DNAFor all you seq...
DNA Rearrangements and Markers
RAD
PE RAD-Seq

GenomeBisulfite conversion
NH2

Restriction sites

Restriction
digestion

GenomeddRADseq

Add barcoded adapters

Add P2
adapter

Shear andsize selectAmplifyDNA

Single cell RNA

NH2N

Restriction-site associated DNA marker
generation (RAD)DNA-Protein Interactions
CH3

N
N

O

scM&T-seq

Double digest restriction-site associatedDNA marker generation (ddRADseq)

Restriction
digestion

Second restriction digest

Add barcoded adapters

Add P2
adapter

AmplifyDNA

GenomeBsaXI restriction sites

Restriction-site associated DNA marker
generation (RAD) with type IIB restriction
endonucleases (2b-RAD)
GenomeAmplify

Add restrictionsite-specific adapters

Add sample-specificadapters

AmplifyNO

Add MseI
adaptors

Digest with AluI

5-Methyl Cytosine

DNA

Single cell

Add sequencing
adaptors

Purify andpool

PCR with barcoded
MseI primer

PCR

Biotinylated
probes

Shotgun library

Well 1
Well 2
Add well-specificbarcodesDigest withrestriction
enzymeHybridize to probes

Streptavidin
pull down

Pool wells
and shear

Library prep with Pool Hybridize Biotinylated
capture bait
plate barcodes

Restriction
enzyme digest

Ribonucleotides
R
R

Streptavidin
pull downN

Sequence

Alkalinehydrolysis (KOH)

R

Ligate oligo with 5’-aminoterminated C6 spacer

Phosphorylation (T4 PNK
3-phosphatase minus)

P 5’

P 5’

A

mA

dA tailingG4-seqAm

mA

Adaptor ligation

A

A

T

Am
T

A
A

mA

Alkali treatment

Am

R Am
T

mA

Self-ligation by
AtRNL

A

Hibridizesequencing
primerRead reference
sequence

CXXC

CXXC

X

CXXC

R

T

N
R

CH3

Polymerase stalls

Comparesequence reads
1 and 2

Read stabilized
squenceO

HO

Elute unmethylated CpG
enriched fragments

Indexed transposome reactions

Dilute and divide
into 96 reactions

EDTA
Pool

Retrotransposon
binding sites

R

Fractionate

Hybridize Microarray with transposon
binding sites

DNAfragments

Transposon
Inverted MmeI
recognition site

Transposon sequencing (TN-Seq)
and insertion sequencing (INSeq)

NH2

N
O

Add adapters

R

Isolate Nuclei

SplitsampleCreate sequencing
library

Sequence capture

N3

Acylation

Enriched libraryDNA

X-ChIP

Split sample

Pool anddiluteBarcode each wellwith Tn5 transposase

Pool for libraryprep

PCR-barcode
every wellDNAO

ORGANICDNA

Isolatesingle cell

Lyse and introduce Insert in regions of open chromatin
Tn5 transposase

Amplify withcell-specificbarcodesFragmented and primed

Pool libraries
from all cells

N

DNA

N

N

N

TC-SeqI-SceI site

TC-Seq: translocation
capture sequencing

Ig-seqRep-Seq
MAF

Genomic DNA

CDR3 junction region

+AID

A

Fuse droplets

Pool all droplets

DNA templates

Template amplification

Single template
encapsulation

Sequence

Chromatin immunoprecipitation

Template fragmentation

Barcoded sequencesfrom single cells

Barcode every droplet

C

T

-AID

A
Sonicate, bluntand A-tailInfect I-Sel

V D J

A
Ligate linkers
Cut I-Scel

Constant

Semi-nested PCR
Linker cleavage

Purification

8N UID

Reverse transcription

Extracted RNA

DNA sequencing of immunoglobulin genes(Ig-seq), repertoire sequencing (Rep-Seq), andmolecular amplification fingerprinting (MAF)

Second strand synthesis

PCR

Purify

DNA

H2N

O
NH

O

H2N

O

N

NH

Pool for library
prep

Shear DNA

T

C

G

U

T

C

G

Bisulfite conversion
C

T

C

G

T

C

G

T

C

G

T

T

T

C

DNA

G

TTTT

TT

TTTT

TT

TT

TT

Ligate primer

DNA isolation andrandom shearing

Pyridostatin (PDS)DistalprimerCapture on streptavidin
beads

Methyl-Seq
MRE-Seq

TT

TT

TTTT

Biotinylated
proximal primer

Genome DNA

TT

Digest with
I-SceI and PCR

DNA

T

U
PCR

Bisulfite conversion

T

T

Converted single-strandedfragments

PCR

3’ tagging

Random priming
DNA synthesis

DNA

FAIRE-seq
Sono-Seq

Random primer 2
AdaptorBisulfite
conversion

First randompriming

Capture first strand on Streptavidin coated magnetic beads

Second randompriming

ElutionGenerate second strand

DNA with adaptors

Genome DNA

Map DNA double-strand
breaks (DSB-Seq)

dsOligo insert

Oligo insert

GUIDE-seq

DNA isolation and random shearing

TdT and biotinylated-dUTP

MethylatedDNA

Bisufite-converted
DNA

UMI

Extensionand ligation

Hybridize

Methylated
adapter

Bisufite-converted DNA

Bisulfite
conversion

Methylated sites
in the genome

Split sample

Genome DNA

Genome-wide, unbiased identification of
DSBs enabled by sequencing (GUIDE-seq)

dsOligo tag integrated in live cells

DNA isolation
Random shearing

Exonuclease
digestion

Bisulfite
conversion

End repair
and ligation

End repair
and adaptor
ligation

PCR

DNA with
adaptors

PCR

dsOligo-specific amplification

PCR

Converted fragments

DNA
HO
HO

OH

Amplicons

PCR with primers specific forbisulfite converted DNA

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

Transposome withadaptor

P

P

O

N
O

O

O

OH

OH

DNA

Tagmentation

ChIPmentation

NH
O-

O-

J2

J1

V1

V2

V3

J5

J4

J3

J2

J1

Excision circle sequencing(EC-seq)

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

J3

V2

V1

V1

J2

V2 J3

J2

J1

V1

J1

V3

J3

Synapsis, cleavage and coding
end hairpin formation

J4

J4

V2

V3

J5

J4

J3

J2

J1

J5

J5

V3

V3

J4

V2

J5

V1

Resolved genomic Liberated excision circle withcoding junction
resolved signal junction

Align read pairs toreference genome

HO

HO

O
HO

G
G G

Align sequences anddetermine undigested sites

Sequence

Bubble-Seq

CH3

Libraries of restriction fragmentsthat contain replication initiationsites (bubbles) in vivo

Bubble-containing
fragment

Restriction
digestCast fragments intrapping gelO

Recover bubble-containing plug

Run gelDNA extraction

Add sequencing
primersDNA

HN

BiotinNSCR

RNA
primerNascent strand capture andrelease (NSCR)

Repli Seq—to map temporally orderedreplicating DNA

Run-on with analog

Streptavidin pull-down

RNase I
digestion

Bead coated with
anti-BrdU antibody

Sort cells and lyse

O
O

O

DNA

Isolate andamplify SNS

O-

N

O

Elute
End Repair

TAmC-Seq

Digoxigenin

DNA replication origin

Digestion

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

Degenerate
molecular tag

Single Molecule Molecular
Inversion Probes (smMIPs) for
detecting low frequency targets

