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DNA
DNA Rearrangements and Markers DNA-Protein Interactions
Bisulfite conversion

Genome
NH2 NH2 Single cell RNA RNA On-bead transcriptome H2 N NH2 ChIP-Seq
RAD Restriction sites AA(A)n AA(A)n AAAAAAA
amplification with Smart-seq2
ChIP-exo
CH3
scM&T-seq
AAAAAAA TTTTTTTTTT N
N N TTTTTTTTTT
PE RAD-Seq DNA DNA Whole genome bisulfite Me
Restriction-site associated DNA marker Restriction Add barcoded adapters Shear and Add P2 Amplify DNA O N O N sequencing with scBS-seq HT-ChIP Chromatin immune precipitation (ChIP-Seq), DNA-protein complex Crosslink proteins and DNA Sample fragmentation Exonuclease digestion Immunoprecipitate DNA DNA
generation (RAD) digestion size select adapter O
Cytosine 5-Methyl Cytosine
Methylome and transcrip-
tome sequencing from a
Cell
suspension
Isolate single
cell
Lyse cell Streptavidin magnetic bead
with mRNA capture primer
Separate the DNA and the RNA Sequence Align RNA and
methylome O Mint-ChIP High-throughput chromatin immunoprecipi-
tation (HT-ChIP))
extraction
single cell (scM&T-seq) O- O
NH
Genome Restriction sites
ddRADseq Single cell RNA
N
Fe
O-
RNA On-bead transcriptome O-
AA(A)n AA(A)n AAAAAAA
amplification with Smart-seq2
N DNase-Seq
Double digest restriction-site associated Restriction Add barcoded adapters Second restriction digest Add P2 Amplify
scMT-seq
NH2 TTTTTTTTTT
DNA O AAAAAAA
TTTTTTTTTT
DNA marker generation (ddRADseq) digestion adapter
N N
CH3 DNA DNA Whole genome amplification N
H
O- DNaseI-Seq
with RRBS DNase I hypersensitive sites sequencing Active chromatin DNase I digestion Isolate trimmed complexes DNA extraction DNA
Methylome and transcrip- Cell Isolate single Lyse cell Streptavidin magnetic bead Separate the DNA and the RNA Sequence Align RNA and O (DNase-Seq, DNaseI-Seq)
Genome BsaXI restriction sites N N
O O Methidiumpro-
2b-RAD Uracil 5-Methyl Cytosine
tome sequencing from a
single cell (scMT-seq)
suspension cell with mRNA capture primer methylome
pyl-EDTA (MPE)
Supernatant Nucleus
Restriction-site associated DNA marker
generation (RAD) with type IIB restriction
Restriction digestion with type IIB
restriction endonucleases e.g. BsaXI
Add restriction-
site-specific adapters
Amplify Add sample-specific
adapters
Amplify DNA
Single cell
RNA
AA(A)n AA(A)n
DNaseI-SIM
RNA T T (T)n AA(A)n
endonucleases (2b-RAD) AA(A)n
scTrio-seq DNA
Supernatant
DNA
Add carrier RNA Hybridize oligo cDNA synthesis Add poly A with TDT PCR and sequence
Nuclei
Genome Restriction sites
SLAF-seq DNA methylation Add sequencing adaptors PCR and sequence Dnase I simplified in-nucleus
method (Dnase I SIM) for plants
Cells Lyse and
centrifuge
Sort
nuclei
Nucleus DNase I digestion Terminate DNase I Polish ends DNA DNA
Nucleus digestion extraction
Methylated DNA
Specific locus amplified fragment sequenc- Digest with MseI Add MseI Digest with AluI PCR with barcoded Purify and Add sequencing PCR DNA
ing (SLAF-seq) for large scale genotyping adaptors MseI primer pool adaptors
Single-cell triple omics Cell Isolate Lyse and MspI Methylated Methylated End repair Bisulfite Converted PCR and sequence Align sequences
sequencing (scTrio-seq) suspension single
cell
centrifuge digestion regions adapter and ligation conversion fragments MAINE-Seq
Genome High quality DNA DNA to be captured MNase-Seq
hyRAD scBS-seq MNase-assisted isolation of nucleosomes (MAINE-Seq).
N
N Random primer 1
Adaptor Random primer 2
Adaptor
Nucleo-Seq Also Micrococcal nuclease sequencing (MNase-Seq)
Open chromatin MNase digestion Isolate trimmed complexes DNA extraction DNA
Hybridization RAD (hyRAD) Prepare RAD-seq Adaptor removal Biotinylated Shotgun library Hybridize to probes Streptavidin DNA N N
for degraded DNA library and size select and labeling probes pull down Single-cell bisulfite Isolated Lyse Methylated DNA Bisulfite First random Repeat Extend Exo I and Second random PCR Align fragments from every Sequence
sequencing (scBSBS-seq) single cell conversion priming 4 times purify priming unique molecular tag
Plate 1 Plate 2
Genome Restriction sites
Rapture
Well 1
Well 2
H3C Single cell 5hmc residues FiT-seq
scAba-seq
CH3 Adaptor with cell-specific barcode
O Illumina 5’ adaptor Fixed-tissue chromatin immuno- FFPE-fixed Deparaffination Heat 40°C in SDS Proteinase K Enzyme Sonicate Soluble Immunoprecipitate Cromatin Purification DNA
Restriction-site associated Streptavidin T7 promoter precipitation sequencing (FiT-Seq) chromatin and rehydration digestion inactivation extract elution
Digest with Add well-specific Pool wells Restriction Library prep with Pool Hybridize Biotinylated Streptavidin DNA
DNA marker generation restriction barcodes and shear pull down enzyme digest plate barcodes capture bait pull down Detect 5hmC marks in single cells Hydroxy-methyl- T4-βGT Glucosylated 5-hmC AbaSI Primer Ligate Pool T7 amplification DNA
(RAD) capture (Rapture) enzyme with AbaSI nuclease (scAba-seq) ated DNA
Display methods on O
mobile device
Dige- Genome Cas9 target Deletion
Novel retrotrans- Retrotransposon N
Wild type DNA + N
PAT-ChIP
nome-seq Insert
scRC-Seq
position events binding site
N Pathology tissue chromatin FFPE-fixed Deparaffination MNase digestion Sonicate Soluble Immunoprecipitate Reverse crosslink and Purification DNA
In vitro Cas9-digested whole-ge- Target site digestion Sequence Align and determine immunoprecipitation (PAT-ChIP) chromatin and rehydration extract and chromatin elution proteinase K digestion
nome sequencing (Digenome-seq) sequence breaks N3
Single cell retrotransposon Genomic DNA Cell FACS Pick Nucleus Whole genome Create sequencing Sequence capture Enriched library Acylation
Ribonucleotides capture sequencing (scRC-Seq) suspension isolation nuclei amplification library
Genome 2’,3’-cyclic phosphate
HydEn-seq 5’
3’
R R
3’
5’ 5’HO 5’P-O
R R O
DNA
Single cell
Hydrolytic end sequencing (HydEn-seq) to reveal replicase- and Alkaline Phosphorylation (T4 PNK Ligate oligo with 5’-amino- Add sequencing primers, Map locations
scATAC-Seq O X-ChIP
strand-specific patterns of ribonucleotides in the genome hydrolysis (KOH) 3-phosphatase minus) terminated C6 spacer PCR and sequence on the genome High resolution of mapping of in vivo Crosslink Lyse cells Cross-linked chromatin MNase digestion Sonicate Soluble Immunoprecipitate DNA
NH2 (Cell index) N
chromatin associated proteins (X-ChIP) cells in vivo extract and DNA extraction

