DNA · oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937 PAT–ChIP Fanelli M et al. (2011)...
Transcript of DNA · oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937 PAT–ChIP Fanelli M et al. (2011)...
FDNA
or all you seq...
2b-RAD Wang S et al. (2012) Nat Methods 9:808-8103C Duan Z. et al. (2012) Methods 58: 277-2884-C Zhao Z. et al. (2006) Nat Genet 38: 1341-13475-C Dostie J. et al. (2007) Nat Protoc 2: 988-1002Aba-seq Sun Z. et al. (2013) Cell Rep 3: 567-576ATAC-Seq Buenrostro J. D. et al. (2013) Nat Methods 10:
1213-1218BisChIP-Seq Statham A. L. et al. (2012) Genome Res 22:
1120-1127Bisulfite-seq Berman B. P. et al. (2012) Nat Genet 44: 40-46BLESS Crosetto, N. et al. Nat. Methods 10, 361–365 (2013).Break-seq Hoffman E. A. et al. (2015) Genome Res 25: 402-412BSAS Masser D. R. et al. (2013) Epigenetics Chromatin 6:
33BSPP Deng J. et al. (2009) Nat Biotechnol 27: 353-360BS-Seq Lister R. et al. (2009) Nature 462: 315-322
Bubble-Seq Mesner L. D. et al. (2013) Genome Res 23: 1774-1788
CAB-Seq Lu X. et al. (2013) J Am Chem Soc 135: 9315-9317caMAB-seq Wu H et al. (2016) Nat Protoc 11:1081-100CAP-seq Illingworth R. S. et al. (2010) PLoS Genet 6:
e1001134Capture-C Hughes J. R. et al. (2014) Nat Genet 46: 205-212CATCH-IT Deal R. B. et al. (2010) Science 328: 1161-1164Chem-Seq Anders L. et al. (2014) Nat Biotechnol 32: 92-96Chia-PET Li G. et al. (2010) Genome Biol 11: R22ChIP-BS-seq Brinkman A. B. et al. (2012) Genome Res 22:
1128-1138ChIP-exo Yen K. et al. (2013) Cell 154: 1246-1256ChIPmentation Schmidl C et al. (2015) Nat Methods 12:963-5ChIP-Seq Barski A. et al. (2007) Cell 129: 823-837CPT-seq Amini S. et al. (2014) Nat Genet 46: 1343-1349DamID Vogel M. J. et al. (2007) Nat Protoc 2: 1467-1478
ddRADseq Peterson B. K. et al. (2012) PLoS One 7: e37135Digenome-seq Kim D et al. (2015) Nat Methods 12:237-43DIP-seq Shen L. et al. (2013) Cell 153: 692-706Dnase I SIM Cumbie JS et al. (2015) Plant Methods 11:42DNaseI-Seq Hesselberth J. R. et al. (2009) Nat Methods 6:
283-289DNase-Seq Boyle A. P. et al. (2008) Cell 132: 311-322Drop-ChIP Rotem A et al. (2015) Nat Biotechnol 33:1165-72DR-Seq Dey SS et al. (2015) Nat Biotechnol 33:285-9DSB-seq Baranello L et al. (2014) Int J Mol Sci 15:13111-22Duplex-Seq Schmitt M. W. et al. (2012) Proc Natl Acad Sci U S A
109: 14508-14513EC-seq Parkinson N. J. et al. (2015) Genome Res 25: 226-234EpiRADSeq Schield DR et al. (2016) Methods in Ecology and
Evolution 7:60-69FAIRE-Seq Gaulton K. J. et al. (2010) Nat Genet 42: 255-259fCAB-Seq Song C. X. et al. (2013) Cell 153: 678-691
fC-CET Xia B et al. (2015) Nat Methods 12:1047-50fC-Seal Song C. X. et al. (2013) Cell 153: 678-691FiT-Seq Cejas P et al. (2016) Nat Med G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522G4-seq Chambers V. S. et al. (2015) Nat Biotechnol 33:
877-881GUIDE-seq Tsai SQ et al. (2015) Nat Biotechnol 33:187-97HELP-Seq Oda M. et al. (2009) Nucleic Acids Res 37: 3829-3839Hi-C Lieberman-Aiden E. et al. (2009) Science 326:
289-293Histone meth. Barski A. et al. (2007) Cell 129: 823-837HiTS-Flip Nutiu R. et al. (2011) Nat Biotechnol 29: 659-664hMeDIP-seq Xu Y et al. (2011) Mol Cell 42:451-64HT-ChIP Blecher-Gonen R. et al. (2013) Nat Protoc 8: 539-554HTGTS Chiarle, R. et al. (2011) Cell 147, 107–119 HT-SELEX Jolma A. et al. (2010) Genome Res 20: 861-873
HydEn-seq Clausen A. R. et al. (2015) Nat Struct Mol Biol 22: 185-191
hyRAD Suchan T et al. (2016) PLoS One 11:e0151651Ig-Seq Vollmers C. et al. (2013) Proc Natl Acad Sci U S A
110: 13463-13468IMS-MDA Seth-Smith H. M. et al. (2013) Nat Protoc 8:
