1975;14:2915-2920.
248. Axel R. Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease. Biochemistry. 1975;14:2921-2925.
249. Schones D. E., Cui K., Cuddapah S., et al. Dynamic regulation of nucleosome positioning in the human genome. Cell. 2008;132:887-898.
250. Kuan P. F., Huebert D., Gasch A. and Keles S. A non-homogeneous hidden-state model on first order differences for automatic detection of nucleosome positions.
Stat Appl Genet Mol Biol. 2009;8:Article29.
251. Cusick M. E., Herman T. M., DePamphilis M. L. and Wassarman P. M. Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal
deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease. Biochemistry. 1981;20:6648-6658.
252. Ponts N., Harris E. Y., Prudhomme J., et al. Nucleosome landscape and control of transcription in the human malaria parasite. Genome Res. 2010;20:228-238.
253. Valouev A., Johnson S. M., Boyd S. D., Smith C. L., Fire A. Z. and Sidow A. Determinants of nucleosome organization in primary human cells. Nature. 2011;474:516520.
254. Chodavarapu R. K., Feng S., Bernatavichute Y. V., et al. Relationship between nucleosome positioning and DNA methylation. Nature. 2010;466:388-392.
255. Zentner G. E., Kasinathan S., Xin B., Rohs R. and Henikoff S. ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.
Nat Commun. 2015;6:8733.
256. Schones D. E., Cui K., Cuddapah S., et al. Dynamic regulation of nucleosome positioning in the human genome. Cell. 2008;132:887-898.
257. Zentner G. E. and Henikoff S. Surveying the epigenomic landscape, one base at a time. Genome Biol. 2012;13:250.
258. Henikoff J. G., Belsky J. A., Krassovsky K., MacAlpine D. M. and Henikoff S. Epigenome characterization at single base-pair resolution. Proc Natl Acad Sci U S A.
2011;108:18318-18323.
259. Kensche P. R., Hoeijmakers W. A., Toenhake C. G., et al. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics ofregulatory sequences. Nucleic Acids Res. 2016;44:2110-2124.

An overview of recent DNA-seq publications featuring Illumina technology

109

References
Lavender C. A., Cannady K. R., Hoffman J. A., et al. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin
Environment at Mammalian Promoters. PLoS Genet. 2016;12:e1006224.
The authors performed MNase-Seq on T47D/A1-2 cells to investigate the interplay between antisense transcription and nucleosome positioning. The results showedthat nucleosomes were regularly positioned relative to all 3 classes of identified TSSs. Like gene TSSs, MNase-Seq read density was consistent with “+1” nucleosomes
placed immediately downstream of daTSSs and uaTSSs, though MNase-Seq peaks were less sharp when centered on daTSS positions.
Illumina Technology: HiSeq System, MiSeq System
Rube H. T., Lee W., Hejna M., et al. Sequence features accurately predict genome-wide MeCP2 binding in vivo. Nat Commun. 2016;7:11025.
The authors used MNase-Seq to investigate genomic locations of nucleosomes in wild-type olfactory epithelial tissue in vivo. The expressed genes had deep
nucleosome-depleted regions around the TSS and distinct +1 nucleosomes. The transcription termination sites were also depleted of nucleosomes. Overlaying the
MNase-Seq and methyl-CpG binding protein 2 (MeCP2) ChIP-Seq data showed preferential colocalization of MeCP2 peaks with nucleosomes.
Illumina Technology: HiSeq 2000 System
de Dieuleveult M., Yen K., Hmitou I., et al. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells. Nature. 2016;530:113116.
This study used MNase-Seq to develop genome-wide remodeler–nucleosome interaction profiles for the chromatin remodelers Chd1, Chd2, Chd4, Chd6, Chd8, Chd9,
Brg1, and Ep400 in mouse ESCs. After Ep400 depletion, the authors detected an increased MNase resistance, particularly at the −1 nucleosome, where Ep400 wasenriched at both positively and negatively regulated genes. This effect was most evident at H3K4me3 promoters, which are bound by high Ep400 levels, compared tobivalent promoters. The authors suggest that Ep400 may therefore alter the structure of the −1 nucleosome.
Illumina Technology: Genome AnalyzerII System
Cole H. A., Cui F., Ocampo J., et al. Novel nucleosomal particles containing core histones and linker DNA but no histone H1. Nucleic Acids Res. 2016;44:573-581.
Deniz O., Flores O., Aldea M., Soler-Lopez M. and Orozco M. Nucleosome architecture throughout the cell cycle. Sci Rep. 2016;6:19729.
Devaiah B. N., Case-Borden C., Gegonne A., et al. BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nat Struct Mol Biol. 2016;23:540-548.
Johnson G. D., Jodar M., Pique-Regi R. and Krawetz S. A. Nuclease Footprints in Sperm Project Past and Future Chromatin Regulatory Events. Sci Rep.
2016;6:25864.
Kensche P. R., Hoeijmakers W. A., Toenhake C. G., Bras M., Chappell L., et al. The nucleosome landscape of Plasmodium falciparum reveals chromatin architectureand dynamics of regulatory sequences. Nucleic Acids Res. 2016;44:2110-2124.
Lombrana R., Alvarez A., Fernandez-Justel J. M., et al. Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major. Cell Rep.
2016;16:1774-1786.
Maehara K. and Ohkawa Y. Exploration of nucleosome positioning patterns in transcription factor function. Sci Rep. 2016;6:19620.
Matveeva E., Maiorano J., Zhang Q., et al. Involvement of PARP1 in the regulation of alternative splicing. Cell Discov. 2016;2:15046.
Mieczkowski J., Cook A., Bowman S. K., et al. MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat Commun.
2016;7:11485.
Ramakrishnan S., Pokhrel S., Palani S., et al. Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription.
Nat Commun. 2016;7:11949.
Wang M., Wang P., Tu L., et al. Multi-omics maps of cotton fibre reveal epigenetic basis for staged single-cell differentiation. Nucleic Acids Res. 2016;44:4067-4079.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit

110

DNA Sequencing Methods Collection

FiT-Seq: Fixed-Tissue Chromatin Immunoprecipitation Sequencing
FiT-seq is a method to extract soluble chromatin from FFPE tissue samples for the detection of histone-binding sites.260 The
distinguishing feature of this method is a proteinase K digestion step to reverse the effects of heavily crosslinked fixed chromatin in
FFPE tissues, before the sonication step.

FFPE-fixedchromatin

Deparaffination Heat 40°C in SDS
and rehydration

Proteinase K
digestion

Enzymeinactivation

Sonicate

Solubleextract

Immunoprecipitate

Cromatin Purification
elutionDNA

A schematic overview of FiT-seq.

Advantages

Disadvantages

•
•

•
•

Works on FFPE samples
Higher resolution and sensitivity than pathology tissue chromatin
immunoprecipitation (PAT-ChIP)

Not tested in other laboratories
Sonication may introduce sequence bias261

Reviews
None available yet.

References
Cejas P., Li L., O’Neill N. K., Duarte M., Rao P., et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat
Med. 2016;22:685-691.
The authors tested FiT-seq on human clinical samples that had been preserved between 2 and 15 years (median 8 years). They were able to assess various histonemarks readily and to generate chromatin-state maps comparable to those from cell lines and fresh-frozen tumors.
Illumina Technology: NextSeq 500 System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

260. Cejas P., Li L., O’Neill N. K., et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med. 2016;22:685-691.
261. Teytelman L., Ozaydin B., Zill O., et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One. 2009;4:e6700.

An overview of recent DNA-seq publications featuring Illumina technology

111

PAT-ChIP: Pathology Tissue Chromatin Immunoprecipitation
PAT-ChIP is a variation of ChIP-Seq, optimized for the analysis of chromatin derived from FFPE samples. The distinguishingfeatures of this method are dewaxing with a xylene substitute (Histolemon, Carlo Erba) followed by both MNase digestion andsonication.262 FiT-seq introduces further refinements to this method.263

FFPE-fixedchromatin elution

Deparaffination
and rehydration

MNase digestion

Sonicate

Solubleextract

Immunoprecipitateand chromatin elution

Reverse crosslink and Purification DNA
proteinase K digestion

A schematic overview of PAT-ChIP.

Advantages

Disadvantages

•

•
•
•

Works on FFPE samples

Long protocol takes 4 days
Low yield259
Sonication may introduce sequence bias264

Reviews
Mimura I., Kanki Y., Kodama T. and Nangaku M. Revolution of nephrology research by deep sequencing: ChIP-seq and RNA-seq. Kidney Int. 2014;85:31-38.

References
Cejas P., Li L., O’Neill N. K., Duarte M., Rao P., et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med.
2016;22:685-691.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

262. Fanelli M., Amatori S., Barozzi I. and Minucci S. Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue.
Nat Protoc. 2011;6:1905-1919.
263. Cejas P., Li L., O’Neill N. K., et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med. 2016;22:685-691.
264. Teytelman L., Ozaydin B., Zill O., et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One. 2009;4:e6700.

112

DNA Sequencing Methods Collection

X-ChIP-seq: High-Resolution Crosslinking Chromatin
Immunoprecipitation Sequencing
Crosslinking chromatin immunoprecipitation (X-ChIP) is a foundational technique in chromatin research.265,266 With the advent of
NGS this simple technique, now called X-ChIP-seq, is able produce high-resolution results.267
The method involves crosslinking chromatin-bound DNA in cells with 1% formaldehyde for 10 minutes at room temperature.
The cells are washed and resuspended in lysis buffer. After MNase digestion, the chromatin is solubilized by brief sonication andthen subjected to chromatin immunoprecipitation. The DNA is extracted, enriched for short fragments, and used to prepare asequencing library.

Crosslink
cells in vivo

Lyse cells

Cross-linked chromatin

Sonicate

MNase digestion

Solubleextract

Immunoprecipitateand extract DNA

DNA

A schematic overview of X-ChIP-seq.

Advantages

Disadvantages•• Sonication may introduce sequence bias268• Captures protein-DNA interactions regardless of duration269

Single-base resolution

Reviews
Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;15:814-827.

References

Schmidt S. V., Krebs W., Ulas T., et al. The transcriptional regulator network of human inflammatory macrophages is defined by open chromatin. Cell Res.
2016;26:151-170.
To determine how prolonged exposure to typical macrophage stimuli would alter transcription through long-term changes in histone modifications in humanmacrophages, the authors performed transcriptome analysis and ChIP-Seq. They used X-ChIP-seq to study 4 histone modifications: H3K4me1 (enhancers), H3K4me3(promoters), H3K27ac (active chromatin states), and H3K27me3 (poised enhancers and promoters). They were able to define a unique network of transcriptional andepigenetic regulators (TRs), which was characterized by accessible promoters, independent of the activation signal. The results support a model where macrophageactivation during inflammation is regulated transcriptionally mainly by a predefined TR network.
Illumina Technology: HiSeq 1000 System
Elsasser S. J., Noh K. M., Diaz N., Allis C. D. and Banaszynski L. A. Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem
cells. Nature. 2015;522:240-244.
The authors used both native ChiP-Seq and X-ChIP-seq in mouse ESCs to study the silencing of a subset of TEs—endogenous retroviral elements (ERVs)—mediatedby histone modifications. They identified 79,532 regions of H3.3 enrichment across the entire mouse genome, including repetitive regions. H3.3 was associated withboth active and repressed chromatin states. While most H3.3 peaks localized to genic regions and intergenic regulatory regions such as enhancers, 23% intersectedwith heterochromatic regions. The authors conclude that their results establish an important role for H3.3 in control of ERV retrotransposition in the mouse genome.
Illumina Technology: HiSeq 2000 System

Associated Kits
TruSeq ChIP Library Prep Kit

TruSeq DNA PCR-Free Library Prep Kit

TruSeq Nano DNA Library Prep Kit

Nextera DNA Library Prep Kit

TruSeq DNA Sample Preparation Kit

Nextera XT DNA Library Prep Kit

265. Breiling A., Turner B. M., Bianchi M. E. and Orlando V. General transcription factors bind promoters repressed by Polycomb group proteins. Nature. 2001;412:651-655.
266. Negre N. and Cavalli G. Finding Downstream Target DNAs for Chromatin Proteins by X-CHIP in Drosophila. Mapping Protein/DNA Interactions by Cross-Linking. 2001;
267. Skene P. J. and Henikoff S. A simple method for generating high-resolution maps of genome-wide protein binding. Elife. 2015;4:e09225.
268. Teytelman L., Ozaydin B., Zill O., et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One. 2009;4:e6700.
269. Zentner G. E., Kasinathan S., Xin B., Rohs R. and Henikoff S. ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.
Nat Commun. 2015;6:8733.