R Single-cell assay for transposase Cell suspension Isolate Nuclei Split Barcode each well Pool and Split sample PCR-barcode Pool for library DNA N3
Genome with ribonucleotides R mA R Am R N accessible chromatin (scATAC-Seq) sample with Tn5 transposase dilute every well prep
T
2’P

T
R R R R R DIBO-biotin “click”
Ribose-seq
T

2’P

A Am A Am A R
5’ P 5’ P A
T

T A Am
T

ORGANIC
A T
P 5’ A P 5’ mA A T mA A T mA A T N O Microfluidics device
Detect ribonucleotides Fragmented dA tailing Adaptor ligation Alkali treatment Self-ligation by Degradation of Remove 2’-phos- DNA for Single cell O Occupied regions of genomes from Cell Isolate nucei Isolated chromatin Soluble extract Immunoprecipitate
DNA
MNase digestion DNA
embedded in DNA genomic DNA AtRNL linear ssDNA phate and PCR sequencing R affinity-purified naturally isolated and DNA extraction
(Ribose-seq) Cytosine scATAC-Seq O chromatin (ORGANIC)
Sequencing Primer NH2 (Microfluidics) Single-cell assay for transposase Cell Isolate Lyse and introduce Insert in regions of open chromatin Fragmented and primed Amplify with Pool libraries DNA N
CH3 accessible chromatin (scATAC-Seq) suspension single cell Tn5 transposase cell-specific from all cells N
Genome Potential G-quadruplex K+ or PDS Polymerase stalls
N barcodes N N
G4-seq ATAC-Seq
N O
Single cell Assay for transposase accessible Open DNA Tn5 Transposome Insert in regions of open chromatin Fragmented and primed DNA purification DNA
Droplet with
Determine the location of Target sequence Fragment and Hibridize Read reference Denature and remove Hibridize Add K+ or PDS to Read stabilized Compare
5-Methyl Cytosine
R Drop-ChIP unique oligos
chromatin (ATAC-Seq) Amplification
potential G-quadruplexes in create library on sequencing sequence read fragment sequencing stabilize G4 regions squence sequence reads
DNA (G4-seq) flow cell primer primer 1 and 2 scChIP-seq Biotin Read primer
NH2 T7 promoter
Droplet-based single-cell Cell Load single cells into droplets Fuse droplets Pool all droplets Chromatin immuno- Sequence Barcoded sequences Preparation of acylated THS-seq
XC
CX

X CXXC CXXC CXXC ChIP-seq (Drop-ChIP) suspension with lysis buffer and MNase precipitation from single cells
XC