2404-2412INSeq Goodman AL et al. (2009) Cell Host Microbe 6:279-89JBP1-seq Cui L. et al. (2014) Genomics LAM-HTGTS Hu J et al. (2016) Nat. Protocols 11:853-871MAB-seq Wu H. et al. (2014) Nat Biotechnol MAF Khan TA et al. (2016) Sci Adv 2:e1501371MAINE-Seq Ponts N. et al. (2010) Genome Res 20: 228-238MALBAC Zong C. et al. (2012) Science 338: 1622-1626MBDCap-Seq de Assis S. et al. (2012) Nat Commun 3: 1053MBD-Seq Nair S. S. et al. (2011) Epigenetics 6: 34-44MDA Dean F. B. et al. (2001) Genome Res 11: 1095-1099
MeDIP-Seq Down T. A. et al. (2008) Nat Biotechnol 26: 779-785MethylCap-Seq Brinkman A. B. et al. (2010) Methods 52: 232-236Methyl-seq Brunner A. L. et al. (2009) Genome Res 19:
1044-1056MIDAS Gole J et al. (2013) Nat Biotechnol 31:1126-32MiGS Serre D. et al. (2010) Nucleic Acids Res 38: 391-399MINCE-seq Ramachandran S et al. (2016) Cell 165:580-92Mint-ChIP van Galen P et al. (2016) Mol Cell 61:170-80MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761MIRA Rauch T. A. et al. (2010) Methods 52: 213-217MNase-Seq Schones D. E. et al. (2008) Cell 132: 887-898MPE-seq Ishii H et al. (2015) Proc Natl Acad Sci U S A
112:E3457-65MRE-Seq Maunakea A. K. et al. (2010) Nature 466: 253-257NG Capture-C Davies JOJ et al. (2016) Nat Meth 13:74-80NOME-Seq Han H. et al. (2011) Hum Mol Genet 20: 4299-4310NSCR Kunnev D. et al. (2015) Genome Res 25: 558-569
NS-seq Foulk MS et al. (2015) Genome Res 25:725-35Nucleo-Seq Valouev A. et al. (2011) Nature 474: 516-520nuc-seq Wang Y. et al. (2014) Nature 512: 155-160ORGANIC Zentner G. E. et al. (2013) PLoS Genet 9: e1003317OS-Seq Myllykangas S. et al. (2011) Nat Biotechnol 29:
1024-1027oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937PAT–ChIP Fanelli M et al. (2011) Nat Protoc 6:1905-19PBAT Miura F. et al. (2012) Nucleic Acids Res 40: e136PB-seq Guertin M. J. et al. (2012) PLoS Genet 8: e1002610PD-Seq Arango D. et al. (2013) Proc Natl Acad Sci U S A 110:
E2153-2162PDZ-Seq Ernst A. et al. (2010) Mol Biosyst 6: 1782-1790PE RAD-Seq Willing E. M. et al. (2011) Bioinformatics 27:
2187-2193ProP-PD Ivarsson Y. et al. (2014) Proc Natl Acad Sci U S A 111:
2542-2547
Pu-seq Daigaku Y. et al. (2015) Nat Struct Mol Biol 22: 192-198
RAD Baird N. A. et al. (2008) PLoS One 3: e3376Rapture Ali OA et al. (2016) Genetics 202:389-400RBBS Meissner A et al. (2008) Nature 454:766-70RC-Seq Baillie J. K. et al. (2011) Nature 479: 534-537RedBS-Seq Booth M. J. et al. (2014) Nat Chem 6: 435-440Repli-Seq Hansen R. S. et al. (2010) Proc Natl Acad Sci U S A
107: 139-144Rep-Seq Benichou J. et al. (2012) Immunology 135: 183-191Ribose-seq Koh K. D. et al. (2015) Nat Methods 12: 251-257RRBS Meissner A. et al. (2008) Nature 454: 766-770RRMAB-seq Neri F. et al. (2015) Cell Rep 10: 674-683Safe-SeqS Kinde I et al. (2011) Proc Natl Acad Sci U S A
108:9530-5scABA-seq Mooijman D et al. (2016) Nat Biotechnol advance
online publication
scATAC-seq Cusanovich DA et al. (2015) Science 348:910-4(Cell index)scATAC-seq Buenrostro J. D. et al. (2015) Nature 523: 486-490(Microfluidics) scBS-seq Smallwood S. A. et al. (2014) Nat Methods 11:
817-820scChIP-seq Rotem A et al. (2015) Nat Biotechnol 33:1165-72scM&T-seq Angermueller C et al. (2016) Nat Methods advance
online publicationscMT-Seq Hu Y et al. (2016) Genome Biol 17:88scRC-Seq Upton KR et al. (2015) Cell 161:228-39scRRBS Guo H. et al. (2015) Nat Protoc 10: 645-659scTrio-seq Hou Y et al. (2016) Cell Res 26:304-19SELEX Jolma A. et al. (2010) Genome Res 20: 861-873SELEX-seq Slattery M. et al. (2011) Cell 147: 1270-1282SLAF-Seq Sun X et al. (2013) PLoS One 8:e58700SMDB Lan F et al. (2016) Nat Commun 7:11784
smMIP Hiatt J. B. et al. (2013) Genome Res 23: 843-854SNES Leung M. L. et al. (2015) Genome Biol 16: 55Sono-Seq Auerbach R. K. et al. (2009) Proc Natl Acad Sci U S A