Copy target sequence

MDA
IMS-MDA
MIDAS

Copy target STR

PCR amplification

Multiple annealing and looping-based
amplification cycles (MALBAC)

Hybridize primers

N

Phi 29Synthesis

Phi 29Synthesis

S1 nuclease

O

SynthesisN

Bst DNA
polymerase

Denature

Cycles of quasilinear
amplification

Partial amplicons

N

Looped full
amplicons

PCR

Lyse cell

Cell sorting from
G2/M distribution

Nucleus

5fC-AI

Limited amplification

CC

Synthesis

S1 nucleaseTarget sequence

C

C

T

C

C

C

C

C

C

T

C

C G

DNA

PCR

C

C

C

C

C

C

C

C

Extend and
Denature

Extend and
Denature

HybridizeExtend and
Denature

Hybridize

C

C

C

C

C

Nu

C

OH

O

DNA

Randomly shearedends serve as UMIs

Shear

Very rare mutation

Duplex sequencing detects rare A mutation occurs
on both strands
mutations by sequencing andaligning both strands of the DNA

RNA

AA(A)nP5αP7

12 randombase index

Adaptor ligation

P7

β

Add
adaptors

Amplify and solid
phase capture

12 randombase index

C

Random error

C

C

C

C

Sequence

DNADNA

RT with barcoded primer

Genome DNA and mRNA Single Lyse cell
sequencing (DR-Seq)
cell

Ad-2
primerQuasilinear
amplification

Splitsamples

5fC-AI-SH

S-S

C
C mC
5m 5h 5fc 5ca
C

C

C

C

C

C

C

C

C

C

AA(A)nAA(A)n

O

C

C

C

C

C

C

C

C

C

C

C

C

C

C

C

T

C

C

C

AAAAAAA
TTTTTTTTTT

DNA

DNA
Genome and transcriptome
sequencing from a single
cell (G&T-seq)Cellsuspension

Lyse cell

Isolate single
cell

Streptavidin magnetic beadwith mRNA capture primer

Separate the DNA and the RNA

O

C

C

C

C

C

C

O-

O
O-

M

TET-assisted bisulfite sequencing, (TAB-Seq)
to map 5-hydroxymethylcytosine

Label with ethynyl
deoxyuridine (EdU)

Isolatenucei

Cycloaddition
reaction

PurifyDNA purification
AmplificationDNA

Add barcodes andsequencing adaptors

dsDNA synthesis

End fill-in and IVT
amplification

Remove of H2A–H2B dimers
and non-histone proteins

MNase digestionDNA
extraction

DNA

DNA
extraction

DNA

Streptavidin

Crosslink with formaldehyde

Chase with
Thymidine

Formaldehyde-assisted isolation of regulatory elements (FAIRE-Seq) and sonication ofcross-linked chromatin (Sono-Seq)

Click reaction
to attach biotin

Crosslink protein and DNA with formalin

Open DNAStreptavidin
capturePhenol extract and purify DNA
from the aquous phase
Protected

CpG dinucleotides
Methylated CpG

GpC methyltransferase (M.CviPI) and
S-Adenosyl methionine (SAM)

Open DNA

Sonication and
MNase digestion

Sonicate

CpG dinucleotides
Methylated CpG

Control
M.CviPI

Bisulfite conversion BS-Seq

Crosslink proteinsand DNA

DNA-protein complex

Chromatin immunoprecipitation
with sequencing library preparation
by Tn5 transposase (ChIPmentation)

Chromatin

Samplefragmentation

Exonuclease digestion

Tn5 Transposome

Chromatin immunoprecipitation

Sample fragmentation

Chromatin conformation capture(3-C, Hi-C and Capture-C)

Crosslink proteins and DNA

Formaldehyde fixation

DNA

Protected

Protected
methylated

Unprotected
methylated

Protected
unmethylated

Unprotected
unmethylated

Immunoprecipitate

DNA extraction

DNA

Fragmented
and primed

DNA purification
Amplification

DNA

Adaptor insertion

Immunoprecipitate

Ligation

Sample fragmentation

Restriction
enzyme digestion

Ligation De-crosslink andextract DNA

Crosslink proteins and DNA

CCRestriction enzyme digestion

DNA

Ligation

PCR amplify ligated junctions

DNA

Sonicate to 200
bp fragments

Add indexed
PCR
sequencing adaptors

Hybridize biotinylated
capture bait

Streptavidin
pull downDNA

Sample fragmentation

Self-circularization
and Reverse PCR

DNA

Ligation

Restriction digest

C G C G C G CC G C G

C

T

C

C

T

C

DNA

Pu-seqOrigin of replication

O

C

T

T

Bisulfite treatment
PCR amplification

T

C

C

T

T

Bisulfite treatment
PCR amplification

T

C

C

C

T

Bisulfite treatment
PCR amplification

T

C

C

T

T

Bisulfite treatment
PCR amplification

T

Bisulfite treatment
PCR amplification

T

Bisulfite treatment
PCR amplificationC

C

Control
M.SssI

C

C

C

C

C
C

C

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

TET
C

C

C

C

C

C

C

C

C

Bisulfite treatment
PCR amplification

C

C

T

T

C

LAM-HTGTS

Biotin

DNA

C

OO

DNA

C

T

C

DNA

DNA

Nested PCR amplification

T3

DNA

O

OH

NH2

N

Hybridize with
DNA-binding protein

U

5’
3’

Attachadaptors

Index 2Elution andfragmentation

LAM-PCR withbiotinilated primer

SELEX
SELEX-seq
HT-SELEX
HiTS-FLIP

Protein binding site

Targetsequence

PCR andpurifyDNA

NestedPCR

Enzymeblocking

Tagged PCR

PurifyDNA

PurifyDNA

A

3’ A addition

Adaptor ligation
and PCR

pD40htSELEX

FusionproteinDNA binding region
Luciferase
Streptavidin binding peptide

LigandIndex 1A

Forward sequencing adaptor
Sequencing primer
Barcode to identify sample
14N with all possible combinations
Reverse sequencing adaptor

High-throughput systematic
evolution of ligands by exponential
enrichment (HT-SELEX)

DNA

A
A

End repair

PCR andpurifyNNNN

Adaptor ligation

Streptavidin magnetic
bead pull downA

Fragmentation bysonication

DNA

Attachadaptors

Streptavidin magnetic
bead pull down

NNNN

Fragmentation bysonication

Uracil DNA glycosylaseand DNA lyase (USER)

3’
5’

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

DNA trapped inagarose gelDNAextraction

U

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

3’5’

Isolate protein-boundDNA

Index 1

U

Breakage in genome

Transcription factor binding site

OR

N3-5GMC

DNA

T

Break

5’
3’

Shear DNAStreptavidin magnetic
bead pull down

Tagged PCR

Luciferase

Fusion protein
immobilized in well

Expression vector

NestedPCRMatching ligand
binds

Wash and
eluteBindingsite

Recovered
PCR andmatching ligand Sequence

O

HO
HO

T

T

Universal primer

LMA: Ligation-mediated amplification

Index 2

3’
5’