An overview of recent DNA-seq publications featuring Illumina technology

113

ORGANIC: Occupied Regions of Genomes from Affinity-Purified Naturally
Isolated Chromatin
ORGANIC is a gentle protocol that avoids crosslinking and sonication.270 The method is a combination of MNase-Seq and native
ChIP that provides accurate maps of chromatin-occupied regions in complex eukaryotic genomes.

Cell

Isolate nucei

Isolated chromatin

MNase digestion

Soluble extract

Immunoprecipitateand extract DNA

DNA

A schematic overview of ORGANIC.

Advantages

Disadvantages• Avoids sonication bias271• Avoids crosslinking artifacts272• Potentially poor solubilization of proteins273• MNase sequence bias274• Nuclei isolation may introduce artifacts• Not replicated in other laboratories

Reviews
Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;15:814-827.

References
Kasinathan S., Orsi G. A., Zentner G. E., Ahmad K. and Henikoff S. High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods.
2014;11:203-209.
Zentner G. E. and Henikoff S. Mot1 redistributes TBP from TATA-containing to TATA-less promoters. Mol Cell Biol. 2013;33:4996-5004.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

270. Kasinathan S., Orsi G. A., Zentner G. E., Ahmad K. and Henikoff S. High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods.
2014;11:203-209.
271. Teytelman L., Ozaydin B., Zill O., et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One. 2009;4:e6700.
272. Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;15:814-827.
273. Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;15:814-827.
274. Kensche P. R., Hoeijmakers W. A., Toenhake C. G., et al. The nucleosome landscape of Plasmodium falciparum reveals chromatin architecture and dynamics ofregulatory sequences. Nucleic Acids Res. 2016;44:2110-2124.

114

DNA Sequencing Methods Collection

ATAC-Seq: Assay for Transposase-Accessible Chromatin Sequencing
Fast-ATAC: ATAC-seq Optimized for Blood Cells
ATAC-Seq uses the Tn5 transposome to detect nucleosome-free regions of the genome.275 The method is commonly used, andoptimized protocols are available for tissues, such as blood (Fast-ATAC),276 neurons,277 biobank specimens,278 and single cells(scATAC-seq279 and single-cell ATAC-seq280).
In this method, gDNA is incubated with Tn5 transposomes, which fragments it and adds adapters simultaneously, in open
chromatin regions. Deep sequencing of the purified regions provides base-pair resolution of nucleosome-free regions in
the genome.

Open DNA

Tn5 Transposome

Insert in regions of open chromatin

Fragmented and primed

DNA purification
AmplificationDNA

A schematic overview of ATAC-seq.

Advantages

Disadvantages

•

•

•

Two-step protocol with no adapter ligation steps, gel purification, or
crosslink reversal
High signal-to-noise ratio compared to FAIRE-Seq••

During mechanical sample processing, bound chromatin regions
might open and be tagged by the transposome
Only half of the molecules contain the adapters in the orientation
required for PCR amplification
Distance between adapter sites may not be optimal for PCR
amplification281

Reviews
Chaitankar V., Karakulah G., Ratnapriya R., Giuste F. O., Brooks M. J., et al. Next generation sequencing technology and genomewide data analysis: Perspectives forretinal research. Prog Retin Eye Res. 2016;55:1-31.
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

275. Buenrostro J. D., Giresi P. G., Zaba L. C., Chang H. Y. and Greenleaf W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open
chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10:1213-1218.
276. Corces M. R., Buenrostro J. D., Wu B., et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat Genet.
2016;48:1193-1203.
277. Milani P., Escalante-Chong R., Shelley B. C., et al. Cell freezing protocol suitable for ATAC-Seq on motor neurons derived from human induced pluripotent stem cells.
Sci Rep. 2016;6:25474.
278. Scharer C. D., Blalock E. L., Barwick B. G., et al. ATAC-seq on biobanked specimens defines a unique chromatin accessibility structure in naive SLE B cells. Sci Rep.
2016;6:27030.
279. Buenrostro J. D., Wu B., Litzenburger U. M., et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature. 2015;523:486-490.
280. Cusanovich D. A., Daza R., Adey A., et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science. 2015;348:910-914.
281. Sos B. C., Fung H. L., Gao D. R., et al. Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay.
Genome Biol. 2016;17:20.

An overview of recent DNA-seq publications featuring Illumina technology

115

References
Bogdanovic O., Smits A. H., de la Calle Mustienes E., Tena J. J., Ford E., et al. Active DNA demethylation at enhancers during the vertebrate phylotypic
period. Nat Genet. 2016;48:417-426.
The authors studied epigenetic modifications during the phylotypic stage in zebrafish embryos. They observed widespread, Tet-dependent DNA demethylation of
thousands of enhancers associated with conserved regulatory pathways. They used ATAC-seq and whole-genome methylome profiling of tet1-tet2-tet3 morphantzebrafish embryos and demonstrated an upstream regulatory role for DNA methylation in these conserved genomic elements.
Illumina Technology: HiSeq 1500 System

Corces M. R., Buenrostro J. D., Wu B., Greenside P. G., Chan S. M., et al. Lineage-specific and single-cell chromatin accessibility charts humanhematopoiesis and leukemia evolution. Nat Genet. 2016;48:1193-1203.
This study used Fast-ATAC to create a comprehensive map of chromatin accessibility and transcriptional landscapes of 13 human primary blood cell types in a totalof 137 samples. Single-cell ATAC-seq of acute myeloid leukemia (AML) cells showed distinctive, mixed-regulome profiles corresponding to disparate developmental
stages, possibly caused by HOX factors. The authors suggest that these results could provide insights into hematopoietic development and disease.
Illumina Technology: HiSeq 2000 System, HiSeq 2500 System, NextSeq 500 System
Miller C. L., Pjanic M., Wang T., et al. Integrative functional genomics identifies regulatory mechanisms at coronary artery disease loci. Nat Commun.
2016;7:12092.
The authors examined the regulatory roles of epigenetic mechanisms in human coronary artery smooth muscle cells (HCASMCs) to gain insight into the developmentof coronary artery disease. They used ATAC-seq to generate epigenomic profiles in primary cultured HCASMCs stimulated with various growth factors, as well as innormal and atherosclerotic human coronary artery tissues. They integrated the data with transcription-factor binding and H3K27ac ChIP-Seq data to define HCASMCenriched cis-regulatory mechanisms.
Illumina Technology: HiSeq 2500 System
Wu J., Huang B., Chen H., et al. The landscape of accessible chromatin in mammalian preimplantation embryos. Nature. 2016;534:652-657.
The authors used ATAC-seq with CRISPR/Cas9-assisted mtDNA depletion to produce a genome-wide map of accessible chromatin in mouse preimplantationembryos. They found that, despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable
after major zygotic genome activation.
Illumina Technology: HiSeq 1500 System, HiSeq 2500 System

Ackermann A. M., Wang Z., Schug J., Naji A. and Kaestner K. H. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes.
Mol Metab. 2016;5:233-244.
Atianand M. K., Hu W., Satpathy A. T., et al. A Long Noncoding RNA lincRNA-EPS Acts as a Transcriptional Brake to Restrain Inflammation. Cell. 2016;165:1672-1685.
Boukhaled G. M., Cordeiro B., Deblois G., et al. The Transcriptional Repressor Polycomb Group Factor 6, PCGF6, Negatively Regulates Dendritic Cell Activation andPromotes Quiescence. Cell Rep. 2016;16:1829-1837.
de Dieuleveult M., Yen K., Hmitou I., Depaux A., Boussouar F., et al. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells. Nature.
2016;530:113-116.
Flynn R. A., Do B. T., Rubin A. J., et al. 7SK-BAF axis controls pervasive transcription at enhancers. Nat Struct Mol Biol. 2016;23:231-238.
George J., Uyar A., Young K., et al. Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells. Nat Commun. 2016;7:12166.
Han D., Lu X., Shih A. H., et al. A Highly Sensitive and Robust Method for Genome-wide 5hmC Profiling of Rare Cell Populations. Mol Cell. 2016;63:711-719.
Hay D., Hughes J. R., Babbs C., Davies J. O., Graham B. J., et al. Genetic dissection of the alpha-globin super-enhancer in vivo. Nat Genet. 2016;48:895-903.
Kaaij L. J., Mokry M., Zhou M., Musheev M., Geeven G., et al. Enhancers reside in a unique epigenetic environment during early zebrafish development. Genome Biol.
2016;17:146.
Kaufman C. K., Mosimann C., Fan Z. P., et al. A zebrafish melanoma model reveals emergence of neural crest identity during melanoma initiation. Science.
2016;351:aad2197.
Koues O. I., Collins P. L., Cella M., et al. Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells. Cell. 2016;165:1134-1146.
Lu F., Liu Y., Inoue A., Suzuki T., Zhao K., et al. Establishing Chromatin Regulatory Landscape during Mouse Preimplantation Development. Cell. 2016;165:1375-1388.
Proudhon C., Snetkova V., Raviram R., et al. Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation. Cell Rep.
2016;15:2159-2169.
Rendeiro A. F., Schmidl C., Strefford J. C., et al. Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtype-specific epigenome signatures andtranscription regulatory networks. Nat Commun. 2016;7:11938.
Sebe-Pedros A., Ballare C., Parra-Acero H., et al. The Dynamic Regulatory Genome of Capsaspora and the Origin of Animal Multicellularity. Cell. 2016;165:1224-1237.

116

DNA Sequencing Methods Collection

Shih H. Y., Sciume G., Mikami Y., et al. Developmental Acquisition of Regulomes Underlies Innate Lymphoid Cell Functionality. Cell. 2016;165:1120-1133.
Smith J. D., Suresh S., Schlecht U., et al. Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design.
Genome Biol. 2016;17:45.
Wang L., Siegenthaler J. A., Dowell R. D. and Yi R. Foxc1 reinforces quiescence in self-renewing hair follicle stem cells. Science. 2016;351:613-617.
Wang W., Org T., Montel-Hagen A., et al. MEF2C protects bone marrow B-lymphoid progenitors during stress haematopoiesis. Nat Commun. 2016;7:12376.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

117

THS-seq: Transposome Hypersensitive Sites Sequencing
THS-seq is a method for highly sensitive characterization of chromatin accessibility. This variation on ATAC-seq uses linear
amplification of accessible DNA ends, in vitro transcription, and an engineered Tn5 super-mutant.282
Read primer
T7 promoter

Open DNA

Tn5 Transposome

Insert in regions of open chromatin

PurifyEnd fill-in and IVT
amplification

dsDNA synthesis

Add barcodes andsequencing adapters

A schematic overview of THS-seq.