106: 14926-14931SSB-Seq Baranello L et al. (2014) Int J Mol Sci 15:13111-22TAB-Seq Yu M. et al. (2012) Cell 149: 1368-1380TAmC-Seq Zhang L. et al. (2013) Nat Commun 4: 1517TC-Seq Klein I. A. et al. (2011) Cell 147: 95-106THS-seq Sos BC et al. (2016) Genome Biol 17:20TN-Seq van Opijnen T. et al. (2013) Nat Rev Microbiol 11:
435-442T-WGBS Wang Q. et al. (2013) Nat Protoc 8: 2022-2032UMI-4C Schwartzman O et al. (2016) Nat Methods advance
online publicationWGBS Lister R. et al. (2009) Nature 462: 315-322X-ChIP-seq Skene P. J. et al. (2014) Elife 3: e02042
References
TruSeq RNA
AAAAA mRNA
T T T T TAAAAA polyA select
Fragment
Illumina, Inc. • 5200 Illumina Way, San Diego, CA 92122 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • [email protected] • illumina.com
FOR RESEARCH USE ONLY
© 2015 Illumina, Inc. All rights reserved.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
MoleculoSheared genomic DNA
End repair
Adapter ligation
Prepared fragments
~10kb
P5
Index 2
Index 1
P7
P5
Index 2
Index 1
P7
Transposase
Tagmentation
~600bpP5
Index 2
Index 1
P7
P5 P7P5 P7P5 P7
Generate clonal poolsAmplify
Add indices
Pool and purify
Sequencing by Synthesis
TruSeq PCR Free
Double-stranded DNA
FractionateSize select
A-overhang
End repairPhosphorylate
PP
A
AP
P
T
P
P5
P7
IndexT
P
P5
P7
Index Adaptor ligation
P5
P5P7Index
P7
Index
Add Adaptors
Product ready for cluster generation
TruSeq NanoDouble-stranded DNA
FractionateSize select
A-overhang
End repairPhosphorylate
PP
A
AP
P
T
P
P5
P7
Index 1Index 2
Index 2Index 1
T
P
P5
P7
Adaptor ligation
Denature and amplify
Add Adaptors
P5
P7
Index 1Index 2
Index 2Index 1
P5
P7
P5 P7Index 1Index 2
Double-stranded DNA
Product ready for cluster generation
TruSeq Custom Amplicon
Add custom probes
Region of interest
Double-stranded DNADouble-stranded DNA
Add custom probes
Extension and ligation
Add sequencing primers
Index 2Index 1
P5
P7
Index 1P7
Index 2P5
Custom Probe 1
Custom Probe 2
PCR
Product ready for cluster generation
TruSeq Small RNA
3’5’ Small RNA fragment
Ligate adaptors
Add primer
Reverse transcription
Denature and amplify
5’ Adapter 3’ Adapter
P7
Index 1
P5
P5 P7Index
Product ready for cluster generation
TruSeq RNA Stranded5’ 3’ RNA
Random primer
cDNACreate cDNA
Create second strand cDNA
dUTP + dCTP + dATP + dGTP
dT TP + dCTP + dATP + dGTP
End repairPhosphorylateA-overhang
Adaptor ligation
Denature and amplify
P5 P7
P5 P7Index 1Index 2
U U U UUUUUUUUSense strand
A
AP
PU U U UUUUUUUSense strand
P7
Index 1
P5Index 2
U U U UUUUUUU Index 2
P5
Index 1
P7
Sense strand
P5P7U U U UUUUUUU
Sense strand Block polymerase
Product ready for cluster generation
Nextera Library Preparation
Transposase
DNA
~300bp
Tagmentation
Ampli�cation
P5 P7Index 1Index 2
P5
Index 2
Index 1
P7
Product ready for cluster generation
Nextera Mate Pair
Adaptor ligation
Isolate biotinylatedfragment
Denature and amplify
P5 P7
Transposase
DNA
Tagmentation
Circularize
R R R RBiotinylated junction adapter
R
R
R
R
R
R
Fragment
R
R
R
R
P5
P7 P5
P7R
R
Product ready for cluster generation
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
Synthesize second strand
The second read is sequenced
Sequence Index2
AATTCGC
Deblock P5 primer and add unlabeled bases
Read 2 primer
The forward-strand is cleaved and washed away
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
AATTCGC
Adapter hybrid-izes to flowcell
Reverse strand syntesis
Reverse strand
Forward strand
Remove forward strand
Fold over and hybridize to second primer
Synthesize second strand
The reverse strand is cleaved and washed away
With each cycle, four fluores-cently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template.
