Illumina Sequencing Methods

Field Guide to Methylation Methods
GlossaryMethylation and Cellular Processes
Cytosine methylation (5-mC) has a significant effect on gene expression and chromatin remodeling.
Cytosine methylation and hydroxymethylation (5-hmC) regulate spatial and temporal gene
expression, and are critical for embryonic development and cellular differentiation processes.
linked to chromosomal instability and loss ofimprinting. Controlled changes in chromatin
structure are responsible for selective
X chromosome inactivation and suppression oftransposable elements.
• Cellular differentiation.
Methylation change usually drives one-way
differentiation; that is, differentiated cells do not
typically revert to stem cells.
• Onset of diseases.
Perturbation of any of the above processes
may result in disease.

• Modulation of gene expression.
Cytosine methylation in promoter regions
often reduces gene expression. 88% of active
promoters are associated with unmethylated
CpGs in mammals. Hydroxymethylation often
correlates with increased gene expression• Change in chromatin structure.
Heterochromatin formation is initiated byhypermethylation. Methyl binding domain(MBD) proteins specific for 5-mC recruit
histone-modifying and chromatin-remodelingproteins. Abnormal hypomethylation ofheterochromatin (normally hypermethylated) is

5-Formylcytosine Oxidation product of 5-hmC.
Detected in mouse embryonic stem cells.
Base modification Addition of methyl- or othergroups to nucleic acid bases. In addition to
5-methylcytosine, other common modifications
are 7-methylguanosine (5’-cap for RNA), and6-methyladenosine (common RNA modification.)
CpG island Defined as regions > 500 bp,> 55% GC and expected/observed CpG ratio of >0.65. 40% of gene promoters contain islands.
CpG shelves ~4Kb from islands.

Perturbation of Methylation
Heterochromatin

EuchromatinHypermethylated

Hypomethylated
Gene body
Exon
Shore

Exon

PromoterIf hypermethylated

Genomic instability

Transcriptional repression

Expected/observed CpG ratio The humangenome contains 25% of the expected number of
C-G pairs due to spontaneous deamination of meC
to T over evolutionary time scales.
Genomic imprinting An epigenetic process
causing genes to be expressed only from one of theparental chromosomes.

DiseaseFragile X, Rett syndrome, autism schizophrenia, dementia,
suicide risk, Alzheimers, Prader-Willi, Angelman,
Beckwith-Wiedemann Syndromes

Differentially methylated regions (DMR)
Cell-, tissue-, and condition- specific differences inmethylation.
Enhancer A short region of DNA that can activatetranscription and is often regulated by methylation.

CpG Island(>500 bp >55% GC)

If hypomethylated

CpG shores ~2Kb from islands, > 75% of tissuespecific differentially methylated regions foundin shores. Methylation in shores shows higher
correlation with gene expression than CpG islands.

= methylated cytosine

Methylation Enzymes
Mammalian
DNMT1 (DNA methyltransferase) isoforms methylate hemimethylated CpGs, maintaining methylation
patterns during DNA replication.
DNMT3a and DNMT3b, de novo methyltransferases that set up DNA methylation patterns early indevelopment; can methylate unmethylated and methylated DNA.
DNMT3L unknown; facilitates de novo methyltransferase activity.
TET (Ten eleven translocation) enzymes involved in methyl group oxidation with the production of
5-hydroxymethylation as an intermediate.

Hypermethylation Most cytosines are methylated
Hypomethylation Most cytosines do not have
5-mC. Euchromatin and active gene promoters arehypomethylated.

Cytosine Modifications
TetPlant

DNMT

CytosineBacteria

Decarboxylase?

-O-OH

For Research Use Only. Not for use in diagnostic procedures.

Tet-HDam (DNA adenine methyltransferase), methylates ‘A’ in GATC, key role in mismatch repair, DNAreplication timing, and gene regulation. Independent of restriction modification systems.
Dcm (DNA cytosine methylase) produces 5-mC in CCAG and CCTGG sites.
EcoKI methylates adenine in AAC(N6)GTGC and GCAC(N6)GTT.

5-hmC

-O

DRM2 (RNA directed DNA methylation) homologous to DNMT3.
MET1 (methyltransferase 1) homologous to DNMT1.
CMT3 unique to plants. Function for other methyl transferases unknown.

5-mC-OHShelf

5-Methylcytosine (5mC) Formed by addition of-CH3 to N5 position of cytosine. 70–80% of CpG
dinucleotides are methylated (high proportion inrepetitive elements). In plants, cytosine methylation
is observed at CpG, CpHpG, and CpHpH (H =nucleotide other than guanine).

5-caC

Tet5-fC

Illumina Sequencing Methods
Techniques in Brief

Primary Methods For Detecting Cytosine Modifications
MethodWGBS-Seq
Whole-Genome
Bisulfite Sequencing withTruSeq® DNA Methylation

Methylation Array
Infinium® MethylationEPIC

RRBS-Seq
ReducedRepresentation Bisulfite
Sequencing

H3C

NH2N

MeDIP

Methylated DNAN
O Immunoprecipitation
DNA5-mC

NH2NHONDNA

Tab-Seq
Tet-Assisted
O Bisulfite
Sequencing

5-hmC

Tab/OxBS ArrayhmC analysis on amethylation array

DNA Preparation

Considerations

Convert nonmethylated Cs to U
with sodium bisulfite, which areread as T. 5-mC and 5-hmC areprotected from conversion andread as C.
Sequence with random-primer
extension, 2 × 75–100 bp reads.