RNA for biotin–streptavidin

CX
XC

CpG island HO N
CX

Methylated CpG CXXC CXXC

Add barcodes and
CAP-seq CXXC CXXC
purification. Transposome hypersensitive site Open DNA Tn5 Transposome Insert in regions of open chromatin Purify End fill-in and IVT dsDNA synthesis sequencing adaptors
Unmethylated CpG N O SMDB DIBO, dibenzocyclooxtyne sequencing (THS-seq) amplification
CXXC affinity purification plus CXXC bound to nickel-charged Hybridize to sepharose column Elute unmethylated CpG DNA
deep sequencing (CAP-seq) sepharose beads enriched fragments R Single-molecule droplet DNA templates Single template Template amplification Template fragmentation Barcode every droplet Pool for library DNA
5-hydroxymethylcytosine (5hmc) Biotin
barcoding (SMDB) encapsulation prep
Streptavidin
O NH2
CATCH-IT
Genome H N Epigenetics Covalent attachment of tags to
capture histones and identify
Cells starved of
methionine
Add methionine analogue
l-azidohomoalanine (AHA)
Isolate
nucei
Cycloaddition
reaction
MNase digestion Remove of H2A–H2B dimers
and non-histone proteins
DNA
extraction
DNA
CPT-Seq
T C T C G
Bisulfite turnover (CATCH-IT)
N O
BS-Seq T U T C G Newly-formed chromatin
Streptavidin
Bisulfite-seq Bisulfite conversion of genomic DNA (bs-Seq)
PCR
R Polymerase
Methylated DNA Shear DNA Bisulfite conversion DNA
MINCE-seq
T T T C G
Contiguity-preserving Divide sample Indexed transpo- EDTA Dilute and divide SDS Indexed PCR DNA 5-formylcytosine (5fC)
transposition sequenc- into 96 reactions some reactions Pool into 96 reactions PCR primers WGBS or whole-genome bisulfite sequencing (WGBS)
ing (CPT-seq) O NH2
T C T C G Mapping in vivo nascent Label with ethynyl Chase with Crosslink with formaldehyde Click reaction Sonication and Streptavidin DNA DNA
Novel retrotrans- Retrotransposon Bisulfite chromatin with EdU and deoxyuridine (EdU) Thymidine to attach biotin MNase digestion capture extraction
HO N
RC-Seq
position events binding sites
Read1
Sequenced fragment Known
retrotrans-poson EpiGnomeTM T U
PCR
T C G
sequencing (MINCE-seq)

Reference sequence
N O HELP-Seq Bisulfite conversion of genomic DNA without Methylated DNA Bisulfite conversion T T T C G Converted single-stranded Random priming 3’ tagging PCR DNA
RC-Seq: Retrotransposon Genomic DNA Fractionate DNA Hybridize Microarray with transposon
Read2
Transposon sites
Align
Novel retrotransposition
events R
shearing. HpaII tiny fragment enrichment by
ligation-mediated PCR (HELP-Seq)
fragments DNA synthesis FAIRE-seq
capture sequencing binding sites
fragments
5-carboxylcytosine (5caC)
Streptavidin
Sono-Seq Formaldehyde-assisted isolation of regula- Crosslink protein and DNA with formalin Sonicate Phenol extract and purify DNA
Open DNA DNA
Biotin
Random primer 2 tory elements (FAIRE-Seq) and sonication of from the aquous phase
Transposon Transposon 20bp MmeI Adaptor cross-linked chromatin (Sono-Seq)
Adaptor
TN-Seq MmeI 20bp PBAT
Post-bisulfite adaptor Methylated DNA Bisulfite First random Capture first strand on Streptavi- Second random Generate second strand Elution DNA with adaptors
INSeq Transposon sequencing (TN-Seq) Inverted MmeI MmeI Transposon Protected Protected
recognition site recognition site MmeI digestion Add adapters PCR and sequence
tagging (PBAT) conversion priming din coated magnetic beads priming CpG dinucleotides CpG dinucleotides
and insertion sequencing (INSeq) insertion sites
NOMe-Seq Methylated CpG Methylated CpG
Control
Protected
methylated
Unprotected
methylated
MmeI AluI MmeI
Nucleosome Occupancy Methylome- Open DNA GpC methyltransferase (M.CviPI) and Bisulfite conver- M.CviPI
+AID H1P1 P2 H2 Sequencing (NOMe-Seq), a single-molecule S-Adenosyl methionine (SAM) Protected Unprotected
AID-dependent rearrangement A A sion BS-Seq
TC-Seq
I-SceI site BSPP nucleosome positioning assay unmethylated unmethylated

-AID A A Bisulfite sequencing with CpG island Bisulfite Bisufite-converted Padlock Hybridize Extension Exonuclease PCR End repair DNA with
padlock probes (BSPP) conversion DNA probe and ligation digestion and adaptor adaptors
TC-Seq: translocation
capture sequencing
Genomic DNA Infect I-Sel Sonicate, blunt
and A-tail
Ligate linkers
Cut I-Scel
Purification Semi-nested PCR
Linker cleavage
DNA ligation Histone
methylation
Ig-seq CDR3 junction region V D J Constant 8N UID O
RRBS
H2N
ChIP-Seq of methylated histones DNA-protein complex Crosslink proteins Sample Exonuclease digestion Immunoprecipitate DNA extraction DNA
Rep-Seq 8N UID (Histone methylation) and DNA fragmentation
DNA sequencing of immunoglobulin genes Extracted RNA Reverse transcription Second strand synthesis PCR Purify DNA H2N H2N scRRBS Reduced representation bisulfite Methylated MspI Methylated Methylated End repair Bisulfite Converted fragments PCR DNA
MAF
O O
(Ig-seq), repertoire sequencing (Rep-Seq), and N sequencing (RRBS-Seq). DNA digestion regions adapter and ligation conversion HO OH
molecular amplification fingerprinting (MAF) NH NH Single cell RRBS (scRRBS) HO O
O O