Advantages

Disadvantages•••

Requires very little input material
Can detect small regions near distal enhancers

Requires an engineered transposase

Reviews
None available yet.

References
Sos B. C., Fung H. L., Gao D. R., Osothprarop T. F., Kia A., et al. Characterization of chromatin accessibility with a transposome hypersensitive sites
sequencing (THS-seq) assay. Genome Biol. 2016;17:20.
The authors developed THS-seq to address the limitations of ATAC-seq. They demonstrated the improved sensitivity of THS-seq in 100 GM12878 lymphoblastoid
cells, which yielded approximately 110,000 unique transposition events per cell. THS-seq showed an approximate 500-fold improvement in sensitivity over published
ENCODE ATAC-seq data.
Illumina Technology: MiSeq System, HiSeq 2500 System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

282. Sos B. C., Fung H. L., Gao D. R., et al. Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay.
Genome Biol. 2016;17:20.

118

DNA Sequencing Methods Collection

CATCH‑IT: Covalent Attachment of Tags to Capture Histones and Identify Turnover
CATCH-IT is a direct method for measuring nucleosome turnover dynamics genome-wide.283
In this method, cells are treated briefly with the methionine surrogate azidohomoalanine (Aha), which couples biotin to
nucleosomes containing newly incorporated histones. The labeled chromatin is affinity-purified with streptavidin, washedstringently to remove nonhistone proteins, and analyzed using tiling microarrays.
BiotinStreptavidin

Cells starved ofmethionine

Add methionine analogue
l-azidohomoalanine (AHA)

Isolatenucei

Cycloaddition
reaction

Remove of H2A–H2B dimers
and non-histone proteins

MNase digestionDNA
extractionDNA

A schematic overview of CATCH-IT.

Advantages

Disadvantages••

Can determine differences in nucleosome turnover acrossthe genome

Potential artifacts

Reviews
Zentner G. E. and Henikoff S. High-resolution digital profiling of the epigenome. Nat Rev Genet. 2014;15:814-827.

References
Skene P. J., Hernandez A. E., Groudine M. and Henikoff S. The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin
remodeler Chd1. Elife. 2014;3:e02042.
This study examined the interactions of the chromatin-remodeling protein Chd1 with regions of active transcription in mouse embryonic fibroblasts. The authors
used CATCH-IT to show that histone turnover was most rapid at the nucleosomes flanking either side of the promoter and progressively decreased toward the genebody. Upon expression of a dominant-negative mutant (K510R-Chd1), nucleosome turnover was significantly reduced on either side of the TSS but significantly
increased within the gene body. The authors conclude that, through both positively and negatively impacting histone dynamics, Chd1 can regulate pluripotency
and reprogramming.
Illumina Technology: HiSeq 2000 System
Weber C. M., Ramachandran S. and Henikoff S. Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase. Mol Cell. 2014;53:819-830.
The authors used CATCH-IT to determine how the histone variant H2A.Z could modulate RNAPII kinetics. They found that H2A.Z occupancy anticorrelated withcomplete nucleosome turnover. CATCH-IT measured nucleosome turnover as the replacement of H3 and H4, but not H2A or H2B, which suggests that H2A.Z-H2B
dimers facilitate retention of (H3-H4)2 tetramers.
Illumina Technology: HiSeq 2000 System

Perez-Lluch S., Blanco E., Tilgner H., et al. Absence of canonical marks of active chromatin in developmentally regulated genes. Nat Genet. 2015;47:1158-1167.
Teves S. S. and Henikoff S. Transcription-generated torsional stress destabilizes nucleosomes. Nat Struct Mol Biol. 2014;21:88-94.
Yildirim O., Hung J. H., Cedeno R. J., et al. A system for genome-wide histone variant dynamics in ES cells reveals dynamic MacroH2A2 replacement at promoters.
PLoS Genet. 2014;10:e1004515.

Associated Kits
TruSeq Nano DNA Library Prep Kit

Nextera DNA Library Prep Kit

TruSeq DNA Sample Preparation Kit

Nextera XT DNA Library Prep Kit

TruSeq DNA PCR-Free Library Prep Kit

283. Deal R. B., Henikoff J. G. and Henikoff S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones. Science. 2010;328:1161-1164.

An overview of recent DNA-seq publications featuring Illumina technology

119

MINCE-seq: Mapping in Vivo Nascent Chromatin with EdU
MINCE-seq was developed to characterize the genome-wide location of nucleosomes and other chromatin proteins behind
replication forks at high temporal and spatial resolution.284
In this method, newly replicated DNA is labeled with the nucleotide analog EdU, which is coupled with biotin using click chemistry.
Coupling with biotin ensures highly specific purification of newly replicated DNA from asynchronous cells, even if it is only afraction of a percentage of total DNA. Subsequent MNase treatment recovers DNA fragments bound both by nucleosomes and bynonhistone DNA-binding proteins, which enables the mapping of newly replicated chromatin at near base-pair resolution.283
Newly-formed chromatin

Streptavidin

Polymerase

Label with ethynyl
deoxyuridine (EdU)

Chase withthymidine

Crosslink with formaldehyde

Click reaction
to attach biotin

Sonication and
MNase digestion

Streptavidin
captureDNA
extractionDNA

A schematic overview of MINCE-seq.

Advantages

Disadvantages••

Maps newly replicated chromatin at near base-pair resolution

Robustness unknown

Reviews
None available yet.

References

Ramachandran S. and Henikoff S. Transcriptional Regulators Compete with Nucleosomes Post-replication. Cell. 2016;165:580-592.
The authors used MINCE-seq to show that the characteristic chromatin landscape at Drosophila promoters and enhancers is lost upon replication. Promoters that hadhigh levels of RNAPII stalling and DNA accessibility showed specific enrichment for the Brahma (BRM) remodeling factor. Enhancer chromatin was also disrupted during
replication, suggesting a role for transcription factor competition in nucleosome re-establishment.
Illumina Technology: HiSeq 2500 System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

284. Ramachandran S. and Henikoff S. Transcriptional Regulators Compete with Nucleosomes Post-replication. Cell. 2016;165:580-592.

120

DNA Sequencing Methods CollectionFAIRE-seq: Formaldehyde-Assisted Isolation of Regulatory Elements
Sono-Seq: Sonication of Crosslinked Chromatin
FAIRE-seq285,286 and Sono-Seq287 are based on differences in crosslinking efficiencies between DNA and nucleosomes orsequence-specific DNA-binding proteins.
In this method, DNA-protein complexes are crosslinked briefly in vivo using formaldehyde. The sample is then lysed and sonicated.
After phenol/chloroform extraction, the DNA in the aqueous phase is purified and sequenced. Sequencing provides information forregions of DNA that are not occupied by histones.283

Open DNA

Crosslink protein and DNA with formalin

Phenol extract and purify DNA
from the aquous phase

SonicateDNA

A schematic overview of FAIRE-seq.

Advantages

Disadvantages

•
•
•

•
•

•

Simple and highly reproducible protocol
Does not require antibodies
Does not require enzymes, such as DNase or MNase, avoiding the
optimization and extra steps necessary for enzymatic processing
Does not require a single-cell suspension or nuclear isolation, so it
is easily adapted for use on tissue samples288

Cannot identify regulatory proteins bound to DNA
DNase-Seq may be better at identifying nucleosome-depleted
promoters of highly expressed genes289

Reviews
Yan H., Tian S., Slager S. L., Sun Z. and Ordog T. Genome-Wide Epigenetic Studies in Human Disease: A Primer on -Omic Technologies. Am J Epidemiol.
2016;183:96-109.

References
Lavender C. A., Cannady K. R., Hoffman J. A., Trotter K. W., Gilchrist D. A., et al. Downstream Antisense Transcription Predicts Genomic Features That
Define the Specific Chromatin Environment at Mammalian Promoters. PLoS Genet. 2016;12:e1006224.
The authors used FAIRE-seq to characterize accessible regions of DNA in T47D/A1-2 cells and revealed an open genomic region at downstream antisense transcription
start site (daTSS) positions. ChIP-Seq data revealed that the daTSS coincides with the binding of trans-regulatory factors, consistent with these areas being open andenriched in protein-binding motifs.
Illumina Technology: HiSeq 2500 System, MiSeq System
Behura S. K., Sarro J., Li P., et al. High-throughput cis-regulatory element discovery in the vector mosquito Aedes aegypti. BMC Genomics. 2016;17:341.
The authors used FAIRE-seq to assess genetic variation in the regulatory elements of mosquito strains susceptible (Moyo-S) and refractory (Moyo-R) to dengue virus.
Known transcription factor consensus binding sites were enriched in the FAIRE peaks and, of these, FoxA1, Hunchback, Gfi, Klf4, MYB/ph3, and Sox9 were the most
predominant. All of the elements tested in vivo were confirmed to drive gene expression in transgenic Drosophila reporter assays. Of the > 13,000 SNPs recently
identified in Moyo-S and Moyo-R mosquito strains, 3365 mapped to FAIRE peaks.
Illumina Technology: HiSeq 2000 System

285. Giresi P. G. and Lieb J. D. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements).
Methods. 2009;48:233-239.
286. Hogan G. J., Lee C. K. and Lieb J. D. Cell cycle-specified fluctuation of nucleosome occupancy at gene promoters. PLoS Genet. 2006;2:e158.
287. Auerbach R. K., Euskirchen G., Rozowsky J., et al. Mapping accessible chromatin regions using Sono-Seq. Proc Natl Acad Sci U S A. 2009;106:14926-14931.
288. Simon J. M., Giresi P. G., Davis I. J. and Lieb J. D. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nat Protoc.
2012;7:256-267.
289. Song L., Zhang Z., Grasfeder L. L., et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res.
2011;21:1757-1767.