The read product is washed away
Thousands of molecules are amplified in parallel
Reverse strand
Forward strand
Bridge amplification
Sequence primer
Fold over and hybridize to first primer
Fold over and hybridize to first primer
Sequence Index1
Index 1 primer
The read product is washed away
CH3O CH3
Nextera Rapid Capture
Elute
Target
TargetP5 P7
Index 1Index 2 Product ready for cluster generation
Denatured and pooled fragments from Nextera library
Capture on magnetic beads
Hybridize probes to targets
Biotinylated target probe
TruSeq RNA Access
Elute
Target
TargetP5 P7
Index 1Index 2 Product ready for cluster generation
Pool stranded RNA-Seq libraries
Biotinylated target probe
Hybridize probes to targets
Capture on streptavidin magnetic beads
Random hexam-er
First and second strand synthesis
TruSeq Targeted RNA ExpressionTarget
ULSO DLSO
Total RNA
cDNA
Hybridization
P7
Index 1
P5
P5 P7
5’ P
5’ P
Index 2
Target Index 1Index 2 Product ready for cluster generation
Add custom primers
Denature and amplify
Extension-Ligation
O O
OH
NH
HN
H
H S
Biotin
This poster was compiled by the Illumina Scienti�c A�airs. 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 Scienti�c A�airs with any questions, comments, or suggestions.
EDTAO
O
O-O
O
O-
O-
O-
N
NM
Display methods on mobile device
N
NN
N
CH3O CH3
Display methods on mobile device
N
NN
N
DNA Low-Level Detection
DNA Rearrangements and Markers
Duplex-Seq α βVery rare mutation
Duplex sequencing detects rare mutations by sequencing and aligning both strands of the DNA
P5
P7 P5
P7
A mutation occurs on both strands
12 random base index
12 random base index
True variantRandom error
Ligate and PCR Rare variantSequence Create single strand consensus sequence from every unique molecular tag
ConsensusCreate duplex sequences based on molecular tags and sequencing primers
Add adaptors
Ig-seqRep-SeqMAF
CDR3 junction region V D J Constant
DNADNA sequencing of immunoglobulin genes (Ig-seq), repertoire sequencing (Rep-Seq), and molecular ampli�cation �ngerprinting (MAF)
Extracted RNA Reverse transcription8N UID
8N UID
Second strand synthesis PCR Purify
CpG island
CAP-seqDNACXXC a�nity puri�cation plus
deep sequencing (CAP-seq)CXXC bound to nickel-charged sepharose beads
Hybridize to sepharose column
CXXC
CXXC
CXXC
CXXC
CXXC
Methylated CpG
Unmethylated CpG
CXXC
CXXC
CXXC
CXXC CXXCX
Elute unmethylated CpG enriched fragments
TC-SeqAID-dependent rearrangement
Infect I-Sel Puri�cation
I-SceI site+AID
-AID AA
AA
DNAGenomic DNATC-Seq: translocation capture sequencing
Sonicate, blunt and A-tail
Ligate linkersCut I-Scel
Semi-nested PCRLinker cleavage
RC-Seq
Genomic DNA Fractionate DNA fragments
Hybridize
Read1
Read2Transposon sites
Sequenced fragment
Reference sequence Align
Novel retrotrans-position events
Retrotransposon binding sites Known
retrotrans-poson
Novel retrotransposition events
Microarray with transposon binding sites
RC-Seq: Retrotransposon capture sequencing
TN-SeqINSeq
Transposon Transposon
20bp
MmeI
MmeIMmeI digestion Add adapters
20bp
PCR and sequenceTransposon sequencing (TN-Seq) and insertion sequencing (INSeq)
Inverted MmeI recognition site
MmeI recognition site Transposon
insertion sites
MALBACGenome
Hybridize primers PCR
27-bp common sequence8 random nucleotides
Partial amplicons
Template
Denature