Single-strand break HO
O
O- O-
NH
ChIPmenta-
SSB-Seq tion
N O
BSAS O P O P O O
Chromatin immunoprecipitation Chromatin Chromatin immunoprecipitation Tn5 Transposome Adaptor insertion Fragmented DNA purification DNA
Map DNA single-strand Genome DNA DNA Pol I, dU-digoxigenin, and dNTPs DNA isolation and random shearing Immunoprecipitate with DNA Bisulfite amplicon Bisulfite Bisufite-con- PCR with primers specific for Amplicons Transposome with
OH OH
with sequencing library preparation and primed Amplification
Methylated DNA Tagmentation DNA
breaks (SSB-Seq) anti-digoxigenin antibody Pyridostatin (PDS) sequencing (BSAS) conversion verted DNA bisulfite converted DNA adaptor HO HO by Tn5 transposase (ChIPmentation)
Uridine diphosphate
glucose (UDP-Glu)
MspI
Double-strand break TT TT
TT C C G G C C G G Chia-PET
TTTT
TTTT

TTTT

TT

BLESS
C C G G C C G G
Methyl-Seq
TT
TT
TT
TTTT

TT TT TT TT
TTTT

TTTT
TTTT

TT TT TT

HpaII
MRE-Seq
C C G G C C G G
Breaks labeling and enrichment on Genome DNA Biotinylated Ligate primer DNA isolation and Capture on streptavidin Distal Digest with DNA Methyl-seq and MRE-Seq use C C G G C C G G Chromatin interaction analysis by Sample fragmentation Immunoprecipitate Ligation Restriction enzyme digestion DNA
streptavidin and sequencing (BLESS) proximal primer random shearing beads primer I-SceI and PCR methyl-sensitive enzymes to Methylated sites Split sample Restriction Sequence Align sequences and Identified paired-end tag sequencing (ChIA-PET)
identify methylation patterns in the genome enzyme digest determine undigested sites methylation site

Double-strand break Hi-C

DSB-Seq Genome 3-C
Restriction sites
Map DNA double-strand
breaks (DSB-Seq)
Genome DNA TdT and biotinylated-dUTP DNA isolation and random shearing Capture on streptavidin beads DNA EpiRADseq Capture-C
Chromatin conformation capture Crosslink proteins and DNA Sample fragmentation Ligation PCR amplify ligated junctions DNA
dsOligo insert UMI Oligo tag Double digest restriction-site associated DNA Restriction Add barcoded adapters Second restriction digest with Add P2 Amplify DNA (3-C, Hi-C and Capture-C)
Oligo insert P5 Index 2 marker generation (ddRADseq) with a methyla- digestion methylation-sensitive HpaII adapter
GUIDE-seq Index 1 P7
tion-sensitive restriction enzyme (EpiRADseq)
Genome-wide, unbiased identification of Genome DNA dsOligo tag integrat- DNA isolation End repair UMI
PCR dsOligo-specific amplification DNA NG Cap-
DSBs enabled by sequencing (GUIDE-seq) ed in live cells Random shearing Adaptor ligation Displaced oligo ture-C
T-WGBS Next-generation Capture-C Formaldehyde fixation Restriction Ligation De-crosslink and Sonicate to 200 Add indexed PCR Hybridize biotinylated Streptavidin DNA
V(D)J antigen receptor region Tagmentation-based Methylated DNA Tagmentation Oligo with Displace oligo Bisulfite PCR DNA (NG Capture-C) capture bait pull down
Transposome with enzyme digestion extract DNA bp fragments sequencing adaptors
whole-genome bisulfite methylated Hybridize methylated conversion
EC-seq methylated adaptor
V3 J4 J5
V1 V2 V3 J5 J4 J3 J2 J1 V1 V2 V3 J5 J4 J3 J2 J1 V1 V2 J3 J2 J1
J3
V1 V2 V3 J5 J4 J3 J2 J1 sequencing (T-WGBS) adaptor adaptor and gap repair
V1 V2 J2 J1
V3 J4 J5

Excision circle sequencing Lymphocyte-specific recombination activat- Synapsis, cleavage and coding Resolved genomic Liberated excision circle with Align read pairs to
(EC-seq) ing gene (RAG) recombinase, bound genome end hairpin formation coding junction resolved signal junction reference genome 5hmc residues
4-C
O
O
JBP1-seq OH
HO
CH3 J-binding protein 1 sequencing Hydroxy-methyl- Transposomes Tagmentation T4-βGT Glucosylated 5-hmC JBP1-magnetic bead PCR DNA HO
O

Bubble-Seq CH3
(JBP1-seq), for genome-wide profiling
of 5-hydroxy-methylcytosine (5hmC)
ated DNA pull down HO
OH
O NH2 Chromatin conformation
capture circular (4-C)
Crosslink proteins and DNA Sample fragmentation Ligation Restriction digest Self-circularization
and Reverse PCR
DNA