An overview of recent DNA-seq publications featuring Illumina technology

121Verfaillie A., Imrichova H., Atak Z. K., et al. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state.
Nat Commun. 2015;6:6683.
To determine if cis-regulatory regions underlie invasive and proliferative transcriptional states in melanoma, the authors used FAIRE-seq and ChIP-Seq against activated(H3K27ac) and repressed (H3K27me3) chromatin marks in melanoma cell cultures. Clustering on the basis of H3K27ac and FAIRE-seq tracks indicated an active andopen SOX10 promoter in the 9 proliferative samples with high SOX10 expression. In the 2 invasive cultures, the SOX10 promoter lacked activating marks but carried
repressing H3K27me3 marks.
Illumina Technology: HiSeq 2000 System

Ackermann A. M., Wang Z., Schug J., Naji A. and Kaestner K. H. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes.
Mol Metab. 2016;5:233-244.
de Dieuleveult M., Yen K., Hmitou I., Depaux A., Boussouar F., et al. Genome-wide nucleosome specificity and function of chromatin remodellers in ES cells. Nature.
2016;530:113-116.
Du J., Leung A., Trac C., et al. Chromatin variation associated with liver metabolism is mediated by transposable elements. Epigenetics Chromatin. 2016;9:28.
Fabrizius A., Andre D., Laufs T., et al. Critical re-evaluation of neuroglobin expression reveals conserved patterns among mammals. Neuroscience. 2016;337:339-354.
Nevedomskaya E., Stelloo S., van der Poel H. G., et al. Androgen receptor DNA binding and chromatin accessibility profiling in prostate cancer. Genom Data.
2016;7:124-126.
Bicker A., Brahmer A. M., Meller S., et al. The Distinct Gene Regulatory Network of Myoglobin in Prostate and Breast Cancer. PLoS One. 2015;10:e0142662.
Davie K., Jacobs J., Atkins M., et al. Discovery of transcription factors and regulatory regions driving in vivo tumor development by ATAC-seq and FAIRE-seq open
chromatin profiling. PLoS Genet. 2015;11:e1004994.
Naval-Sanchez M., Potier D., Hulselmans G., Christiaens V. and Aerts S. Identification of Lineage-Specific Cis-Regulatory Modules Associated with Variation inTranscription Factor Binding and Chromatin Activity Using Ornstein-Uhlenbeck Models. Mol Biol Evol. 2015;32:2441-2455.
Reschen M. E., Gaulton K. J., Lin D., et al. Lipid-induced epigenomic changes in human macrophages identify a coronary artery disease-associated variant that
regulates PPAP2B Expression through Altered C/EBP-beta binding. PLoS Genet. 2015;11:e1005061.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

122

DNA Sequencing Methods Collection

NOMe-Seq: Nucleosome Occupancy Methylome-Sequencing
NOMe-Seq is a single-molecule, high-resolution nucleosome positioning assay.290 This method is based on the ability of the GpC
methyltransferase M.CviPI to methylate GpC sites that are not bound by nucleosomes, to create a digital footprint of nucleosome
positioning. M.CviPI can map nucleosome positions at CpG-poor promoters, irrespective of their endogenous methylation status.
In this method, native chromatin is treated with M.CviPI, following which the DNA is treated with sodium bisulfite and subjected to
WGBS. From these data, CpG methylation patterns as well as nucleosome-free regions (GpC methylation) can be identified.291
CpG dinucleotides
Methylated CpG

Open DNACpG dinucleotides
Methylated CpG

GpC methyltransferase (M.CviPI) and
S-Adenosylmethionine (SAM)

Bisulfite conversion BS-Seq

Protected
Control
M.CviPI

Protected

Protected
methylated

Unprotected
methylated

Protected
unmethylated

Unprotected
unmethylated

A schematic overview of NOMe-Seq.

Advantages

Disadvantages••

High resolution

Relies on the presence of GpC residues292

Reviews
Shull A. Y., Noonepalle S. K., Lee E. J., Choi J. H. and Shi H. Sequencing the cancer methylome. Methods Mol Biol. 2015;1238:627-651.

References

Wallner S., Schroder C., Leitao E., Berulava T., Haak C., et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics Chromatin.
2016;9:33.
The authors used NOMe-Seq to investigate whether the chromatin accessibility of DMRs change during differentiation. Monocytes had 89,212 NOMe-Seq peaks
covering ~21 Mbp, and macrophages had 127,267 peaks covering ~42 Mbp, which demonstrates that macrophages had more nucleosome-depleted regions.
More detailed analysis of DMRs and NOMe-Seq peaks that were gained or lost during differentiation indicated that most DMRs become nucleosome-depleted
during differentiation.
Illumina Technology: HiSeq 2000 System

Lay F. D., Liu Y., Kelly T. K., et al. The role of DNA methylation in directing the functional organization of the cancer epigenome. Genome Res. 2015;25:467-477.
Statham A. L., Taberlay P. C., Kelly T. K., Jones P. A. and Clark S. J. Genome-wide nucleosome occupancy and DNA methylation profiling of four human cell lines.
Genom Data. 2015;3:94-96.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

290. Han H., Cortez C. C., Yang X., Nichols P. W., Jones P. A. and Liang G. DNA methylation directly silences genes with non-CpG island promoters and establishes anucleosome occupied promoter. Hum Mol Genet. 2011;20:4299-4310.
291. Wallner S., Schroder C., Leitao E., et al. Epigenetic dynamics of monocyte-to-macrophage differentiation. Epigenetics Chromatin. 2016;9:33.
292. Ishii H., Kadonaga J. T. and Ren B. MPE-seq, a new method for the genome-wide analysis of chromatin structure. Proc Natl Acad Sci U S A. 2015;112:E3457-3465.

An overview of recent DNA-seq publications featuring Illumina technology

123

ChIPmentation: Chromatin Immunoprecipitation with Sequencing Library
Preparation by Tn5 Transposase
ChIPmentation combines ChIP with sequencing library preparation by Tn5 transposase (tagmentation).293 Both ATAC-seq and
ChIPmentation can be combined usefully in the same experiment to assay open chromatin and protein binding, respectively.294
This combination of methods takes advantage of the flexibility and efficiency provided by using Tn5 transposasein library preparation.
Tagmentation is performed directly on bead-bound, immunoprecipitated chromatin, followed by standard library
preparation methods.

Chromatin

Chromatin immunoprecipitation

Tn5 Transposome

Adapter insertion

Fragmented
and primed

DNA purification
AmplificationDNA

A schematic overview of ChIPmentation.

Advantages

Disadvantages•••

Can generated accurate profiles from as little as 10,000 cells
Simple, one-step reaction

None reported

Reviews
None available yet.

References
Rendeiro A. F., Schmidl C., Strefford J. C., Walewska R., Davis Z., et al. Chromatin accessibility maps of chronic lymphocytic leukaemia identify subtypespecific epigenome signatures and transcription regulatory networks. Nat Commun. 2016;7:11938.
The authors studied epigenetic deregulation in chronic lymphocytic leukemia (CLL). Chromatin profiles created by ChIPmentation and RNA-Seq accurately predicted
CLL. The authors also examined the mutation status of IGHV genes, a clinical biomarker for CLL. Gene regulatory networks inferred for IGHV-mutated vs IGHVunmutated samples identified characteristic differences between less aggressive and more aggressive CLL subtypes, respectively.
Illumina Technology: HiSeq 3000/4000 System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

293. Schmidl C., Rendeiro A. F., Sheffield N. C. and Bock C. ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nat Methods.
2015;12:963-965.
294. Chaitankar V., Karakulah G., Ratnapriya R., Giuste F. O., Brooks M. J. and Swaroop A. Next generation sequencing technology and genomewide data analysis:
Perspectives for retinal research. Prog Retin Eye Res. 2016;55:1-31.

124

DNA Sequencing Methods Collection

ChIA-PET: Chromatin Interaction Analysis by Paired-End Tag Sequencing
ChIA-PET features an immunoprecipitation step to map long-range DNA interactions, similar to Hi-C.295,296 In this method, DNAprotein complexes are crosslinked and fragmented. Specific antibodies are used to immunoprecipitate proteins of interest. Twosets of linkers, with unique barcodes, are ligated to the ends of the DNA fragments in separate aliquots, which then self-ligate
based on proximity. The DNA aliquots are precipitated, digested with restriction enzymes, and sequenced. Deep sequencing
provides base-pair resolution of the ligated fragments. Hi-C and ChIA-PET currently provide the best balance of resolution andreasonable coverage in the human genome to map long-range interactions.297
A modified protocol, called advanced or long-read ChIA-PET, has been published by Tang et al. 298 This method replaces the
2 separate ligation reactions with 2 half linkers and a single biotinylated linker ligation. Next, the de-crosslinked, purified DNA isfragmented and adapters are ligated using Tn5 transposase in a single step. Finally, the DNA is PCR-amplified and sequenced.299

Sample fragmentation

Immunoprecipitation

Ligation

Restriction enzyme digestionDNA

A schematic overview of ChIA-PET.

Advantages

Disadvantages

•

•

•
•
•
•

Suitable for detecting a large number of both long-range andshort-range chromatin interactions globally300
Studies the interactions made by specific proteins or protein
complexes
Public ChIA-PET datasets are available through the ENCODE
Project301
Removes background generated during traditional ChIP assays
Immunoprecipitation step reduces data complexity113

Requires a large amount of starting material required, generally atleast 100 million cells302• Nonspecific antibodies can pull down unwanted protein complexes
and contaminate the pool
• Linkers can self-ligate, generating ambiguity about true DNA
interactions• Limited sensitivity; may detect as little as 10% of interactions113

Reviews
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;.

References
Ricano-Ponce I., Zhernakova D. V., Deelen P., et al. Refined mapping of autoimmune disease associated genetic variants with gene expression suggests animportant role for non-coding RNAs. J Autoimmun. 2016;68:62-74.
The authors performed a systematic analysis of transcriptomic data from 629 blood samples and linked 460 SNPs that are associated with 14 autoimmune diseases.
The SNPs were linked to functional regulatory elements, which suggests a model where autoimmune disease genes are regulated by a network of chromatin-looping/noncoding RNA interactions. Data from the ChIA-PET assay in B lymphoblastoid cells indicated that multigene SNPs were more often (70%) involved in looping
interactions in B cells than single-gene SNPs (55%).
Illumina Technology: HiSeq 2000 System

295. Li G., Fullwood M. J., Xu H., et al. ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol. 2010;11:R22.
296. Fullwood M. J., Liu M. H., Pan Y. F., et al. An oestrogen-receptor-alpha-bound human chromatin interactome. Nature. 2009;462:58-64.
297. Dekker J., Marti-Renom M. A. and Mirny L. A. Exploring the three-dimensional organization of genomes: interpreting chromatin interaction data. Nat Rev Genet.
2013;14:390-403.
298. Tang Z., Luo O. J., Li X., et al. CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription. Cell. 2015;163:1611-1627.
299. Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;
300. Sajan S. A. and Hawkins R. D. Methods for identifying higher-order chromatin structure. Annu Rev Genomics Hum Genet. 2012;13:59-82.
301. Consortium E. P. A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol. 2011;9:e1001046.
302. Mumbach M. R., Rubin A. J., Flynn R. A., et al. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat Methods. 2016;13:919-922.

An overview of recent DNA-seq publications featuring Illumina technology

125

Chang H., Liu Y., Xue M., et al. Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition. Nucleic Acids Res. 2016;44:2514-2527.
Darabi H., Beesley J., Droit A., et al. Fine scale mapping of the 17q22 breast cancer locus using dense SNPs, genotyped within the Collaborative Oncological GeneEnvironment Study (COGs). Sci Rep. 2016;6:32512.
Fujimoto A., Furuta M., Totoki Y., et al. Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer. Nat Genet.
2016;48:500-509.
Tewhey R., Kotliar D., Park D. S., et al. Direct Identification of Hundreds of Expression-Modulating Variants using a Multiplexed Reporter Assay. Cell. 2016;165:15191529.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

126

DNA Sequencing Methods Collection

3-C, Capture-C and Hi-C: Chromatin Conformation Capture Sequencing
3C-Seq,303 Capture-C, and Hi-C304 comprise a family of methods for analyzing chromatin interactions. Capture-C adds anadditional pull-down of the biotinylated fragments with magnetic beads to the 3C method. A new refinement of the Capture-C
method (NG Capture-C) is available.305 The Hi-C approach extends 3C-Seq to map chromatin contacts genome-wide, and it has
also been applied to studying in situ chromatin interactions.306,307
In this method, DNA-protein complexes are crosslinked with formaldehyde. The sample is fragmented, and the DNA is extracted,
ligated, and digested with restriction enzymes. The resulting DNA fragments are PCR-amplified and sequenced. Deep sequencing
provides base-pair resolution of the ligated fragments.