R

A

N3

T

Breakage at replication fork

Linear amplification-mediatedhigh-throughput genome-wide
sequencing (HTGTS)

Klenow

Target sequence

T

Prepare sequencing libraries andsequence first strand
Remove second strand DNA

DNA-protein interaction

DNA adenine
methyltransferase (DAM)

Hybridizeprimer

Add fluorescent- Hybridize
ly labeled ligand

DpnI

Elute with increasing stringency

DpnII

Scan flowcell

PCR

Non-targeted methylation

DAM

Create fusion
protein

Synthesize second
strand with unmodified nucleotides

Specific and non-targeted
methylation

Fusion protein

Protein of interest

DNA adenine methyltransferaseinteraction detection (DamID)

Binding site

FlowcellHigh-throughput sequencing:fluorescent ligand interaction
profiling (HiTS-FLIP)

DamID

Split sample

DpnI digestion

Unmethylated GATCs
are cut by DpnII

Adaptor ligation

Align sequences and determine
differentially digested sites

C G C
C G C G

MBD2B/MBD3L1
protein complex

Isolate on glutathione-coatedbeads

DNA purification
PCR amplification

MPE-seqDNA

Methidiumpropyl-EDTA sequencing(MPE-seq)

Active chromatin

Isolated nuclei

MPE digestion

Add bathophenanthroline

DNA extraction

DNA

Streptavidin

Methylated DNA Immunoprecipitation(MeDIP-Seq), DNA immunoprecipitation followedby high throughput sequencing (DIP-seq))

Methylated DNA

Extract DNA

Fractionate
Denature

Immunoprecipitate

DNA purification

DNA

Chem-seq

Pyridine

Hydroxymethylated
DNA

Extract DNA

Fractionate
Denature

MBD

Methylated
DNA

Extract DNA

Immunoprecipitate

N

T

BisChIP-Seq
ChIP-BS-seq

Yellow highlights indicate the target of the protocol

C

T

C

G

U

T

C

G

T

T

C

G

Bisulfite

T

PCR

T

Bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq) and
ChIP-BS-seq), to correlate protein
modifications with DNA methylation

DNA-protein complex withmethylated histones andmethylated DNA

Sonicate

Immunoprecipitate

Protein target

NH2

DNA
purification

Purify DNA
and shearN

DNA

Bisulfite conversion

Add biotinylated
compoundin vitro

Crosslink

Harvest cells andfragment DNA
Enrich DNA
fragments

Add biotinylated
compoundN

ProP-PDPDZ-Seq

N

K2CO3

NH2

NH2

HybridizeNHDNA
extraction

DNA

NH2
Wash

Polyethylene glycol
linker added

Beads

Immobilized
proteinRNA from cells

Create phage display
cDNA library

Repeat cycle threetimes

Protein-protein
interaction

PCR phage
insertDNA

HybridizeProtein target

Proteomic peptide-phagedisplay (ProP-PD) to identify
short linear motif (SLiM) interactions or PDZ domains (PDZ-Seq)

CH3

O

Pyridine

DMF

PD-Seq identifies candidate
cellular targets for proteins

BiotinStreptavidin

Elute with increasing
salt concentrationin vivo

Protein-Protein InteractionsDNA

PD-SeqCapture biotinylated MBD on
Streptavidin coated magnetic beads

Fractionate

DNA purification

H3C

EDTA

Single end
adaptor ligation

Ligation

Purify DNA

DNA-protein complex

Breakage in genome

HTGTS

DNA

T

C

Sample fragmentation

Alkalitreatment

Linear amplification-mediatedhigh-throughput genome-wide
sequencing (LAM-HTGTS)DNAC G C G

Fractionate

Sonicate

Reverse crosslink andproteinase K digestion

OH
S

H

T

T

O

NH H

HN

T

T

C

T

Bisulfite treatment
PCR amplification

CC

Bisulfite treatment
PCR amplification

C

5m

C

C

C

Ligation

HC DpnII
digestion

Crosslink proteins and DNA

5’
3’

Double-stranded breaklabeling to map chromosome
breaks (Break-seq)

N

C
C mC
5m 5h 5fc 5ca
C

Break-seq
SequenceT

C G C

Methylated-CpG island
recovery assay (MIRA)

T

DS
US
primer primer

PB-seqOxidation

C G C

5m

T

C

C

C

C

T

C

C

C

C

C

C

C

Crosslink proteinsand DNA

Protein/DNA binding (PB–seq), to
determine the binding energy landscapeC

Bisulfite treatment
PCR amplification

C

C

C

Glucosylation

C

C

5m

5mc CpG island

C

C

C

O

Key

Fragmented and primed

T7

C

T

T

C

C

C

C

C

T

C

C

C

mC
5g

C

C

C

5m

βGT

C

C

Add methylated
adapters

CC mC
5m 5h 5fc 5ca

C

T

C

C

C

C

C

Control

Digestwith MspI

MBDCap-seq
MethylCap-seq
Methyl-CpG binding domain-based capture and
MBD-Seq
sequencing (MBDCap-seq). Capture of methylated
DNA using the MBD domain of MeCP2 (MethylMiGS
Cap-Seq). MBD-isolated Genome Sequencing (MiGS)

gDNA amplification

Align RNA andgenomeDNA

Polymerase

Polymerase usage sequencing(Pu-seq)

Bisulfite treatment
PCR amplification

CC mC
5m 5h 5fc 5ca

5hmc residue

cDNA amplification

O-

Immunoprecipitate
and DNA extraction

DNA

PurifyCC
C mC
5m 5h 5fc 5ca

M.SssI

Hydroxymethylated DNA immunoprecipitation combined with next generation DNA sequencing (hMeDIP-seq)

N

Insert in regions of open chromatin

Tn5 Transposome

Add methionine analogue
l-azidohomoalanine (AHA)

Cells starved ofmethionine

Chromatin conformation capture
carbon copy (5-C)

hMeDIP-Seq

Rare variant

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

DNA

Pad

C

Bisulfite treatment
PCR amplification

BlockedCC mC
5m 5h 5fc 5ca
C

C

5h

Control

C

C

C
C mC
5m 5h 5fc 5ca

C

C

C

mC

O-ethylhydroxylamine (EtONH2)

C

C

C

C

C

ControlCC mC
5m 5h 5fc 5ca
C

C

C

4

4

Reduced representation M.SssI
methylase-assisted bisulfite
sequencing (RRMAB-seq)

MeDIP-Seq
DIP-seq

Sequence

Insert in regions of open chromatin

5-C

DNA

5caC S

5fc

C

MIRA

True
mutantCreate duplex sequences
based on molecular tags
and sequencing primers

2nd strand
synthesis
PCR and Remove
adaptors

AAAAAAA
TTTTTTTTTT

Covalent attachment of tags tocapture histones and identify
turnover (CATCH-IT)

Targeted chromosome conformation
capture (4C) with unique molecular
identifiers (UMI-4C)

Streptavidin pulldown
and DTT cleavage

C

Streptavidin pulldown
and DTT cleavage

C

NaBH

C

C

C

Linker5fC/5caC residues

DNA

N

RNA

Tn5 Transposome

DNA-protein complex with
Identify sites bound by small
chemical molecules (Chem-seq) putative drug binding site