OH N
O O O
Libraries of restriction fragments Bubble-containing Restriction Cast fragments in Run gel Recover bubble-con- DNA extraction Add sequencing DNA 5hmc residues N O US DS
that contain replication initiation fragment digest trapping gel taining plug primers
Aba-seq
HN Universal primer
Adaptor primer primer
UMI-4C
O O
sites (bubbles) in vivo R
N HN Pad
H AbaSI coupled with sequencing Hydroxy-methyl- T4-βGT Glucosylated 5-hmC AbaSI Biotinylated Ligate Fragment Streptavidin magnetic DNA Glucosylated 5hmc
Biotin (Aba-seq) to map high-resolution ated DNA primers bead pull down
O- O- O- O N O
gDNA gDNA O- O- O- O hydroxymethylome (5hmC) Targeted chromosome conformation Crosslink proteins HC DpnII Ligation Reverse crosslink and Sonicate Single end Nested PCR amplification DNA
NSCR RNA
primer SNS SNS O O O O
5mC 5hmC 5mC g5hmC 5cmC g5hmC N3 N3S-S Biotin
capture (4C) with unique molecular
identifiers (UMI-4C)
and DNA digestion proteinase K digestion adaptor ligation
g5hmC
5mc residue βGT TET g5hmC
Nascent strand capture and DNA replication bubble Denaturation and 5’-biotinylation Streptavidin pull-down RNase I Isolate and DNA
release (NSCR) size-selection digestion amplify SNS Digoxigenin TAmC-Seq C T C C G C T C C G C T C C G C T C C G C T C C G
T7 T3
Tet-assisted 5-methylcytosine βGT-catalyzed Biotinylation Streptavidin pulldown
sequencing (TAmC-Seq)
Glucosylation Oxidation
UDP-6-N3 glucosylation and DTT cleavage
DNA
5-C
BrdU
Nascent DNA
Repli Seq 5fc residue
C mC
5m 5h 5fc 5ca
C Blocked 5hmC residue 5hmC residue N3 N3S-S Biotin
Chromatin conformation capture Crosslink proteins and DNA Sample fragmentation Ligation LMA: Ligation-mediated amplification DNA
fC-Seal C C C C C C C C C C C C C C C C C C C C C C C C C carbon copy (5-C)
Repli Seq—to map temporally ordered Run-on with analog Sort cells and lyse Bead coated with Elute Purify DNA A 5-formylcytosine-selective βGT-catalyzed NaBH4 Convert 5fc to βGT-catalyzed Biotinylation Streptavidin pulldown DNA
replicating DNA anti-BrdU antibody End Repair chemical labeling (fC-Seal) approach glucosylation 5hmc UDP-6-N3 glucosylation and DTT cleavage
for genome-wide profiling of 5fC PB-seq
C mC C
5m 5h 5fc 5ca
Biotin
Nascent DNA 5fc residue N3 N 3S-S N 3SH Protein/DNA binding (PB–seq), to DNA-protein complex Purify DNA Shear DNA Hybridize with Isolate protein-bound DNA DNA
NS-seq fC-CET C C C C C C C C C C C C C C C C C C C C C C C T C determine the binding energy landscape DNA-binding protein DNA extraction
5fC based on selective chemical Azido 1,3-indandione DBCO-S-S-PEG3-biotin Pulldown, Adaptor ligation Purify DNA
Pu-seq Origin of replication Index 2 Index 1
labeling of 5fC and subsequent (AI) NaOH and DTT and PCR 5’ 3’
Nascent strand sequencing (NS-seq) to discover Digestion Purify C-to-T transition during PCR 3’ R 5’ U U U
DNA replication origin Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo) Amplify DNA
DNA replication origins and G4 structures Polymerase usage sequencing Alkali Klenow reaction(+ random Attach Uracil DNA glycosylase- PCR and DNA
C mC C
5m 5h 5fc 5ca (Pu-seq) treatment primer, dATP, dGTP,dCTP, dUTP) adaptors and DNA lyase (USER) purify
o-acylisourea Control C C C C C T C C T T
C mC C C mC C C mC
DNA Low-Level Detection True variant AI-mediated cyclization CAB-Seq 5caC residue C
5m 5h 5fc 5ca
C C C C C
5m 5h 5fc 5ca
C C C C
Nu S-S
C
5m 5h 5fc 5caC S-S
C C C C
Biotin
C C C C C
5caC S
T C C T C Break-seq
Breakage at replication fork
5’
3’
Break 3’
5’
5’
3’
3’
5’
Index 2 Index 1

Read1 Sample index labeling in fC-CET Chemical modification-assisted 1-ethyl-3-[3-dimethylamino-propyl]-car- Linker Streptavidin pulldown Bisulfite treatment Sequence
Degenerate Double-stranded break DNA trapped in End-repair with dGTP, dCTP, Elution and Streptavidin magnetic Attach PCR and DNA
Gene O bisulfite sequencing (CAB-Seq) bodiimide hydrochloride (EDC) chemistry and DTT cleavage PCR amplification labeling to map chromosome agarose gel dTTP, and biotinylated-dATP fragmentation bead pull down adaptors purify
smMIP molecular tag
Read2
Random error
NH2 for 5caC detection O
NH H O breaks (Break-seq)
C mC C
Single Molecule Molecular Genomic DNA Copy target sequence Exonuclease PCR amplification Align fragments from every Corrected N 5m 5h 5fc 5ca Bisulfite treatment HN OH NNNN
Inversion Probes (smMIPs) for unique molecular tag sequence C mC C T C C T T NNNN
N 5hmc residue 5m 5h 5fc 5ca Control C C C C C PCR amplification Breakage in genome
LAM-HTGTS
detecting low frequency targets O H S