Crosslink proteins and DNA

Sample fragmentation

Ligation

PCR-amplify ligated junctions

DNA

A schematic overview of 3C-Seq.

Advantages

Disadvantages••• Detection may result from random chromosomal collisions• Less than 1% of DNA fragments actually yield ligation products308• Due to multiple steps, the method requires large amounts ofstarting material

Allows detection of long-range DNA interactions
High-throughput method

Reviews
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;.
Turaev D. and Rattei T. High definition for systems biology of microbial communities: metagenomics gets genome-centric and strain-resolved. Curr Opin Biotechnol.
2016;39:174-181.

References

Criscione S. W., De Cecco M., Siranosian B., et al. Reorganization of chromosome architecture in replicative cellular senescence. Sci Adv. 2016;2:e1500882.
The authors used Hi-C, fluorescence in situ hybridization (FISH), and in silico modeling methods to characterize the 3D architecture of interphase chromosomes inproliferating, quiescent, and senescent cells. Direct measurements of distances between genetic loci, chromosome volumes, and chromatin accessibility suggested thatthe Hi-C interaction changes were caused by a significant reduction of the volumes occupied by individual chromosome arms. In contrast, centromeres opposed thisoverall compaction trend and increased in volume.
Illumina Technology: HiSeq 2500 System

Darrow E. M., Huntley M. H., Dudchenko O., et al. Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture.
Proc Natl Acad Sci U S A. 2016;113:E4504-4512.
During interphase, the inactive X chromosome (Xi) is largely silent transcriptionally and adopts an unusual 3D configuration known as the “Barr body.” The authors
constructed a diploid Hi-C map of human GM12878 cells and showed that the Xi chromosome had a distinctive superstructure. It contained superdomains, which
are unusually large contact domains. It also contained unusually large chromatin loops called “superloops.” Both superdomains and superloops can span dozens of
megabases of the genome. Superloop anchors, like the macrosatellite repeat DXZ4, tended to colocate simultaneously. Deleting DXZ4 on Xi led to the disappearance ofsuperdomains and superloops, changes in compartmentalization patterns, and changes in the distribution of chromatin marks.
Illumina Technology: HiSeq 2000 System

303. Duan Z., Andronescu M., Schutz K., et al. A genome-wide 3C-method for characterizing the three-dimensional architectures of genomes. Methods. 2012;58:277-288.
304. Lieberman-Aiden E., van Berkum N. L., Williams L., et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome.
Science. 2009;326:289-293.
305. Davies J. O., Telenius J. M., McGowan S. J., et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat Methods. 2016;13:74-80.
306. Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;307. Rao S. S., Huntley M. H., Durand N. C., et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159:1665-1680.
308. Bourgo R. J., Singhal H. and Greene G. L. Capture of associated targets on chromatin links long-distance chromatin looping to transcriptional coordination.
Nat Commun. 2016;7:12893.

An overview of recent DNA-seq publications featuring Illumina technology

127

Krijger P. H., Di Stefano B., de Wit E., et al. Cell-of-Origin-Specific 3D Genome Structure Acquired during Somatic Cell Reprogramming. Cell Stem Cell.
2016;18:597-610.
The authors used Hi-C contact maps for each of 4 founder cell types and their respective p3 and p20 iPSC derivatives to investigate how nuclear organization changes
during reprogramming. They found that early passage iPSCs carried topological hallmarks that enabled recognition of their cell of origin. These hallmarks were notremnants of somatic chromosome topologies. Instead, the distinguishing topological features were acquired during reprogramming.
Illumina Technology: HiSeq 2000 System

Veluchamy A., Jegu T., Ariel F., et al. LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome.
PLoS One. 2016;11:e0158936.
Like Heterochromatin Protein 1 (LHP1) controls actively transcribed genes and is a member of the plant-specific polycomb-group (PcG) family originally identified in
Drosophila. The authors used Hi-C to map the spatial contacts and distribution of genes in chromatin between different parts of the Arabidopsis genome for wild-type
and lhp1 plants. Chromosomal contact maps at 100 kb resolution showed significant changes between wt and lhp1 plants. Additional experiments showed that LHP1
was responsible for the spreading of H3K27me3 toward the 3’ end of the gene body. The authors also identified a subset of LHP1-activated genes that shape local
chromatin topology to control transcriptional coregulation.
Illumina Technology: HiSeq 2500 System

Acemel R. D., Tena J. J., Irastorza-Azcarate I., et al. A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hoxbimodal regulation. Nat Genet. 2016;48:336-341.
Bigot P., Colli L. M., Machiela M. J., et al. Functional characterization of the 12p12.1 renal cancer-susceptibility locus implicates BHLHE41. Nat Commun.
2016;7:12098.
Capurso D., Bengtsson H. and Segal M. R. Discovering hotspots in functional genomic data superposed on 3D chromatin configuration reconstructions. Nucleic Acids
Res. 2016;44:2028-2035.
Gunnell A., Webb H. M., Wood C. D., et al. RUNX super-enhancer control through the Notch pathway by Epstein-Barr virus transcription factors regulates B cell
growth. Nucleic Acids Res. 2016;44:4636-4650.
Kim K. D., Tanizawa H., Iwasaki O. and Noma K. Transcription factors mediate condensin recruitment and global chromosomal organization in fission yeast. Nat Genet.
2016;48:1242-1252.
Petryk N., Kahli M., d’Aubenton-Carafa Y., et al. Replication landscape of the human genome. Nat Commun. 2016;7:10208.
Pichugina T., Sugawara T., Kaykov A., et al. A diffusion model for the coordination of DNA replication in Schizosaccharomyces pombe. Sci Rep. 2016;6:18757.
Putnam N. H., O’Connell B. L., Stites J. C., et al. Chromosome-scale shotgun assembly using an in vitro method for long-range linkage. Genome Res. 2016;26:342350.
Rocha P. P., Raviram R., Fu Y., et al. A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch Recombination.
Cell Rep. 2016;16:48-55.
Tjong H., Li W., Kalhor R., et al. Population-based 3D genome structure analysis reveals driving forces in spatial genome organization. Proc Natl Acad Sci U S A.
2016;113:E1663-1672.
Ulianov S. V., Khrameeva E. E., Gavrilov A. A., et al. Active chromatin and transcription play a key role in chromosome partitioning into topologically associating
domains. Genome Res. 2016;26:70-84.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

128

DNA Sequencing Methods CollectionNG Capture-C: Next-Generation Capture-C
NG Capture-C is a refinement of 3C-Seq309 and Hi-C.310 It represents a family of methods used to analyze chromatin interactions.
NG Capture-C adds multiple pull-down steps of the biotinylated fragments with magnetic beads to the 3C method.
The protocol uses formaldehyde fixation followed by restriction enzyme digestion and ligation to form ~10 kb concatamers. The
DNA is extracted and sonicated. Indexing adapters are added, and the samples are pooled, purified by pull-down, and PCRamplified. The pull-down and PCR steps can be repeated to yield up to a million-fold enrichment.311

Formaldehyde fixation

Restriction
enzyme digestion

Ligation De-crosslink andextract DNA

Sonicate to 200
bp fragments

Add indexed
PCR
sequencing adapters

Hybridize biotinylated
capture bait

Streptavidin
pull-downDNA

A schematic overview of NG Capture-C.

Advantages

Disadvantages

•
•
•
•

•

High sensitivity to detect cis and trans interactions
Low sample input requirements
Sonicated capture fragments reduces cost compared to capture-C
Sonicated capture fragments act as UMIs to reduce PCR bias

None reported

Reviews
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;.

References
Davies J. O., Telenius J. M., McGowan S. J., Roberts N. A., Taylor S., et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity.
Nat Methods. 2016;13:74-80.
The authors used NG Capture-C to detect ligation junctions, equivalent to the detection of interactions present in 1 in 10,000 cells, at single-restriction-fragment
resolution (~250 bp). This result exceeds the sensitivity and resolution of detection of current 3C methods and complementary methods, such as FISH.
Illumina Technology: HiSeq System, MiSeq System
Hay D., Hughes J. R., Babbs C., Davies J. O., Graham B. J., et al. Genetic dissection of the alpha-globin super-enhancer in vivo. Nat Genet. 2016;48:895-903.
The authors used homologous recombination to generate 7 mouse models in which each constituent of the proposed α-globin superenhancer was deleted, individuallyand in informative pairs, to dissect its function. NG Capture-C detected the only statistically significant changes, which occurred at the α-globin promoters.
Illumina Technology: NextSeq System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

309. Duan Z., Andronescu M., Schutz K., et al. A genome-wide 3C-method for characterizing the three-dimensional architectures of genomes. Methods. 2012;58:277288.
310. Lieberman-Aiden E., van Berkum N. L., Williams L., et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome.
Science. 2009;326:289-293.
311. Davies J. O., Telenius J. M., McGowan S. J., et al. Multiplexed analysis of chromosome conformation at vastly improved sensitivity. Nat Methods. 2016;13:74-80.

An overview of recent DNA-seq publications featuring Illumina technology

1294C-seq: Circular Chromatin Conformation Capture
4C312, also called 4C-seq, is a method similar to 3C and is sometimes called circular 3C. It allows the unbiased detection of all
genomic regions that interact with a particular region of interest.313
In this method, DNA-protein complexes are crosslinked using formaldehyde. The sample is fragmented, and the DNA is ligatedand digested. The resulting DNA fragments self-circularize, followed by reverse PCR and sequencing. Deep sequencing provides
base-pair resolution of the ligated fragments.

Crosslink proteins and DNA

Sample fragmentation

Ligation

Restriction digest

Self-circularization
and reverse PCRDNA

A schematic overview of 4C.

Advantages

Disadvantages

•

•
•

•

Preferred strategy to assess the DNA contact profile of individual
genomic sites
Highly reproducible data

Will miss local interactions (< 50 kb) from the region of interest
Large circles do not amplify efficiently

Reviews
None available yet.

References

Cai M., Kim S., Wang K., et al. 4C-seq revealed long-range interactions of a functional enhancer at the 8q24 prostate cancer risk locus. Sci Rep.
2016;6:22462.
Genome-wide association studies have identified > 100 independent susceptibility loci for prostate cancer, including a “hot spot” at 8q24. To identify genome-wide
partners interacting with this hot spot, the authors coupled 4C-seq to an enhancer at 8q24 as “bait” in cell lines LNCaP and C4-2B. The 4C-identified regions were
distributed in open nuclear compartments, featuring active histone marks H3K4me1, H3K4me2, and H3K27Ac.
Illumina Technology: HiSeq 2000 System

Loviglio M. N., Leleu M., Mannik K., et al. Chromosomal contacts connect loci associated with autism, BMI and head circumference phenotypes.
Mol Psychiatry. 2016;.
The authors used 4C-seq to identify chromosomal regions that physically associate with the promoters of genes linked to autism-spectrum and other disorders,
including MVP, KCTD13, ALDOA, TBX6 and MAPK3. They found that 2 CNV-prone regions at 16p11.2 were reciprocally engaged in complex chromatin
looping, as successfully confirmed by 4C-seq, FISH, and Hi-C. Their results suggest that disruption of chromatin interplays at 16p11.2 could play a role in theobserved phenotypes.
Illumina Technology: HiSeq System

Yang R., Kerschner J. L., Gosalia N., Neems D., Gorsic L. K., et al. Differential contribution of cis-regulatory elements to higher order chromatin structure andexpression of the CFTR locus. Nucleic Acids Res. 2016;44:3082-3094.
The authors used CRISPR/Cas9 editing of cis-regulatory elements and small interfering RNA (siRNA)-mediated depletion of architectural proteins to determine therelative contribution of structural elements and enhancers to the higher order structure and expression of the cystic fibrosis transmembrane conductance regulator(CFTR) locus. They used CRISPR/Cas9-mediated deletion of a CTCF-binding insulator element 5’ to the CFTR locus and a pivotal intronic enhancer, followed by
4C-seq. The results were consistent with a mechanism coordinating regulatory elements across the locus, which senses structural perturbations to maintain normal
gene expression. However, they found that the loss of a key intronic enhancer on CFTR transcription could not be rescued by structural changes in the locus.
Illumina Technology: HiSeq System

312. Zhao Z., Tavoosidana G., Sjolinder M., et al. Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- andinterchromosomal interactions. Nat Genet. 2006;38:1341-1347.
313. Sajan S. A. and Hawkins R. D. Methods for identifying higher-order chromatin structure. Annu Rev Genomics Hum Genet. 2012;13:59-82.