Create single strand
consensus sequence fromevery unique molecular tag

AAAAAAA
TTTTTTTTTTOpen DNA

Adaptor

BiotinAdaptor ligation
and PCR

Control

BiotinCC mC
5m 5h 5fc 5ca

M.SssI methylase-assisted bisulfite
sequencing (MAB-seq) to map 5fC/5caCTrue variant

Consensus

Immunoprecipitate
and DNA extraction

Soluble extract

MNase digestion

UMI-4C

Glucosylated 5hmc

DNA

Streptavidin pulldown
and DTT cleavage
N3S-S

Pulldown,
NaOH and DTT

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

TAB-Seq

Sequence reads
1 and 2

Align sequences anddetermine actual ratio

Solubleextract

O

P5

Ligate and PCR

AA(A)nSequence

Sonicate

MNase digestion

Isolated chromatin

Open DNA

Chromatin conformation
capture circular (4-C)

NH2

Mutation

Safe-sequencing system is a uniquemolecular identifier (UMI) approachto detect rare variants (Safe-SeqS)

Reverse crosslink and Purification DNA
proteinase K digestion

O

HO
HO

N 3SH

C

C
C mC
5m 5h 5fc 5ca

CC mC
5m 5h 5fc 5ca

5fC/5caC residues

Adaptor sequence

Create target-specific oligos

Cross-linked chromatin

Immunoprecipitateand chromatin elution

C G

Biotinylation

BiotinDBCO-S-S-PEG3-biotin

CT

Biotinylation

βGT-catalyzed
UDP-6-N3 glucosylation

N 3S-SCo-acylisourea
CC mC
5m 5h 5fc 5ca

5fC chemically assisted bisulfite
sequencing (fCAB-seq) method forthe base-resolution detection of 5fC

N
N

Sequence

DNA

4-C

Biotin
g5hmC

N3S-S

g5hmC

N3

C

Convert 5fc to
5hmc

NaBH4

C

C

C G

N3

C

N3

βGT-catalyzed
UDP-6-N3 glucosylationCC

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

RRMAB-seq

N
N N

Single adaptor library

Target sequenceCC

5fc residue

N

DNA

Cromatin Purification
elutionStreptavidin

Next-generation Capture-C(NG Capture-C)

DNA

Streptavidin magnetic
bead pull down

Fragment

5hmC residue

C

O

H
N

Sequencing Primers

Fragment and add single adaptors

Flow cell

G&T-seq

Ligateg5hmC

OxidationCCC

Reduced bisulfite sequencing(redBS-Seq), to map 5-formylcytosine (5fC) in DNA

MAB-seq

Denature

Phi 29

C

C

C

DNA

DNA

O

Oligonucleotide-selective
sequencing (OS-Seq) capturesand sequence gene targets on
the flow cell

Single cell RNA

C G

O

N

DR-Seq

5cmC

TET

Azido 1,3-indandione(AI)

5fc residue

fCAB-Seq

O

Single cell RNA

C

Oxidative bisulfite sequencing(oxBS-Seq) to map 5-methylcytosine
and 5-hydroxymethylcytosine

N

Cell 3

Duplex-Seq

C

C

C

C

RedBS-Seq
caMAB-seq

O

Cell 2

Safe-SeqS

CC

5hmc residue

N3

H
S

Very rare mutation

C

T

C
C mC
5m 5h 5fc 5ca

5caC residue

oxBS-Seq

O

Amplified DNA

Cell 1

Gene

Biotinylated
primers

Blocked 5hmC residue

CC mC
5m 5h 5fc 5ca

5fC

N3

Strain I
Somatic variation within an individualSingle cell genome

OS-SeqC

g5hmC

βGT-catalyzed
glucosylation

Chemical modification-assisted
bisulfite sequencing (CAB-Seq)
for 5caC detectionDNA

O

Hybridize primers

Hybridize primers

Single G2/M nucleus sequencing ofcells in S phase (nuc-seq). Single
nucleus exome sequencing (SNES)C G

N

Nascent replication fork

Template

nuc-seq
SNES

5mC

βGT

C
C mC
5m 5h 5fc 5ca

C

NH2

Corrected
sequence

27-bp common sequence
8 random nucleotides

GenomeJBP1-magnetic beadpull down

Glucosylated 5-hmC

T4-βGT

AbaSI

Glucosylation

5fc residue

CAB-Seq

AI-mediated cyclization
labeling in fC-CETStrain I

Strain II
Natural variation between individuals

Amplify and sequence

3’ blocked random hexamer primers

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

C

T

C

DNA

Random error
Align fragments from every
unique molecular tag

Strain I

Targeted STR

GenomeMALBAC

Exonuclease

Degenerate
molecular tag

Targeted capture of STRloci by smMIPs (MIPSTR)

C

5hmC

5fC based on selective chemicallabeling of 5fC and subsequent
C-to-T transition during PCR

O

Read2

Genomic DNA

Short tandem repeat (STR)

MIPSTR

Tagmentation

Glucosylated 5-hmC

T4-βGTA 5-formylcytosine-selective
chemical labeling (fC-Seal) approachfor genome-wide profiling of 5fC

True variant

Sample index

Read1

smMIP

PCR

R

Hydroxy-methylated DNA

5fc residue

DNA

Amplify

Bisulfite
conversion

Displace oligo
Hybridize methylated
adaptor and gap repair

CC mC
5m 5h 5fc 5ca

DNA Low-Level Detection
Gene

PurifyTransposomes

Tet-assisted 5-methylcytosine
sequencing (TAmC-Seq)

fC-CET

Nascent strand sequencing (NS-seq) to discoverDNA replication origins and G4 structures

Oligo withmethylated
adaptor

Tagmentation

Transposome withmethylated adaptor

Hydroxy-methylated DNA

5mC

5mc residue

fC-SealPurifySolubleextract

Immunoprecipitate

NG Capture-C

OO

Nascent DNA

NS-seqDNA

N

AbaSI coupled with sequencing(Aba-seq) to map high-resolutionhydroxymethylome (5hmC)

BrdU

Nascent DNA

Repli Seq

5’-biotinylation

HN

O-

OO

SNS

Denaturation andsize-selection

DNA replication bubble

Amplify

5hmc residues

Aba-seqOO-O-

Solubleextract

N

O

O-

gDNA

SNS

Add P2
adapter

Second restriction digest withmethylation-sensitive HpaII

Add barcoded adapters

OH

O

O

N
H

gDNA

Restriction
digestion

Methylated DNA

J-binding protein 1 sequencing(JBP1-seq), for genome-wide profilingof 5-hydroxy-methylcytosine (5hmC)

Sonicate

MNase digestion

Isolate nucei

Chromatin interaction analysis bypaired-end tag sequencing (ChIA-PET)

Identified
methylation site

OH

CH3

Sonicate

Chia-PET

G
G

5hmc residues

JBP1-seq

O

Enzymeinactivation

Uridine diphosphate
glucose (UDP-Glu)

Restriction sites

Tagmentation-based
whole-genome bisulfite
sequencing (T-WGBS)

Proteinase K
digestionO

Displaced oligo

V(D)J antigen receptor region

EC-seqCell

ChIP-Seq of methylated histones(Histone methylation)DNA

T-WGBSDNAHistonemethylation

Hi-C
3-C
Capture-C

Double digest restriction-site associated DNA
marker generation (ddRADseq) with a methylation-sensitive restriction enzyme (EpiRADseq)