Compatible with mostspecies, covers all genes
and > 38 M CpGs inhumans
Requires > 90 Gb
sequencing
Cost: US $5000/sample*

Bisulfite convert DNA to findC-to-T changes at defined
genomic positions by theInfinium assay

Samples 95% of CpG
islands, > 850K CpGs,
99% RefSeq promoters,
coverage across the gene
Cost: US $330/sample*

Digest DNA with MspI andisolate 100–150 bp fragments,
representing> 85% CpG islands. Bisulfite
treat fragments and sequence
using 1 × 75 bp reads.

Samples 50% of RefSeq
promoters with a focus on
CpG-rich regions for ~ 85%of CpG islands
Requires 3-5 Gb/sample
Cost: US $300–350/sample*

Sonicate DNA to 100–300 bp,
end-repair, ligate to adapters,
and denature. Capture 5-mC
containing fragments with
anti-5-mC magnetic beads, andsequence using 1 × 75–100 bp
reads. May be combined withbisulfite conversion.

5-mC detected at ~150 bp
resolution. Bias towards
hypermethylated regions.
Requires ~60 M reads,~5 Gb/sample
Cost: US $300–360/sample*β-glucosyltransferase adds
glucose to 5-hmC but not
5-mC. Tet enzymes oxidize
5-mC to 5-caC, and converts
to U on bisulfite treatment but
glucosylated 5-hmC cannot beoxidized. hmC will be read as C
and mC will be read as T.

Detects genome-wide
5-hmC at single-baseresolution
Requires > 90 Gb sample
Cost: US $5,000–6,000/sample*

TAB or OxBS converted DNA
is hybridized to the Infinium
Methylation array for targeted
analysis of > 850K genomic
locations.

Detects 5-hmC at singlebase resolution at 95% ofCpG islands and 99% ofRefSeq promoters
Cost: US $430/sample*

COBRA Combined Bisulfite followed by Restriction
Analysis. RE recognition affected bybisulfite treatment.
C-Subtraction MseI (methylation independent)
digestion, ligation to linkers, then digestion withmethylation sensitive BstUI or HpaII to reduce
unmethylated DNA.
HELP HpaII enrichment by ligation PCR. Differential
restriction of methylated and unmethylated
CpG sites.
MCA Methylated CpG island amplification. DNA cutwith SmaI (blunt ends, cannot cut meC) followedwith XmaI (sticky ends, cuts meC and C). Sticky end
adapters select for meC fragments.
MeDIP Methylated DNA immunoprecipitation with
anti-5-mC methylcytosine antibody
Methyl Light Methylation specific qPCR (see MSP).
Methylation Trapping of methyltransferases onto
DNA with 5-Aza- 2'-deoxycytidine (decitabine) followed by immunoprecipitation.
MIRA Methylated CpG island recovery assay. Capture with MBD2b and MBD3L1 protein heterodimer.
MSDK Methylation-specific digital karyotyping.
Cleavage with methylation sensitive AscI. Sequencetags sequenced and mapped like SAGE.
MSP Methylation-specific PCR on bisulfite converted
DNA with 2 sets of primers to amplify
either -C or -T base.
OxBS K-perruthenate oxidizes only 5-hmC to 5fC
that is converted to U by bisulfite
RLGS Restriction Landmark Genome Scanningwith 2D gel of methylation-sensitive NotI and AscI
restricted DNA*Cost is provided for guidance only, may not reflect your actual cost.

RRHP Reduced Representation
Hydroxymethylation Profiling.
RRBS Reduced Representation
Bisulfite Sequencing.
1.
2.
3.
4.
5.
6.
7.

Guibert S and Weber M. Functions of DNA methylation and hydroxymethylation in mammalian development.
Curr Top Dev Biol. 2013;104:47–83.
Stevens M, Cheng JB, Li D, et al. Estimating absolute methylation levels at single CpG resolution from methylationenrichment and restriction enzyme sequencing methods. Genome Res. 2013;23(9):1541-1553.
Xie W, Schultz MD, Lister R, et al. Epigenomic analysis of multileaneage differentiation of human embryonic stem cells.
Cell. 2013;153(5):1134–1148.
Yu P, Xiao S, Xin X, et al. Spatiotemporal clustering of the epigenome reveals rules of dynamic gene regulation.
Genome Res. 2013;23(2):352–364.
Yu M, Hon GC, Szulwach KE, et al. Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine.
Nat Protoc. 2012;7(12):2159–2170.
Stewart SK, Morris TJ, Guilhamon P, et al. oxBS-450K: A method for analysing hydroxymethylation using 450K BeadChips.
Methods. 2015;72:9-15.
Nazor KL, Boland MJ, Bibikova M, et al. Application of alow cost array-based technique - TAB-Array - for quantifying andmapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells. Genomics. 2014;104(5):358-367.

Illumina • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
For Research Use Only. Not for use in diagnostic procedures.
© 2016 Illumina, Inc. All rights reserved. Illumina, TruSeq, Infinium, and the pumpkin orange color are trademarks of Illumina, Inc. and/or itsaffiliate(s) in the U.S. and/or other countries. Pub. No. 270-2013-001 Current as of 10 June 2016

Tab-Seq Tet-assisted bisulfite sequencingof 5-hmC.
WGBS-Seq Sodium bisulfite conversion of C to T.
mC and hmC are not converted and are read as C
by whole-genome, next-generation sequencing.