130

DNA Sequencing Methods Collection

Acemel R. D., Tena J. J., Irastorza-Azcarate I., Marletaz F., Gomez-Marin C., et al. A single three-dimensional chromatin compartment in amphioxus indicates a
stepwise evolution of vertebrate Hox bimodal regulation. Nat Genet. 2016;48:336-341.
De S., Mitra A., Cheng Y., Pfeifer K. and Kassis J. A. Formation of a Polycomb-Domain in the Absence of Strong Polycomb Response Elements. PLoS Genet.
2016;12:e1006200.
Eckart N., Song Q., Yang R., et al. Functional Characterization of Schizophrenia-Associated Variation in CACNA1C. PLoS One. 2016;11:e0157086.
Kaaij L. J., Mokry M., Zhou M., Musheev M., Geeven G., et al. Enhancers reside in a unique epigenetic environment during early zebrafish development. Genome Biol.
2016;17:146.
Kandaswamy R., Sava G. P., Speedy H. E., et al. Genetic Predisposition to Chronic Lymphocytic Leukemia Is Mediated by a BMF Super-Enhancer Polymorphism.
Cell Rep. 2016;16:2061-2067.
Lin C. Y., Erkek S., Tong Y., et al. Active medulloblastoma enhancers reveal subgroup-specific cellular origins. Nature. 2016;530:57-62.
Proudhon C., Snetkova V., Raviram R., Lobry C., Badri S., et al. Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene
Regulation. Cell Rep. 2016;15:2159-2169.
Rocha P. P., Raviram R., Fu Y., Kim J., Luo V. M., et al. A Damage-Independent Role for 53BP1 that Impacts Break Order and Igh Architecture during Class Switch
Recombination. Cell Rep. 2016;16:48-55.
Vermunt M. W., Tan S. C., Castelijns B., et al. Epigenomic annotation of gene regulatory alterations during evolution of the primate brain. Nat Neurosci. 2016;19:494-503.
Wang Q., Sawyer I. A., Sung M. H., et al. Cajal bodies are linked to genome conformation. Nat Commun. 2016;7:10966.
Wani A. H., Boettiger A. N., Schorderet P., et al. Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nat Commun. 2016;7:10291.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology131

UMI-4C: Circular Chromosome Conformation Capture with Unique
Molecular IdentifiersThis variation on the 4C approach uses UMIs to derive high-complexity quantitative chromosome contact profiles with controlled
signal-to-noise ratios.314 It is an efficient and accurate method for analyzing targeted loci. The method is paired with software to
analyze the data (https://bitbucket.org/tanaylab/umi4cpackage).

AdapterDS
USprimer primer

Universal primer

Pad

Crosslink proteinsand DNAHC DpnII
digestion

Ligation

Sonicate

Reverse crosslink andproteinase K digestion

Single-end
adapter ligation

Nested PCR amplificationDNA

A schematic overview of UMI-4C.

Advantages

Disadvantages•••

Improved sensitivity and specificity
Multiplexing allows robust comparison of contact distributions
between loci and conditions

None reported

Reviews
None available yet.

References
Schwartzman O., Mukamel Z., Oded-Elkayam N., Olivares-Chauvet P., Lubling Y., et al. UMI-4C for quantitative and targeted chromosomal contact profiling.
Nat Methods. 2016;13:685-691.
The authors describe the 4C-UMI method and show that it requires modest sequencing depth (100,000 reads per bait). The method can be multiplexed easily, allowing
the selection of multiple viewpoints.
Illumina Technology: MiSeq System, NextSeq System, HiSeq System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

314. Schwartzman O., Mukamel Z., Oded-Elkayam N., et al. UMI-4C for quantitative and targeted chromosomal contact profiling. Nat Methods. 2016;13:685-691.

132

DNA Sequencing Methods Collection

5C: Chromatin Conformation Capture Carbon Copy
5C315 allows concurrent determination of interactions among multiple sequences and is a high-throughput version of 3C.316
In this method, DNA-protein complexes are crosslinked using formaldehyde. The sample is fragmented and the DNA ligated anddigested with restriction enzymes. The resulting DNA fragments are amplified using ligation-mediated PCR and sequenced. Deep
sequencing provides base-pair resolution of the ligated fragments.
T7

Crosslink proteins and DNA

Sample fragmentation

Ligation

T3

LMA: Ligation-mediated amplificationDNA

A schematic overview of 5C.

Advantages

Disadvantages•• Requires a priori information of the regulatory sites318• Detection may not necessarily mean an interaction, resulting fromrandom chromosomal collisions• Cannot scale to genome-wide studies that would require a large
amount of primers

Different from 4C, 5C provides a matrix of interaction frequencies
for many pairs of sites317• Can be used to reconstruct the (average) 3D conformation of larger
genomic regions120

Reviews
Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;.
Reuter J. A., Spacek D. V. and Snyder M. P. High-throughput sequencing technologies. Mol Cell. 2015;58:586-597.

References
Smith E. M., Lajoie B. R., Jain G. and Dekker J. Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal
Elements around the CFTR Locus. Am J Hum Genet. 2016;98:185-201.
Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements.
Topologically associating domains (TADs) represent an intermediate level of organization. The authors designed a 5C experiment to interrogate looping interactions
between HindIII fragments containing TSSs and any other HindIII restriction fragments (distal fragments) in the target region. Their results showed that the same TAD
boundaries were present in all cell types, and they suggest that TADs represent a universal chromosome architecture.
Illumina Technology: Genome AnalyzerIIx System

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Prep Kit
TruSeq DNA PCR-Free Library Prep Kit

315. Dostie J. and Dekker J. Mapping networks of physical interactions between genomic elements using 5C technology. Nat Protoc. 2007;2:988-1002.
316. Sajan S. A. and Hawkins R. D. Methods for identifying higher-order chromatin structure. Annu Rev Genomics Hum Genet. 2012;13:59-82.
317. de Wit E. and de Laat W. A decade of 3C technologies: insights into nuclear organization. Genes Dev. 2012;26:11-24.
318. Sati S. and Cavalli G. Chromosome conformation capture technologies and their impact in understanding genome function. Chromosoma. 2016;

An overview of recent DNA-seq publications featuring Illumina technology133

Pu-seq: Polymerase Usage Sequencing
Pu-seq provides direct replication-origin location and efficiency data, as well as indirect estimates of replication timing.319
Alkali treatment of duplex ribonucleotide-containing DNA results in phosphate-backbone cleavage 3’ to the ribose, resulting in a
5’-hydroxyl end. If the denatured DNA is used as a template for random-hexamer primer extension, 5’ to 3’ synthesis results in aflush end adjacent to the initial ribose. By generating a library from single-stranded DNA and placing distinct index primers at eachend, sequencing reads can be mapped to individual strands, to determine the original ribonucleotide position with single-baseaccuracy.318
5’
3’

R

Index 2

3’
5’

UAlkalitreatment

Klenow reaction(+ random
primer, dATP, dGTP,dCTP, dUTP)

U

Index 1
U

Attachadapters

Uracil DNA glycosylaseand DNA lyase (USER)

PCR andpurifyDNA

A schematic overview of Pu-seq.

Advantages

Disadvantages••

Single-base accuracy

Only applied to yeast

Reviews
None available yet.

References
Keszthelyi A., Daigaku Y., Ptasinska K., Miyabe I. and Carr A. M. Mapping ribonucleotides in genomic DNA and exploring replication dynamics bypolymerase usage sequencing (Pu-seq). Nat Protoc. 2015;10:1786-1801.
The authors used Pu-seq to define replication dynamics in yeast with a high level of detail. Although other methods that examine replication dynamics provide direct
measures of replication timing and indirect estimates of origin efficiency, Pu-seq directly ascertains origin efficiency.
Illumina Technology: NextSeq System

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit

319. Daigaku Y., Keszthelyi A., Muller C. A., et al. A global profile of replicative polymerase usage. Nat Struct Mol Biol. 2015;22:192-198.

134

DNA Sequencing Methods Collection

PB–seq: Protein/DNA Binding Followed by High-Throughput Sequencing
PB–seq is a DNA-binding assay that allows the DNA-protein binding energy landscape to be characterized genome-wide, in
the absence of chromatin.320 It belongs to the family of methods more commonly known as systematic evolution of ligands byexponential enrichment (SELEX). 321
Genomic DNA is sonicated, size-selected, and purified. After hybridization to the DNA-binding protein the protein-bound DNA isisolated, extracted, and prepared for sequencing.

DNA-protein complex

Purify DNA

Shear DNA

Hybridize with
DNA-binding protein

Isolate protein-boundDNA

DNA
extractionDNA

A schematic overview of PB–seq.

Advantages

Disadvantages••

Determines binding efficiencies independent of chromatin structure

Not yet adopted widely by the scientific community

Reviews
None available yet.

References

Guertin M. J., Martins A. L., Siepel A. and Lis J. T. Accurate prediction of inducible transcription factor binding intensities in vivo. PLoS Genet.
2012;8:e1002610.
The authors used PB–seq to show that DNase I hypersensitivity and tetra-acetylation of H4 are the most influential covariates in predicting changes in Drosophila
heat shock factor (HSF) binding affinity. They developed an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE
interaction and a previously unrecognized substructure within the DNA sequence element.
Illumina Technology: Genome Analyzer II System

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit

320. Guertin M. J., Martins A. L., Siepel A. and Lis J. T. Accurate prediction of inducible transcription factor binding intensities in vivo. PLoS Genet. 2012;8:e1002610.
321. Ozer A., Pagano J. M. and Lis J. T. New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer
Characterization. Mol Ther Nucleic Acids. 2014;3:e183.