Index 1 P7

UMI

End repair
Adaptor ligation

GenomeOligo tagIndex 2

P5

Occupied regions of genomes fromaffinity-purified naturally isolated
chromatin (ORGANIC)

Nucleosome Occupancy MethylomeSequencing (NOMe-Seq), a single-molecule
nucleosome positioning assay

Padlock
probe

Methylated
regions

MspI
digestion

Methylated DNA

Methyl-seq and MRE-Seq use
methyl-sensitive enzymes to
identify methylation patterns

EpiRADseq

DNA

Capture on streptavidin beads

Lyse cells

Crosslink
cells in vivo

NOMe-Seq

Double-strand break

DSB-Seq

Deparaffination
and rehydration

Mapping in vivo nascentchromatin with EdU andsequencing (MINCE-seq)

OBisulfite ampliconsequencing (BSAS)

TTTT

TTTT

TT

TT

FFPE-fixedchromatin

MINCE-seq

BSAS

DNA

DNA

DNA extraction

Newly-formed chromatin

Bisulfite

TT

TTTT

TTTT

Breaks labeling and enrichment on
streptavidin and sequencing (BLESS)

Immunoprecipitate with
anti-digoxigenin antibody

TTTT

Double-strand break

BLESS

DNA isolation and random shearing

DNA Pol I, dU-digoxigenin, and dNTPs

TT

Genome DNA

DNA
extraction

BiotinO

Map DNA single-strand
breaks (SSB-Seq)

Polish ends

Isolate trimmed complexes

DNA

HO

TT

SSB-Seq

Reduced representation bisulfite
sequencing (RRBS-Seq).
Single cell RRBS (scRRBS)OSingle-strand break

Pathology tissue chromatin
immunoprecipitation (PAT-ChIP)PCR

CpG island Bisulfite
conversion

Bisulfite sequencing withpadlock probes (BSPP)

RRBS
scRRBSO

H2N

Deparaffination Heat 40°C in SDS
and rehydration

Transposome hypersensitive site
sequencing (THS-seq)

H1P1 P2 H2DNA

8N UID

MNase digestion

THS-seq

Preparation of acylated
RNA for biotin–streptavidin
purification.
DIBO, dibenzocyclooxtyne

BisulfiteT

BiotinAdaptor

BSPP

Terminate DNase I
digestion

Read primer
T7 promoter

CATCH-IT

Streptavidin

A

Open chromatin

FFPE-fixedchromatin

Assay for transposase accessible
chromatin (ATAC-Seq)

Load single cells into dropletswith lysis buffer and MNase

Methylated DNADNA

ATAC-Seq

Droplet withunique oligos

Post-bisulfite adaptor
tagging (PBAT)DNA extraction

N3

MmeI AluI MmeIAID-dependent rearrangement

DNase I digestion

Nucleus

Fixed-tissue chromatin immunoprecipitation sequencing (FiT-Seq)

High resolution of mapping of in vivo
chromatin associated proteins (X-ChIP)N

DIBO-biotin “click”

5-carboxylcytosine (5caC)

Transposon
insertion sites

PCR and sequence

Cell suspension

shearing. HpaII tiny fragment enrichment byligation-mediated PCR (HELP-Seq)

PBAT

MmeI digestion

PAT-ChIPNO

EpiGnomeTM
Bisulfite conversion of genomic DNA without Methylated DNA
HELP-Seq

20bp

MmeI

Whole genome
amplification

Nucleus

T

MmeI

20bp

MmeI
recognition site

Transposon sites

Pick
nucleiO

BS-SeqBisulfite-seq Bisulfite conversion of genomic DNA (bs-Seq) Methylated DNA
or whole-genome bisulfite sequencing (WGBS)
WGBS

O

N
Novel retrotransposition
eventsFACS
isolation

Epigenetics

N

HO

Known
retrotrans-poson

Align

N

+

NH2

O

Reference sequence

Read2

Cell
suspension

Cellsuspension

Isolate trimmed complexes

DNA

DNA

Cell
suspension

DNA

O

Genomic DNA

Single-molecule droplet
barcoding (SMDB)

5-formylcytosine (5fC)

DNA

Sequenced fragment

Read1

T7 amplification

Pool

SMDB

O

5-hydroxymethylcytosine (5hmc)

PCR

FiT-seq

LigateDNase I digestion

BiotinN

DNA

IndexedPCR primers

SDS

Primer

AbaSI

DNA
extraction

Immunoprecipitate

Align fragments from every Sequence
unique molecular tag

Retrotransposon
binding site

Droplet-based single-cell
ChIP-seq (Drop-ChIP)CXXCHybridize to sepharose column

Glucosylated 5-hmC

T4-βGT

Single cell

Drop-ChIP
scChIP-seq

5-Methyl Cytosine

CXXC

CXXC bound to nickel-charged
sepharose beads

Hydroxy-methylated DNA

accessible chromatin (scATAC-Seq)

R

TN-SeqINSeq

Exo I andpurifyAdaptor with cell-specific barcode
Illumina 5’ adaptor
T7 promoter(Microfluidics) Single-cell assay for transposase

N

Transposon

ExtendAdaptor
PCR
Second randompriming

Exonuclease digestion

Nucleus

Sort
nuclei

Lyse andcentrifuge

Cells

MAINE-Seq
MNase-Seq
isolation of nucleosomes (MAINE-Seq).
Nucleo-Seq MNase-assisted
Also Micrococcal nuclease sequencing (MNase-Seq)

O

N

H

Genomic DNA

Align sequences

Random primer 2

Repeat
4 times

Active chromatin

SupernatantDnase I simplified in-nucleus
method (Dnase I SIM) for plants

PCR and sequence

Convertedfragments

Sample fragmentation

NucleiscATAC-Seq

R

Genome

RC-Seq: Retrotransposon
capture sequencing

First randompriming

Bisulfite
conversion

Bisulfite
conversion

DNase I hypersensitive sites sequencing(DNase-Seq, DNaseI-Seq)

PCR and sequence

Microfluidics device

CytosineO

Novel retrotransposition events

Methidiumpropyl-EDTA (MPE)PCR and sequence
End repair
and ligation

Crosslink proteins and DNA

DNA-protein complex

DNaseI-SIM

Add poly A with TDT

Adaptor5hmc residues

O

N

Unmethylated CpG

Divide sample
into 96 reactions

O

Align RNA andmethylome

Single cell

DNA forsequencing

CXXC

CXXC

Contiguity-preserving
transposition sequencing (CPT-seq)

cDNA synthesis

Methylated Methylated
regions
adapter

MspI
digestion

Methylated DNA

Single-cell assay for transposase
accessible chromatin (scATAC-Seq)

NH2

Add K+ or PDS tostabilize G4 regions

Hibridizesequencing
primerDenature and remove
read fragment

CX
XC

CX
XC

XC
CX

Methylated CpGCXXC affinity purification plus
deep sequencing (CAP-seq)

DNase-Seq
DNaseI-Seq

O-

N
H

Chromatin immune precipitation (ChIP-Seq),
High-throughput chromatin immunoprecipitation (HT-ChIP))