Degenerate DNA
oxBS-Seq C C C C C 5fc Bisulfite treatment T C T T T DNA Biotin
Short tandem repeat (STR) Strain I Strain I Oxidative bisulfite sequencing KRuO PCR amplification Linear amplification-mediated Fragmentation by LAM-PCR with Streptavidin magnetic Adaptor ligation Nested Enzyme Tagged PCR
MIPSTR molecular tag Purify DNA
4 C C C C C
(oxBS-Seq) to map 5-methylcytosine high-throughput genome-wide sonication biotinilated primer bead pull down PCR blocking
Strain II Strain I N3
5fC and 5-hydroxymethylcytosine
Targeted capture of STR Targeted STR Copy target STR Amplify and sequence Natural variation between individuals Somatic variation within an individual C mC C sequencing (LAM-HTGTS)
5m 5h 5fc 5ca
loci by smMIPs (MIPSTR) Bisulfite treatment
C mC C
5m 5h 5fc 5ca C C C C C T C C T T A
5fc residue Control PCR amplification Breakage in genome
A
A

RedBS-Seq mC HTGTS
A
C C C C C
5h
A
A
3’ blocked random hexamer primers Nascent replication fork Reduced bisulfite sequencing NaBH Bisulfite treatment DNA
MDA caMAB-seq
O
Genome O 4 C C C C C T C C C T Linear amplification-mediated Fragmentation by End repair 3’ A addition Adaptor ligation Streptavidin magnetic Nested Tagged PCR Purify DNA
(redBS-Seq), to map 5-formylcyto- PCR amplification
sine (5fC) in DNA high-throughput genome-wide sonication and PCR bead pull down PCR
IMS-MDA Multiple displacement amplification (MDA). Hybridize primers Phi 29 Synthesis Phi 29 Synthesis S1 nuclease Amplified DNA N3 C mC
5m 5h 5fc 5ca
C sequencing (HTGTS)
Immunomagnetic separation for targeted Bisulfite treatment
MIDAS bacterial enrichment for MDA (IMS-MDA) 5fc residue
C mC
5m 5h 5fc 5ca
C
Control
C C C C C
PCR amplification
T C C T T Forward sequencing adaptor
Sequencing primer pD40htSELEX
Fusion
Microwell displacement amplification Transcription factor binding site Barcode to identify sample
fCAB-Seq SELEX
Hybridize primers Synthesis C C C C C protein Luciferase
system (MIDAS) O Blocked 14N with all possible combinations DNA binding region
27-bp common sequence N
5fC chemically assisted bisulfite Bisulfite treatment DNA Reverse sequencing adaptor Luciferase

Genome
8 random nucleotides N sequencing (fCAB-seq) method for
O-ethylhydroxylamine (EtONH2) C C C C C
PCR amplification
T C C C T
SELEX-seq High-throughput systematic Target Ligand
Streptavidin binding peptide
Expression vector Fusion protein Matching ligand Wash and Recovered PCR and Binding
the base-resolution detection of 5fC
MALBAC Cycles of quasilinear
amplification
N O C mC
5m 5h 5fc 5ca
C HT-SELEX evolution of ligands by exponential
enrichment (HT-SELEX)
sequence immobilized in well binds elute matching ligand Sequence site
Partial amplicons C mC C Bisulfite treatment T C C T T N3
5m 5h 5fc 5ca
DNA
Multiple annealing and looping-based Hybridize primers Bst DNA Denature Looped full PCR DNA 5fC/5caC residues Control
C C C C C
PCR amplification
amplification cycles (MALBAC) polymerase amplicons 5fC-AI 5m
C O
MAB-seq
C C C C C HO
Template Bisulfite treatment O
Denature M.SssI methylase-assisted bisulfite M.SssI C C C C C C C C T T DNA HO
OH
NH2
Protein binding site Klenow
Single cell genome sequencing (MAB-seq) to map 5fC/5caC
PCR amplification
N
HiTS-FLIP Target sequence
Binding site
H
C mC C
Cell 1 S
5m 5h 5fc 5ca
nuc-seq Flowcell
O
H
N C mC C Bisulfite treatment T C C T T N O
5m 5h 5fc 5ca
Cell 2 N C C C C C
5fC/5caC residues Control PCR amplification High-throughput sequencing: Prepare sequencing libraries and Hybridize Synthesize second Add fluorescent- Hybridize Elute with increas- Scan flowcell
SNES Cell 3
O
5m
C fluorescent ligand interaction sequence first strand primer strand with unmodi- ly labeled ligand ing stringency
RRMAB-seq
C C C C C R
N Bisulfite treatment DNA N3-5GMC profiling (HiTS-FLIP) Remove second strand DNA fied nucleotides
Reduced representation M.SssI Digest Add methylated M.SssI C C C C C C C C T T
Single G2/M nucleus sequencing of Cell sorting from Lyse cell Nucleus Phi 29 Limited amplification Synthesis S1 nuclease DNA N N
methylase-assisted bisulfite PCR amplification
cells in S phase (nuc-seq). Single G2/M distribution with MspI adapters
sequencing (RRMAB-seq) DNA adenine
nucleus exome sequencing (SNES) DNA-protein interaction methyltransferase (DAM) Specific and non-targeted
C mC C C methylation DpnI DpnII PCR
mC 5ca 5g 5ca 5ca
Fusion protein
O