An overview of recent DNA-seq publications featuring Illumina technology

135

SELEX or SELEX-seq: Systematic Evolution of Ligands by Exponential Enrichment
HT-SELEX: High-Throughput Systematic Evolution of Ligands byExponential Enrichment
From the time that the first SELEX experiments were described by 3 independent groups in 1990322,323,324 the method has been
adapted to a wide range of technologies.325,326 A highly multiplexed, parallel HT-SELEX method was developed for NGS.327
A variation of SELEX-seq328 uses Nextera adapter sequences for efficient library preparation.329
In this method, proteins are expressed as fusions with streptavidin-binding peptide (SBP), conjugated to Gaussia luciferase, in the
pD40htSELEX expression vector. Each DNA ligand contains a 14 bp randomized region (14N), and a 5 bp barcode that uniquely
identifies the individual SELEX sample. Partially nested primers are used in successive SELEX rounds. A double-stranded DNA
mixture containing all possible 14 bp sequences is incubated with a DNA-binding protein immobilized into a well of a 96-wellplate, resulting in binding of DNA to the protein. After washing and elution, the resulting population of more specific sequences is
amplified by PCR and sequenced.326
Forward sequencing adapter
Sequencing primer
Barcode to identify sample
14N with all possible combinations
Reverse sequencing adapter

Targetsequence

LigandpD40htSELEX
DNA binding region
Luciferase
Streptavidin binding peptide

Expression vector

FusionproteinLuciferase

Fusion protein
immobilized in well

Matching ligand
binds

Wash andelute

Recovered
PCR andmatching ligand Sequence

Bindingsite

A schematic overview of HT-SELEX.

Advantages

Disadvantages•••

High-throughput and efficient
Software pipelines are available330,331

Could contain sequence bias332

Reviews
Anzalone A. V., Lin A. J., Zairis S., Rabadan R. and Cornish V. W. Reprogramming eukaryotic translation with ligand-responsive synthetic RNA switches. Nat Methods.
2016;13:453-458.
Jijakli K., Khraiwesh B., Fu W., et al. The in vitro selection world. Methods. 2016;106:3-13.

322. Ellington A. D. and Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990;346:818-822.
323. Tuerk C. and Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990;249:505-510.
324. Sullenger B. A., Gallardo H. F., Ungers G. E. and Gilboa E. Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication.
Cell. 1990;63:601-608.
325. Chen D., Orenstein Y., Golodnitsky R., et al. SELMAP - SELEX affinity landscape MAPping of transcription factor binding sites using integrated microfluidics.
Sci Rep. 2016;6:33351.
326. Takahashi M., Wu X., Ho M., et al. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.
Sci Rep. 2016;6:33697.
327. Jolma A., Kivioja T., Toivonen J., et al. Multiplexed massively parallel SELEX for characterization of human transcription factor binding specificities. Genome Res.
2010;20:861-873.
328. Slattery M., Riley T., Liu P., et al. Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell. 2011;147:1270-1282.
329. Zhang Y., Lee J. K., Toso E. A., et al. DNA-binding sequence specificity of DUX4. Skelet Muscle. 2016;6:8.
330. Hoinka J. and Przytycka T. AptaPLEX - A dedicated, multithreaded demultiplexer for HT-SELEX data. Methods. 2016;106:82-85.
331. Caroli J., Taccioli C., De La Fuente A., Serafini P. and Bicciato S. APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HTSELEX data. Bioinformatics. 2016;32:161-164.
332. Takahashi M., Wu X., Ho M., et al. High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.
Sci Rep. 2016;6:33697.

136

DNA Sequencing Methods CollectionReferences

Urak K. T., Shore S., Rockey W. M., et al. In vitro RNA SELEX for the generation of chemically-optimized therapeutic RNA drugs. Methods. 2016;103:167174.
This publication describes the SELEX process for the rapid identification of RNA aptamers for in vivo applications. It contains a detailed protocol for the selection ofchemically optimized RNA aptamers using the original in vitro SELEX methodology.
Illumina Technology: Various

Ahirwar R., Vellarikkal S. K., Sett A., et al. Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer.
PLoS One. 2016;11:e0153001.
The authors used HT-SELEX to identify a DNA aptamer that selectively binds and facilitates the detection of estrogen receptor alpha (ERα) in human breast cancer
tissue sections. They validated the results of aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients and performed further
validation by immunohistochemistry on the same samples with an ERα antibody. The results demonstrated a significant consistency in the aptamer-assisted detectionof ERα in strong-positive, moderate-positive, and negative breast cancer tissues.
Illumina Technology: HiSeq 2500 System
Iaboni M., Fontanella R., Rienzo A., et al. Targeting Insulin Receptor with a Novel Internalizing Aptamer. Mol Ther Nucleic Acids. 2016;5:e365.
The authors used a cell-based SELEX method to discover a nuclease-resistant RNA aptamer, named GL56, which specifically recognizes the insulin receptor (IR). It
rapidly internalizes into target cells and is able to discriminate IR from the highly homologous insulin-like growth factor receptor 1. When applied to IR-expressing cancer
cells, the aptamer inhibited IR-dependent signaling, suggesting its potential use as a delivery tool for IR-dependent cancers.
Illumina Technology: MiSeq System
Long Y., Qin Z., Duan M., et al. Screening and identification of DNA aptamers toward Schistosoma japonicum eggs via SELEX. Sci Rep. 2016;6:24986.
Janowski R., Heinz G. A., Schlundt A., et al. Roquin recognizes a non-canonical hexaloop structure in the 3’-UTR of Ox40. Nat Commun. 2016;7:11032.
Schneider T., Hung L. H., Schreiner S., et al. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs. Sci Rep. 2016;6:31313.
Stewart H., Bingham R. J., White S. J., et al. Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement ingenome packaging. Sci Rep. 2016;6:22952.
Oakes B. L., Xia D. F., Rowland E. F., et al. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing. Nat Commun.
2016;7:10194.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

An overview of recent DNA-seq publications featuring Illumina technology

137

HiTS-FLIP: High-Throughput Sequencing With Fluorescent Ligand
Interaction Profiling
HiTS-FLIP is a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, theoptics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell.333
A microfluidic flow cell with anchored single-stranded DNA is sequenced by synthesis. Second-strand DNA is stripped and rebuilt
using Klenow polymerase and unmodified dNTPs to form double-stranded DNA clusters. Fluorescently labeled binding protein is
introduced at different concentrations, and binding is imaged.

Klenow

Target sequence

Binding site

Flowcell

Prepare sequencing libraries andsequence first strand
Remove second strand DNA

HybridizeprimerSynthesize second
strand with unmodified nucleotides

Add fluorescent- Hybridize
ly labeled ligand

Elute with increasing stringency

Scan flowcell

A schematic overview of HiTS-FLIP.

Advantages

Disadvantages•••

Quantitative and comprehensive

Requires specialized hardware
Not yet adopted widely by the scientific community

Reviews
None available yet.

References
Nutiu R., Friedman R. C., Luo S., Khrebtukova I., Silva D., et al. Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument.
Nat Biotechnol. 2011;29:659-664.
The authors applied HiTS-FLIP to study the S. cerevisiae protein GCN4, the master regulator of the yeast amino-acid starvation response. The study yielded ~440
million binding measurements, which enabled the determination of dissociation constants for all 12-mer sequences that had a submicromolar affinity for GCN4.
Illumina Technology: Genome Analyzer System

Associated Kits
TruSeq ChIP Library Prep Kit

333. Nutiu R., Friedman R. C., Luo S., et al. Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument. Nat Biotechnol. 2011;29:659-664.

138

DNA Sequencing Methods Collection

DamID: DNA Adenine Methyltransferase Interaction Detection
DamID allows the identification of protein-binding sites in living cells without the need for crosslinking or immunoprecipitation. It
was developed in 2006 as a microarray method before it was adapted to NGS.334
DamID involves the low-level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatinprotein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates
adenines in the surrounding DNA. Subsequently, the methylated DNA fragments are isolated, amplified by selective PCR, andsequenced.333
DNA adenine
methyltransferase (DAM)

Fusion protein

Protein of interest

Create fusion
protein

DAM

Specific and non-targeted
methylation

DpnI

DpnII

PCR

Nontargeted methylation

Split sample

DpnI digestion

Adapter ligation

Unmethylated GATCs
are cut by DpnII

Align sequences and determine
differentially digested sites

A schematic overview of DamID.

Advantages

Disadvantages

•

•
•

•

Allows the identification of protein-binding sites in living cells
without the need for crosslinking or immunoprecipitation
Dedicated algorithms are available335

Dam can be toxic
Limited to kilobase-sized regions

Reviews
None available yet.

References
Pindyurin A. V., Pagie L., Kozhevnikova E. N., van Arensbergen J. and van Steensel B. Inducible DamID systems for genomic mapping of chromatin proteinsin Drosophila. Nucleic Acids Res. 2016;44:5646-5657.
The authors used an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. With thismethod, they generated a glia-specific map of Polycomb binding sites in small samples of brain tissue.
Illumina Technology: HiSeq 2000 System
Mitchell A. C., Javidfar B., Bicks L. K., et al. Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex. Nat Commun. 2016;7:12743.
The authors introduce “Neuro-Dam” for assessing epigenome status retrospectively. They showed that short-term expression of Dam, tethered to the Gad1 genepromoter in mouse prefrontal cortex neurons, resulted in stable GMeATC tags at Gad1-bound chromosomal contacts. The NeuroDam data revealed that micewith defective cognition 4 months after pharmacological blockade of the N-methyl-D-aspartate (NMDA) receptor already were affected by disrupted chromosomal
conformations, shortly after drug exposure.
Illumina Technology: HiSeq 2000 System
Perovanovic J., Dell’Orso S., Gnochi V. F., et al. Laminopathies disrupt epigenomic developmental programs and cell fate. Sci Transl Med. 2016;8:335ra358.
The authors used DamID-seq with mutated forms of the lamin A protein fused to Dam, to define euchromatic-heterochromatin (epigenomic) transitions at thenuclear envelope during myogenesis. They found that lamin A missense mutations disrupted formation of lamin A–associated heterochromatin domains in an allelespecific manner.
Illumina Technology: HiScan System

334. Vogel M. J., Guelen L., de Wit E., et al. Human heterochromatin proteins form large domains containing KRAB-ZNF genes. Genome Res. 2006;16:1493-1504.
335. Li R., Hempel L. U. and Jiang T. A non-parametric peak calling algorithm for DamID-Seq. PLoS One. 2015;10:e0117415.

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139

McCann T. S., Guo Y., McDonald W. H. and Tansey W. P. Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric genesilencing. Proc Natl Acad Sci U S A. 2016;113:1309-1314.
Carl S. H. and Russell S. Common binding by redundant group B Sox proteins is evolutionarily conserved in Drosophila. BMC Genomics. 2015;16:292.
Kind J., Pagie L., de Vries S. S., et al. Genome-wide maps of nuclear lamina interactions in single human cells. Cell. 2015;163:134-147.
Klocko A. D., Rountree M. R., Grisafi P. L., et al. Neurospora importin alpha is required for normal heterochromatic formation and DNA methylation. PLoS Genet.
2015;11:e1005083.
Steglich B., Stralfors A., Khorosjutina O., et al. The Fun30 chromatin remodeler Fft3 controls nuclear organization and chromatin structure of insulators andsubtelomeres in fission yeast. PLoS Genet. 2015;11:e1005101.

Associated Kits
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit

140

DNA Sequencing Methods Collection

MPE-seq: Methidiumpropyl Ethylenediaminetetraacetic Acid (EDTA) Sequencing
MPE-seq is a method for the genome-wide characterization of chromatin that involves the treatment of nuclei with a complex ofmethidiumpropyl-EDTA (MPE) and ferrous iron. The MPE-Fe(II) complex binds to DNA via intercalation of the methidium moiety andthen generates single- and double-stranded DNA breaks in the presence of oxygen.
MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes with little sequence bias, unlike MNase. For example, DNA
sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). The combined use of MPE-seq
and MNase-Seq should allow the identification of noncanonical chromatin structures.