O-

Fe

N

Single cellN

Remove 2’-phosphate and PCR

Degradation of
linear ssDNA

K+ or PDS

Target sequence Fragment andcreate library on
flow cell

CpG island

O-(Cell index)

NH2

RC-Seq

O- O

NH

N

scATAC-Seq

R

A

T

Potential G-quadruplex

Determine the location ofpotential G-quadruplexes in
DNA (G4-seq)

CPT-Seq

O

Methylated DNA

Lyse andcentrifuge

IsolatedLysesingle cell

Single cell retrotransposon
capture sequencing (scRC-Seq)

N

CAP-seqO

AA(A)nHybridize oligo

Add sequencing adaptors

Nucleus

Novel retrotransposition events

R

T

A
T

T

R

T
A

R

R

T

A

Single cell

scRC-Seq

Sequencing Primer

GenomeAdd carrier RNA

DNA

Detect 5hmC marks in single cellswith AbaSI nuclease (scAba-seq)

Map locations
on the genome

Add sequencing primers,
PCR and sequence

2’P

R

5’ P

Supernatant

Cell
Isolatesuspension single
cellAA(A)nT T (T)nAA(A)nRandom primer 1

scAba-seq

CH3

O

Align and determine
sequence breaks

5’P-O

5’HO

2’P

R

Fragmented
genomic DNA

Detect ribonucleotides
embedded in DNA(Ribose-seq)

AA(A)nSingle-cell bisulfite
sequencing (scBSBS-seq)

NH2

5’ P

RNA

Separate the DNA and the RNA

Streptavidin magnetic beadwith mRNA capture primer

scBS-seqDNA2’,3’-cyclic phosphate

3’5’Hydrolytic end sequencing (HydEn-seq) to reveal replicase- andstrand-specific patterns of ribonucleotides in the genome

Genome with ribonucleotides
R
R

Lyse cell

Isolate single
cell

DNA

Display methods onmobile device
Target site digestion

5’
3’

N

Deletion
Wild type
InsertCas9 target

Genome

DNA

N

H3C

In vitro Cas9-digested whole-genome sequencing (Digenome-seq)

Ribose-seq

Align RNA andmethylome

Plate 1 Plate 2

Restriction sites

GenomeHydEn-seqMeOn-bead transcriptome
amplification with Smart-seq2
Whole genome amplification
with RRBS
Sequence

AAAAAAA
TTTTTTTTTTAAAAAAA
TTTTTTTTTT

RNA

Single-cell triple omics
sequencing (scTrio-seq)

N

Restriction-site associatedDNA marker generation(RAD) capture (Rapture)Cellsuspension

DNA

Streptavidin
pull downAA(A)nDNA

DNA to be captured

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

GenomeAA(A)nDNA methylation

High quality DNA

Hybridization RAD (hyRAD)
for degraded DNA

Digenome-seq

NN

Methylome and transcriptome sequencing from asingle cell (scMT-seq)

Restriction sites

GenomeRaptureSeparate the DNA and the RNA

Streptavidin magnetic beadwith mRNA capture primer

RNA

DNA

scTrio-seq

Specific locus amplified fragment sequenc- Digest with MseI
ing (SLAF-seq) for large scale genotyping

hyRAD

N

UracilRestriction digestion with type IIB
restriction endonucleases e.g. BsaXI

CH3

N

O

scMT-seq

NH2O
N

SLAF-seq

Lyse cell

Isolate single
cell

ChIP-Seq
ChIP-exo
HT-ChIP
Mint-ChIP

NH2

H2 N

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

AAAAAAA
TTTTTTTTTTAAAAAAA
TTTTTTTTTT

Restriction sites

Single cell RNA

2b-RAD

Cell
suspension

Methylome and transcriptome sequencing from asingle cell (scM&T-seq)

Cytosine 5-Methyl Cytosine

AA(A)n

DNA

DNA

N

O

RNA

AA(A)nIdentify C-terminal
sequences

Create oligo
library

Construct phagedisplay library

2
1

Bait proteins immobilized on 96-well plate

Select phages against
baits

Isolate andsequence

Peptidecounts

Display methods onmobile device

DNA

References
2b-RAD

Wang S et al. (2012) Nat Methods 9:808-810

3CDuan Z. et al. (2012) Methods 58: 277-288

4-C

Zhao Z. et al. (2006) Nat Genet 38: 1341-1347

5-C

Dostie J. et al. (2007) Nat Protoc 2: 988-1002

Aba-seq

Bubble-Seq

Clausen A. R. et al. (2015) Nat Struct Mol Biol 22:
185-191

MeDIP-Seq

Down T. A. et al. (2008) Nat Biotechnol 26: 779-785

NS-seqFoulk MS et al. (2015) Genome Res 25:725-35

hyRAD

Suchan T et al. (2016) PLoS One 11:e0151651

MethylCap-Seq

Brinkman A. B. et al. (2010) Methods 52: 232-236

Nucleo-Seq

Valouev A. et al. (2011) Nature 474: 516-520
Wang Y. et al. (2014) Nature 512: 155-160

Chambers V. S. et al. (2015) Nat Biotechnol 33:
877-881

IMS-MDA

Gole J et al. (2013) Nat Biotechnol 31:1126-32

MiGS

Serre D. et al. (2010) Nucleic Acids Res 38: 391-399

Boyle A. P. et al. (2008) Cell 132: 311-322

GUIDE-seq

Tsai SQ et al. (2015) Nat Biotechnol 33:187-97

Seth-Smith H. M. et al. (2013) Nat Protoc 8:2404-2412

MIDAS

ORGANIC

G4-seqVollmers C. et al. (2013) Proc Natl Acad Sci U S A
110: 13463-13468

Brunner A. L. et al. (2009) Genome Res 19:
1044-1056

nuc-seq

Macaulay I. C. et al. (2015) Nat Methods 12: 519-522

Ig-SeqMethyl-seq

MINCE-seq

Drop-ChIP

Rotem A et al. (2015) Nat Biotechnol 33:1165-72

HELP-Seq

Oda M. et al. (2009) Nucleic Acids Res 37: 3829-3839

DR-SeqDey SS et al. (2015) Nat Biotechnol 33:285-9

Hi-C

DSB-seq

Baranello L et al. (2014) Int J Mol Sci 15:13111-22

Lieberman-Aiden E. et al. (2009) Science 326:
289-293

Duplex-Seq

Schmitt M. W. et al. (2012) Proc Natl Acad Sci U S A
109: 14508-14513

Histone meth.