Fragment and add single adaptors Sequencing Primers

N
5hmc residue C mC
5m 5h 5fc 5ca
C βGT 5g TET Bisulfite treatment DamID
OS-Seq Gene Target sequence Single adaptor library N
TAB-Seq C C C C C C C C C C
C C C C C
PCR amplification
T T C T T Protein of interest
DAM
Non-targeted methylation
N O TET-assisted bisulfite sequencing, (TAB-Seq) Glucosylation Oxidation DNA DNA adenine methyltransferase Create fusion Split sample DpnI digestion Adaptor ligation Unmethylated GATCs Align sequences and determine
Target sequence
Oligonucleotide-selective Sequence 5fC-AI-SH DNA to map 5-hydroxymethylcytosine interaction detection (DamID) protein are cut by DpnII differentially digested sites
sequencing (OS-Seq) captures Adaptor sequence C G C
and sequence gene targets on C C
5mc CpG island 5m 5m C G C C G C
the flow cell
Flow cell Create target-specific oligos Extend and Hybridize Extend and Hybridize Extend and Sequence reads
MIRA C G C G C G C C G C G C G C G C G C G
MPE-seq
Denature Denature Denature 1 and 2 Methylated-CpG island Fractionate MBD2B/MBD3L1 Isolate on glutathione-coated DNA purification DNA
recovery assay (MIRA) protein complex beads PCR amplification
Methidiumpropyl-EDTA sequencing Active chromatin Isolated nuclei MPE digestion Add bathophenanthroline DNA extraction DNA
Very rare mutation Mutation (MPE-seq)
Safe-SeqS
Safe-sequencing system is a unique DNA Shear Randomly sheared Adaptor ligation Amplify and solid Sequence Align sequences and True MeDIP-Seq Streptavidin
molecular identifier (UMI) approach ends serve as UMIs phase capture determine actual ratio mutant
to detect rare variants (Safe-SeqS) DIP-seq Add biotinylated Harvest cells and
Methylated DNA Immunoprecipitation
(MeDIP-Seq), DNA immunoprecipitation followed
Methylated DNA Extract DNA Fractionate
Denature
Immunoprecipitate DNA purification DNA Pyridine Chem-seq in vivo
compound fragment DNA
Very rare mutation P5 P7 Random error True variant
α β by high throughput sequencing (DIP-seq))
Duplex-Seq Crosslink
P7 in vitro Enrich DNA DNA
12 random 12 random P5 Identify sites bound by small DNA-protein complex with Add biotinylated DNA
base index base index chemical molecules (Chem-seq) putative drug binding site compound fragments extraction
Consensus
Duplex sequencing detects rare A mutation occurs Add Ligate and PCR Sequence Create single strand Create duplex sequences Rare variant
hMeDIP-Seq
Protein-Protein Interactions
mutations by sequencing and on both strands adaptors consensus sequence from based on molecular tags
aligning both strands of the DNA every unique molecular tag and sequencing primers
Hydroxymethylated DNA immunopre- Hydroxymethylated Extract DNA Fractionate Immunoprecipitate DNA purification DNA
Single cell RNA RNA cipitation combined with next genera- DNA Denature Pyridine
NH2
2nd strand tion DNA sequencing (hMeDIP-seq) Protein target NH2 NH2 K2CO3 NH Hybridize NH2
PD-Seq
AA(A)n AA(A)n AAAAAAA cDNA amplification
DR-Seq synthesis
TTTTTTTTTT
DNA DNA O DMF
PCR and Remove Wash
adaptors gDNA amplification MBDCap-seq
Genome DNA and mRNA Single Lyse cell RT with barcoded primer Ad-2 Quasilinear Split Sequence O- PD-Seq identifies candidate Beads Polyethylene glycol Immobilized RNA from cells Create phage display Repeat cycle three Protein-protein PCR phage DNA
sequencing (DR-Seq) cell primer amplification samples O Methyl- MBD Biotin cellular targets for proteins linker added protein cDNA library times interaction insert
Cap-seq
Streptavidin N
N O- N
Single cell RNA
AA(A)n
RNA
AA(A)n AAAAAAA On-bead transcriptome
N
M
O- MBD-Seq Methyl-CpG binding domain-based capture and Methylated Extract DNA Fractionate Capture biotinylated MBD on Elute with increasing DNA DNA
N N
ProP-PD Protein target Hybridize
2
TTTTTTTTTT amplification with Smart-seq2 sequencing (MBDCap-seq). Capture of methylated DNA Streptavidin coated magnetic beads salt concentration purification
G&T-seq
AAAAAAA
DNA DNA
TTTTTTTTTT
Whole genome amplification MiGS DNA using the MBD domain of MeCP2 (Methyl- PDZ-Seq 1
O Cap-Seq). MBD-isolated Genome Sequencing (MiGS)
with MDA O- Proteomic peptide-phage Identify C-terminal Create oligo Construct phage Bait proteins immobi- Select phages against Isolate and Peptide
H3C display (ProP-PD) to identify sequences library display library lized on 96-well plate baits sequence counts
Genome and transcriptome Cell Isolate single Lyse cell Streptavidin magnetic bead Separate the DNA and the RNA Sequence Align RNA and CH3
sequencing from a single suspension cell with mRNA capture primer genome O O short linear motif (SLiM) interac-
T C T C G tions or PDZ domains (PDZ-Seq)
cell (G&T-seq) Bisulfite
EDTA
BisChIP-Seq
Key
T U T C G
PCR Display methods on
ChIP-BS-seq T T T C G mobile device
Bisulfite-treated chromatin immuno- DNA-protein complex with Sonicate Immunoprecipitate Purify DNA Bisulfite conversion DNA
Yellow highlights indicate the target of the protocol precipitated DNA (BisChIP-seq) and methylated histones and and shear
ChIP-BS-seq), to correlate protein methylated DNA
modifications with DNA methylation