Active chromatin

Isolated nuclei

MPE digestion

Add bathophenanthroline

DNA extractionDNA

A schematic overview of MPE-seq.

Advantages

Disadvantages••

Very little sequence bias

Not replicated in other laboratories

Reviews
None available yet.

References
Ishii H., Kadonaga J. T. and Ren B. MPE-seq, a new method for the genome-wide analysis of chromatin structure. Proc Natl Acad Sci U S A.
2015;112:E3457-3465.
The authors treated mouse ESCs with MPE-Fe(II). For comparison, they also digested nuclei in parallel with MNase. Analysis of the nucleosome positioning analysis with
MPE-seq revealed peaks in active promoter regions that were not seen with MNase-seq. The authors also observed that MPE-seq can be used to detect sequencespecific DNA binding by some transcription factors.
Illumina Technology: HiSeq System

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit
Nextera XT DNA Library Prep Kit

336. Hertzberg R. P. and Dervan P. B. Cleavage of double helical DNA by methidium-propyl-EDTA-iron(II). Journal of the American Chemical Society. 1982;104:313-315.
337. Ishii H., Kadonaga J. T. and Ren B. MPE-seq, a new method for the genome-wide analysis of chromatin structure. Proc Natl Acad Sci U S A. 2015;112:E3457-3465.

An overview of recent DNA-seq publications featuring Illumina technology

141Chem-seq: Identify Sites Bound by Small Chemical Molecules
Chem-seq can be used to detect the binding of small-molecule ligands, such as therapeutic drugs, to proteins associated with the
genome. This information may provide important insights into the perturbation of cellular function by small-molecule drugs.338
The Chem-seq method uses 2 approaches. In living cells, the biotinylated drug is added to allow drug-target binding. The complex
is crosslinked with formaldehyde, the cells are lysed and sonicated, and the complex is captured on streptavidin beads. The
enriched DNA fragments are purified and sequenced. For in vitro analysis, the biotinylated drug is added to a cell extract, and theremaining steps are performed as for the in vivo procedure.
Streptavidin

DNA-protein complex withputative drug binding site

in vivo

Add biotinylated
compoundin vitro

Crosslink

Crosslink, harvest
cells and fragment DNA

Add biotinylated
compoundEnrich DNA
fragments

DNA
extractionDNA

A schematic overview of Chem-seq.

Advantages

Disadvantages••

Can be applied to living, human cells

Creating biotin derivative may alter drug activity

Reviews
None available yet.

References

Anders L., Guenther M. G., Qi J., Fan Z. P., Marineau J. J., et al. Genome-wide localization of small molecules. Nat Biotechnol. 2014;32:92-96.
The authors used Chem-seq to investigate the genome-wide binding of the bromodomain inhibitor JQ1 to the BET bromodomain family members BRD2, BRD3, andBRD4 in MM1.S multiple myeloma cells. They synthesized a biotinylated JQ1 derivative, which had only slightly reduced activity in MM1.S cells. Both in vivo and invitro Chem-seq showed that the genomic sites bound by biotinylated JQ1 are highly similar to the sites occupied by BRD2, BRD3 and BRD4. A functionally inactive
enantiomer of biotinylated JQ1 did not produce significant Chem-seq signals.
Illumina Technology: Genome Analyzer System

Jin C., Yang L., Xie M., et al. Chem-seq permits identification of genomic targets of drugs against androgen receptor regulation selected by functional phenotypic
screens. Proc Natl Acad Sci U S A. 2014;111:9235-9240.

Associated Kits
TruSeq ChIP Library Prep Kit
TruSeq Nano DNA Library Prep Kit
TruSeq DNA Sample Preparation Kit
TruSeq DNA PCR-Free Library Prep Kit
Nextera DNA Library Prep Kit

338. Anders L., Guenther M. G., Qi J., et al. Genome-wide localization of small molecules. Nat Biotechnol. 2014;32:92-96.

142

DNA Sequencing Methods Collection

PROTEIN-PROTEIN INTERACTION
The use of NGS to study protein-protein interactions may seem an unlikely application, but phage display of recombinant proteinsis a well-established method for developing specific affinity reagents for various applications.339 High-throughput sequencing
is poised to change all aspects of the discovery and engineering of antibodies and other protein-binding molecules. Millions ofavailable sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, highquality naïve libraries containing tens of billions of unique molecules. 340

Phage display technology is widely used for high-throughput screening of protein-protein interactions. The gene encoding the protein of interest is inserted into
phages and displayed by the phage. The displayed proteins are screened against target proteins to check for binding.

Reviews
Cariccio V. L., Domina M., Benfatto S., et al. Phage display revisited: Epitope mapping of a monoclonal antibody directed against Neisseria meningitidis adhesin A using
the PROFILER technology. MAbs. 2016;8:741-750.
Jijakli K., Khraiwesh B., Fu W., Luo L., Alzahmi A., et al. The in vitro selection world. Methods. 2016;106:3-13.
Glanville J., D’Angelo S., Khan T. A., Reddy S. T., Naranjo L., et al. Deep sequencing in library selection projects: what insight does it bring? Curr Opin Struct Biol.
2015;33:146-160.

339. Bradbury A. R., Sidhu S., Dubel S. and McCafferty J. Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol. 2011;29:245-254.
340. Glanville J., D’Angelo S., Khan T. A., et al. Deep sequencing in library selection projects: what insight does it bring? Curr Opin Struct Biol. 2015;33:146-160.

An overview of recent DNA-seq publications featuring Illumina technology

143

PD-Seq: Candidate Cellular Protein Target Identification
PD-Seq is a protocol to identify cellular targets for proteins through protein-protein interactions.341
In this method, researchers identified cellular targets for apigenin. They coupled apigenin to amino polyethyleneglycolpolyacrylamide copolymer (PEGA) beads after activation with 4-nitrophenyl bromoacetate. The apigenin-loaded beads were used
for screening phage display cDNA library generated using mRNA from human breast cancer tumor cells. After 3 rounds, thefractions were used to make libraries and sequenced on the Illumina platform.
NH2

Pyridine

NH2

K2CO3

NH2

Hybridize

NH

DMF

Beads

Polyethylene glycol
linker added

NH2
Wash

Immobilized
proteinRNA from cells

Create phage display
cDNA library

Repeat cycle threetimes

Protein-protein
interaction

PCR phage
insertDNA

A schematic overview of PD-Seq.

Advantages

Disadvantages••

Can detect a large number of cellular targets

Not yet adopted widely by the scientific community

Reviews
Arango D., Morohashi K., Yilmaz A., Kuramochi K., Parihar A., et al. Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of
apigenin human targets. Proc Natl Acad Sci U S A. 2013;110:E2153-2162.

References
None available yet.

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit

341. Arango D., Morohashi K., Yilmaz A., et al. Molecular basis for the action of a dietary flavonoid revealed by the comprehensive identification of apigenin human targets.
Proc Natl Acad Sci U S A. 2013;110:E2153-2162.

144

DNA Sequencing Methods Collection

ProP-PD: Proteomic Peptide-Phage Display
PDZ-Seq: PDZ Domains Sequencing
ProP-PD is a protocol that identifies short linear motif (SLiM) interactions or PDZ domains (PDZ-Seq).342,343 ProP-PD can beused for the direct identification of ligands with potential biological relevance.344 This method contrasts with the commonly usedcombinatorial phage display, where highly diverse libraries are used to establish preferred binding motifs.
In this method, C-terminal sequences are first identified and an oligonucleotide library is created. Next, a phage display library isconstructed, which is hybridized to immobilized bait proteins. After multiple rounds of selection, the selected pools that bind to the
PDZ domains are sequenced and counted. Deep sequencing provides detailed information pertaining to the motifs that interact
with specific proteins.
HybridizeIdentify C-terminal
sequences

Create oligo
library

Construct phagedisplay library

Bait proteins immobilized on 96-well plate2
1

Select phages against
baits

Isolate andsequence

PeptidecountsA schematic overview of ProP-PD.

Advantages

Disadvantages••

Direct identification of the ligands

Construction of the oligonucleotide library may be biased

Reviews
None available yet.

References
Garrido-Urbani S., Garg P., Ghossoub R., et al. Proteomic peptide phage display uncovers novel interactions of the PDZ1-2 supramodule of syntenin.
FEBS Lett. 2016;590:3-12.
The authors used ProP-PD to profile syntenin PDZ1-2 by using a library that displays C-terminal regions of the human proteome. The protein recognized a broad rangeof peptides, with a preference for hydrophobic motifs and internal cryptic ligands.
Illumina Technology: Unspecified Illumina system

Ivarsson Y., Arnold R., McLaughlin M., Nim S., Joshi R., et al. Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using humanand viral phage peptidomes. Proc Natl Acad Sci U S A. 2014;111:2542-2547.

Associated Kits
TruSeq DNA PCR-Free Library Prep Kit
TruSeq Nano DNA Library Prep Kit

342. Ivarsson Y., Arnold R., McLaughlin M., et al. Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage
peptidomes. Proc Natl Acad Sci U S A. 2014;111:2542-2547.
343. Ernst A., Gfeller D., Kan Z., et al. Coevolution of PDZ domain-ligand interactions analyzed by high-throughput phage display and deep sequencing. Mol Biosyst.
2010;6:1782-1790.
344. Sundell G. N. and Ivarsson Y. Interaction analysis through proteomic phage display. Biomed Res Int. 2014;2014:176172.

An overview of recent DNA-seq publications featuring Illumina technology

145

Scientific Publication Reviews can be accessed atwww.illumina.com/pubreviews

RNA SEQUENCINGMETHODS COLLECTION
An overview of recent RNA-Seq publications featuring Illumina® technology

RNA Sequencing Methods Collection
This publication is a collection of next-generation sequencing(NGS) methods for RNA sequencing, compiled from the scientific
literature. It is both a tribute to the creativity of the users and theversatility of the technology. We hope it will inspire researchers
to use these methods or to develop new ones to address newscientific challenges.
A method refers to the processing steps between extracting
the nucleic acids (sample preparation) and the addition ofoligonucleotide adapters for sequencing (library preparation).
With a few extra processing steps, a wide range of scientificquestions can be addressed by this technology.

Single-Cell Research
An Overview of Recent Single-Cell Research Publications Featuring Illumina® Technology

Single-Cell Research Review
Most of the impetus for single-cell tissue sequencing has comefrom cancer research, where cell lineage and the detectionof residual disease are of paramount concern. The same
approaches are being used to improve our understandingof massively complex biological systems, such as neuraldevelopment and immunology.
This document highlights recent publications that demonstrate
the use of Illumina technology for single-cell sequencing and very
low input applications and techniques.

Gene Editing Research Review
An Overview of Recent Gene Editing Research Publications Featuring Illumina® Technology

Gene Editing Research Review
CRISPR-Cas9 is a recently developed genome editing techniquethat allows scientists to perform precise genomic manipulation
quickly and conveniently. This technology has a vast spectrum
of applications. As any molecular biology technique, it is crucial
that the obtained results have high levels of specificity. This
review highlights recent publications that demonstrate the useof genomic technologies and high-throughput sequencing in
CRISPR-Cas9 experiments for checking specificity and genomewide off target effects.

This Scientific Publication Review is brought to you by Illumina, Inc.
Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
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