Barski A. et al. (2007) Cell 129: 823-837

HiTS-Flip

Nutiu R. et al. (2011) Nat Biotechnol 29: 659-664

EC-seqParkinson N. J. et al. (2015) Genome Res 25: 226-234

hMeDIP-seq

Xu Y et al. (2011) Mol Cell 42:451-64

EpiRADSeq

Schield DR et al. (2016) Methods in Ecology and
Evolution 7:60-69

HT-ChIP

Blecher-Gonen R. et al. (2013) Nat Protoc 8: 539-554

HTGTS

Chiarle, R. et al. (2011) Cell 147, 107–119

HT-SELEX

Jolma A. et al. (2010) Genome Res 20: 861-873

Mesner L. D. et al. (2013) Genome Res 23:
1774-1788

ddRADseqPeterson B. K. et al. (2012) PLoS One 7: e37135

fC-CET

Xia B et al. (2015) Nat Methods 12:1047-50

CAB-Seq

Lu X. et al. (2013) J Am Chem Soc 135: 9315-9317

Digenome-seq

Kim D et al. (2015) Nat Methods 12:237-43

fC-SealSong C. X. et al. (2013) Cell 153: 678-691

caMAB-seq

Wu H et al. (2016) Nat Protoc 11:1081-100

DIP-seq

Shen L. et al. (2013) Cell 153: 692-706

FiT-Seq

Cejas P et al. (2016) Nat Med

G&T-seq

DNaseI-Seq

ATAC-Seq

Buenrostro J. D. et al. (2013) Nat Methods 10:1213-1218

Illingworth R. S. et al. (2010) PLoS Genet 6:e1001134

Cumbie JS et al. (2015) Plant Methods 11:42

Sun Z. et al. (2013) Cell Rep 3: 567-576

CAP-seq

Dnase I SIM

Capture-C

Hesselberth J. R. et al. (2009) Nat Methods 6:283-289

BisChIP-Seq

Statham A. L. et al. (2012) Genome Res 22:1120-1127

CATCH-IT

Deal R. B. et al. (2010) Science 328: 1161-1164

DNase-Seq

Chem-Seq

Anders L. et al. (2014) Nat Biotechnol 32: 92-96

Bisulfite-seq

Berman B. P. et al. (2012) Nat Genet 44: 40-46

Chia-PET

Li G. et al. (2010) Genome Biol 11: R22

BLESS

Crosetto, N. et al. Nat. Methods 10, 361–365 (2013).

ChIP-BS-seq

Break-seq

Hoffman E. A. et al. (2015) Genome Res 25: 402-412

Brinkman A. B. et al. (2012) Genome Res 22:1128-1138

BSAS

Masser D. R. et al. (2013) Epigenetics Chromatin 6:
33

ChIP-exo

Yen K. et al. (2013) Cell 154: 1246-1256

ChIPmentation

Schmidl C et al. (2015) Nat Methods 12:963-5

BSPP

Deng J. et al. (2009) Nat Biotechnol 27: 353-360

ChIP-Seq

Barski A. et al. (2007) Cell 129: 823-837

BS-SeqLister R. et al. (2009) Nature 462: 315-322

CPT-seq

Amini S. et al. (2014) Nat Genet 46: 1343-1349

FAIRE-Seq

Gaulton K. J. et al. (2010) Nat Genet 42: 255-259

DamID

Vogel M. J. et al. (2007) Nat Protoc 2: 1467-1478

fCAB-Seq

Song C. X. et al. (2013) Cell 153: 678-691

TruSeq PCR Free
Double-stranded DNA

Hughes J. R. et al. (2014) Nat Genet 46: 205-212

TruSeq Nano

TruSeq Custom Amplicon

Double-stranded DNA

Double-stranded DNA

Fractionate
Size selectAdd Adaptors

P

A

P5
Index 2
Index 1

Index
P7

T

P

P

T

Index

Adaptor ligation

P

Index
P7

P

T

P5

P5

Khan TA et al. (2016) Sci Adv 2:e1501371

MAINE-Seq

Ponts N. et al. (2010) Genome Res 20: 228-238

MALBAC

Zong C. et al. (2012) Science 338: 1622-1626

MBDCap-Seq

de Assis S. et al. (2012) Nat Commun 3: 1053

MBD-Seq

Nair S. S. et al. (2011) Epigenetics 6: 34-44

MDA

Dean F. B. et al. (2001) Genome Res 11: 1095-1099

P7

Index 2
P5

Product ready forcluster generation

Cusanovich DA et al. (2015) Science 348:910-4

RAD

Baird N. A. et al. (2008) PLoS One 3: e3376

Buenrostro J. D. et al. (2015) Nature 523: 486-490

Zentner G. E. et al. (2013) PLoS Genet 9: e1003317

RaptureAli OA et al. (2016) Genetics 202:389-400

scATAC-seq(Microfluidics)