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

TruSeq PCR Free TruSeq Nano TruSeq Custom Amplicon TruSeq RNA TruSeq Small RNA TruSeq RNA Stranded TruSeq RNA Access TruSeq Targeted RNA Expression Nextera Library Preparation Nextera Rapid Capture Nextera Mate Pair
Double-stranded DNA Double-stranded DNA 5’ 3’ RNA Target Transposase
Double-stranded DNA 5’ 3’ Small RNA fragment Target Total RNA Target
AAAAA mRNA Random primer Transposase Denatured and R R Biotinylated junction adapter R R
Fractionate Pool stranded pooled fragments DNA
Fractionate Ligate adaptors dT TP + dC TP + dATP + dGTP Create cDNA RNA-Seq libraries from Nextera library
Size select cDNA
Size select Region of interest cDNA DNA Tagmentation
5’ Adapter 3’ Adapter
R R
End repair
End repair AAAAA Create second Biotinylated R R
Phosphorylate TTTTT polyA select dUTP + dC TP + dATP + dGTP ULSO DLSO
Add custom
Phosphorylate Add primer strand cDNA Biotinylated 5’ P target probe
Sense strand U primers
P
P
P
Custom Custom U U U U U U U UU U
target probe R
P Probe 1 Probe 2 Fragment End repair R
A-overhang Add custom probes A Phosphorylate 5’ P
Hybridization
A-overhang P Tagmentation Hybridize probes Circularize
A Reverse transcription Sense strand A-overhang Hybridize probes to to targets R R
P
Random hexam- AU U U U U U U UU U P
targets
A P
P A er ~300bp
R R

Add Adaptors Extension-Ligation Fragment

A P Add Adaptors P5 P7 P5 P7
T P Index 2 Index 1
Index 2 Index 1 Extension and ligation Adaptor ligation
Index 1 P T
Index 2 Index 1 U U U U U U U UU U Index 2
Capture on Capture on
P5 P7
P7 P5
P5
P7 Sense strand P5
streptavidin P5 magnetic beads Isolate biotinylated
T Adaptor ligation magnetic beads P5
Index
P
Index
Adaptor ligation
Index 2 fragment
P T P5 P7 P7 Denature and amplify Index 2
P7 P5 Index 2 Index 1
First and Index 1 P5 P7 Index 1
Denature and Index 1
P5 P7
Index 1 Index 2
Index 1 Add sequencing second strand P7 Denature and amplify P7 R
Index 2
primers synthesis
P7
U U U U U U U UU U P5 P7 Adaptor ligation
P7 P5 P5 Sense strand Block polymerase amplify Amplification Elute
R
Elute P7 P5
Denature and
P5 P7 Denature and amplify PCR amplify
Index Index 2 Target Index 1
Index 2 Target Index 1 Index 2 Index 1
Index
Product ready for Index 2 Index 1
Index 2 Index 1 P5 P7 Product ready for Index 2 Index 1
Product ready for Index 2 Target Index 1
Product ready for Product ready for P5 P7 Product ready for P5 P7 Product ready for Product ready for
Index P5 P7 Product ready for Product ready for
P5 P7 P5 P7 P5 P7
cluster generation
P5 P7
cluster generation
P7 cluster generation P5 P7
cluster generation cluster generation cluster generation cluster generation cluster generation
P5
cluster generation cluster generation

Moleculo
Sequencing by Synthesis
~10kb Sheared genomic DNA

End repair

Sequence A A A A A
Read 2 A A A A A
Adapter ligation
Forward Reverse primer
A
T
T
A
T
T
A
T
T
A
T
T
A
T
T
primer A
T
T
A
T
T
A
T
T
A
T
T
A
T
T Generate clonal pools
Forward Reverse strand strand C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
Amplify
Transposase
C C C C C C C C C C

strand strand

Index 1
A
A
T Tagmentation
primer A
A
T
T
C
G
T C
C
G
C

~600bp
P5 P5 P5 Add indices
Index 2 Index 2 Index 2
Adapter hybrid- Reverse Remove Fold over and Synthesize Thousands of molecules are The reverse With each cycle, four fluores- The read Sequence The read Fold over and Deblock P5 Sequence Synthesize The forward- The second Index 1 Index 1 Index 1
izes to flowcell strand forward hybridize to second strand amplified in parallel strand is cently tagged nucleotides product is Index1 product is hybridize to primer and Index2 second strand is read is P7 P7 P7
syntesis strand second primer Bridge amplification cleaved and compete for addition to the washed away washed away first primer add unlabeled strand cleaved and sequenced P5 P7
washed away growing chain. Only one is bases washed away P5 P7 Prepared fragments Pool and purify
P5 P7
incorporated based on the
sequence of the template.

FOR RESEARCH USE ONLY

This 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