OS-SeqMyllykangas S. et al. (2011) Nat Biotechnol 29:1024-1027

RBBS

Meissner A et al. (2008) Nature 454:766-70

scBS-seq

RC-Seq

Baillie J. K. et al. (2011) Nature 479: 534-537

Smallwood S. A. et al. (2014) Nat Methods 11:
817-820

Ramachandran S et al. (2016) Cell 165:580-92

oxBS-Seq

Booth M. J. et al. (2012) Science 336: 934-937

RedBS-Seq

Booth M. J. et al. (2014) Nat Chem 6: 435-440

Mint-ChIP

van Galen P et al. (2016) Mol Cell 61:170-80

PAT–ChIP

Fanelli M et al. (2011) Nat Protoc 6:1905-19

Repli-Seq

MIPSTR

Carlson K. D. et al. (2015) Genome Res 25: 750-761

PBAT

Miura F. et al. (2012) Nucleic Acids Res 40: e136

Hansen R. S. et al. (2010) Proc Natl Acad Sci U S A
107: 139-144

MIRA

Rauch T. A. et al. (2010) Methods 52: 213-217

PB-seqGuertin M. J. et al. (2012) PLoS Genet 8: e1002610

MNase-Seq

Schones D. E. et al. (2008) Cell 132: 887-898

PD-Seq

MPE-seq

Ishii H et al. (2015) Proc Natl Acad Sci U S A
112:E3457-65

MRE-Seq

Maunakea A. K. et al. (2010) Nature 466: 253-257

NG Capture-C

Davies JOJ et al. (2016) Nat Meth 13:74-80

NOME-Seq

Han H. et al. (2011) Hum Mol Genet 20: 4299-4310

NSCR

Kunnev D. et al. (2015) Genome Res 25: 558-569

dT TP + dC TP + dATP + dGTP

Rep-Seq

Benichou J. et al. (2012) Immunology 135: 183-191

Arango D. et al. (2013) Proc Natl Acad Sci U S A 110:
E2153-2162

Ribose-seq

Koh K. D. et al. (2015) Nat Methods 12: 251-257

RRBS

Meissner A. et al. (2008) Nature 454: 766-770

PDZ-Seq

Ernst A. et al. (2010) Mol Biosyst 6: 1782-1790

RRMAB-seq

Neri F. et al. (2015) Cell Rep 10: 674-683

PE RAD-Seq

Willing E. M. et al. (2011) Bioinformatics 27:
2187-2193

Safe-SeqS

Kinde I et al. (2011) Proc Natl Acad Sci U S A
108:9530-5

Ivarsson Y. et al. (2014) Proc Natl Acad Sci U S A 111:
2542-2547

scABA-seq

ProP-PD

TargetTargetRotem A et al. (2015) Nat Biotechnol 33:1165-72

scM&T-seq

Angermueller C et al. (2016) Nat Methods advanceonline publication

scMT-Seq

Hu Y et al. (2016) Genome Biol 17:88

scRC-Seq

Upton KR et al. (2015) Cell 161:228-39

scRRBS

Guo H. et al. (2015) Nat Protoc 10: 645-659

scTrio-seq

Hou Y et al. (2016) Cell Res 26:304-19

SELEX

Jolma A. et al. (2010) Genome Res 20: 861-873

SELEX-seq

Slattery M. et al. (2011) Cell 147: 1270-1282

SLAF-Seq

Sun X et al. (2013) PLoS One 8:e58700

SMDB

Lan F et al. (2016) Nat Commun 7:11784

smMIP

Hiatt J. B. et al. (2013) Genome Res 23: 843-854

SNES

Leung M. L. et al. (2015) Genome Biol 16: 55

Sono-Seq

Auerbach R. K. et al. (2009) Proc Natl Acad Sci U S A
106: 14926-14931

SSB-Seq

Baranello L et al. (2014) Int J Mol Sci 15:13111-22

TAB-Seq

Yu M. et al. (2012) Cell 149: 1368-1380

TAmC-Seq

Zhang L. et al. (2013) Nat Commun 4: 1517

TC-SeqKlein I. A. et al. (2011) Cell 147: 95-106

THS-seq

Sos BC et al. (2016) Genome Biol 17:20

TN-Seqvan Opijnen T. et al. (2013) Nat Rev Microbiol 11:435-442

T-WGBS

Wang Q. et al. (2013) Nat Protoc 8: 2022-2032

UMI-4C

Schwartzman O et al. (2016) Nat Methods advanceonline publication

WGBS

Lister R. et al. (2009) Nature 462: 315-322

X-ChIP-seq

Skene P. J. et al. (2014) Elife 3: e02042

Nextera Rapid Capture

Nextera Mate Pair
Transposase

TargetTransposase

cDNA

3’ Adapter

scChIP-seq

Nextera Library Preparation

Total RNA

Pool stranded
RNA-Seq libraries

Create cDNA

Mooijman D et al. (2016) Nat Biotechnol advanceonline publication

TruSeq Targeted RNA Expression

TruSeq RNA AccessRNA
Random primer

3’

polyA selectdUTP + dC TP + dATP + dGTP

Add primer

Sense strand U

Reverse transcription

Index 2

Index 1

P7

Sense strand

Index 1P7First andsecond strand
synthesis

Add sequencing
primers

Index 1Denature and amplify

P7

U U U UUUU

U U U U

U

UU

U P

Index

P5

Product ready forcluster generation

Product ready forcluster generation

U U U U U U
Sense strand

U

UU

Index 1
Index 2

U

Denatured andpooled fragmentsfrom Nextera library

DNA

R

R

P7
U U

U U U UU

Index 2UU

P5U
Block polymerase

Index 1

P7

Biotinylated
target probe

Hybridize probes totargets

Tagmentation

Capture on
streptavidin
magnetic beads

P5
Index 2

Capture on
magnetic beads

Denature andamplify

Index 2

TargetIndex 1

P7

Index 2
P5

Product ready forcluster generation

Isolate biotinylated
fragment

Index 1

TargetAmplification

Index 1
P7

Product ready forcluster generation

ForwardstrandSequenceprimerReversestrand

A
A
T
T
C
G
C

A
A
T
T
C
G
C

A
A
T
T
C
G
C

A
A
T
T
C
G
C

Read 2primer

A
A
T
T
C
G
C

Index 1primer

A
A
T
T
C
G
C

A
A
T
T
C
G
C

A
A
T
T
C
G
C

A
A
T
T
C
G
C

P5

Index 2

Index 1

P7

Product ready forcluster generation

P5

TargetIndex 2

Index 1

P7

Elute

P7

Product ready forcluster generation

P5

Adapter ligation

A
A
T
T
C
G
C

Generate clonal pools
Amplify
Transposase

A
A
T
T
C
G
C

A
A
T
T
C
G
C

Tagmentation~600bpRemoveforwardstrandFold over andhybridize tosecond primer

Synthesize
second strand
Bridge amplification

Thousands of molecules areamplified in parallel

The reverse
strand iscleaved andwashed away

With each cycle, four fluorescently tagged nucleotides
compete for addition to thegrowing chain. Only one isincorporated based on thesequence of the template.

The read
product iswashed away

Sequence
Index1

The read
product iswashed away

Fold over andhybridize tofirst primer

Deblock P5
primer andadd unlabeled
bases

Sequence
Index2Synthesize
secondstrandThe forwardstrand iscleaved andwashed away

The second
read issequenced

FOR RESEARCH USE ONLYThis poster was compiled by the Illumina Scientific Affairs. Additional information, the latest version of the poster, and a comprehensive list of *seq methods, are available at http://www.illumina.com/libraryprepmethods. Please contact Scientific Affairs with any questions, comments, or suggestions.

© 2015 Illumina, Inc. All rights reserved. Illumina, Inc. • 5200 Illumina Way, San Diego, CA 92122 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • techsupport@illumina.com • illumina.com
Illumina, HiSeq, MiSeq, MiniSeq, Nextera, NextSeq, TruSeq, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 373-2016-005 Current as of 10 November 2016

P7

P5

R

P7

Elute
P5

R

Fragment

Index 2

P7

Product ready forcluster generation

Circularize

R

R

P5

Index 1Denature andamplify

R

R~300bpExtension-Ligation
Adaptor ligation

Hybridize probes
to targets~10kb Sheared genomic DNA

ReversestrandsyntesisDNA

R

Hybridization

End repair

Adapter hybridizes to flowcell

R

R

R

Moleculo

ReversestrandR

R

Sequencing by Synthesis

ForwardstrandBiotinylated junction adapter

Tagmentation

Add custom
primers

5’ P

P5

P5

P5

DLSO
5’ P

P7

P7

P7

ULSO

Biotinylated
target probe

End repair
Phosphorylate
A-overhangA

AUU

Create second
strand cDNA

U

P

Sense strand

PCR
P5

U U

P5
Index 2

P7
Index 1

P5

Index 1

5’

cDNA

P5

P7

Index 2scATAC-seq(Cell index)

TruSeq RNA Stranded

Small RNA fragment

Random hexamer

Denature and amplify
P5

MAF

mRNA

Adaptor ligation

P7

Product ready forcluster generation

Wu H. et al. (2014) Nat Biotechnol

Extension and ligation

Index 1
Index 2

Index 2
Index 1Index

Add custom probes

Index 1
Index 2

P7
P5

MAB-seq

P7

T

P5

P5

Hu J et al. (2016) Nat. Protocols 11:853-871

Daigaku Y. et al. (2015) Nat Struct Mol Biol 22:
192-198

Fragment

Add Adaptors

P7

P7

CustomProbe 2

P

A

A

P

P5

CustomProbe 1AP

LAM-HTGTS

3’5’ Adapter

AAAAA
TTTTT

A-overhangA-overhang

Cui L. et al. (2014) Genomics

Pu-seq

R

P

P

5’

Region of interest

P

P

Goodman AL et al. (2009) Cell Host Microbe 6:279-89

JBP1-seq

Ligate adaptors

End repair
Phosphorylate

End repair
Phosphorylate

INSeq

TruSeq Small RNA

TruSeq RNA
AAAAAFractionate
Size selectHydEn-seq

P5

P5

P5

Index 2

Index 2

Index 2

Add indices

Index 1

Index 1

Index 1

P7

P7

P7

P5
P5
P5

P7
P7
P7

Prepared fragments

Pool and purify

Adaptor ligation

R

P5

P7

Denature andamplify
Product ready forcluster generation