Denature
Hybridize primers Synthesis
Multiple annealing and looping-based ampli�cation cycles (MALBAC)
DNA
Cycles of quasilinear ampli�cation
Looped full amplicons
Bst DNA polymerase
OS-Seq Gene
Target sequence
Adaptor sequence
Flow cell
Sequencing Primers
Target sequence Single adaptor library
Hybridize Hybridize
SequenceOligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on the �ow cell
Create target-speci�c oligos Extend and Denature
Extend and Denature
Extend and Denature
Sequence reads 1 and 2
Fragment and add single adaptors
CPT-Seq
Divide sample into 96 reactions
Dilute and divide into 96 reactions
SDS PCR DNAIndexed transpo-some reactions
EDTAPool
Contiguity-preserving transposition sequenc-ing (CPT-seq)
Genome
Indexed PCR primers
EC-seqV(D)J antigen receptor region
Excision circle sequencing (EC-seq)
Lymphocyte-speci�c recombination activat-ing gene (RAG) recombinase, bound genome
V1 V2 V3 J5 J4 J3 J2 J1 V1 V2 V3 J5 J4 J3 J2 J1
J5J4
J3 J2 J1V1V2
V3
Synapsis, cleavage and coding end hairpin formation
Resolved genomic coding junction
Liberated excision circle with resolved signal junction
Align read pairs to reference genome
V1 V2 J3 J2 J1
V3 J5J4
V1 V2 V3 J5 J4 J3 J2 J1
HydEn-seqGenome Ribonucleotides
Hydrolytic end sequencing (HydEn-seq) to reveal replicase- and strand-speci�c patterns of ribonucleotides in the genome
Alkaline hydrolysis (KOH)
Phosphorylation (T4 PNK 3-phosphatase minus)
Ligate oligo with 5’-amino- terminated C6 spacer
Add sequencing primers, PCR and sequence
Map locations on the genome
5’ 3’3’ 5’
R R5’HO 5’P-O
2’,3’-cyclic phosphate R R
Genomic DNA
GenesmMIP
Copy target sequence Exonuclease Corrected sequence
Align fragments from every unique molecular tag
Sample indexRead1
Read2
True variant
Random errorSingle Molecule Molecular Inversion Probes (smMIPs) for detecting low frequency targets
PCR ampli�cation
Degenerate molecular tag
Targeted STR
Short tandem repeat (STR)MIPSTRCopy target STR Amplify and sequenceTargeted capture of STR
loci by smMIPs (MIPSTR)
Degenerate molecular tag Strain I
Strain IIStrain I
Strain INatural variation between individuals Somatic variation within an individual
G4-seqPotential G-quadruplexGenome
Target sequence
K+ or PDS
Determine the location of potential G-quadruplexes in DNA (G4-seq)
Hibridize sequencing primer
Hibridize sequencing primer
Read reference sequence
Denature and remove read fragment
Add K+ or PDS to stabilize G4 regions
Compare sequence reads 1 and 2
Fragment and create library on �ow cell
Sequencing Primer
Read stabilized squence
Polymerase stalls
nuc-seqSNES
Cell 1
Cell 2
Cell 3
Cell sorting from G2/M distribution
Lyse cell NucleusSingle G2/M nucleus sequencing of cells in S phase (nuc-seq). Single nucleus exome sequencing (SNES)
Single cell genome
Phi 29 Limited ampli�cation S1 nucleaseSynthesis DNA
Ribose-seqGenome with ribonucleotides
Detect ribonucleotides embedded in DNA (Ribose-seq)
Fragmented genomic DNA
DNA for sequencing
R R
dA tailing Adaptor ligation Alkali treatment Self-ligation byAtRNL
Degradation oflinear ssDNA
Remove 2’-phos-phate and PCR
R5’ P
P 5’
R5’ P
P 5’A
A
RA
AmAT
TAm
AmA TA Am
RT
AmA TA Am
R
T
R
T
R
T
A
mA TA
Am2’P 2’P
RT
Epigenetics
Methylated DNA
DNA
RRBSscRRBS Reduced representation bisul�te
sequencing (RRBS-Seq). Single cell RRBS (scRRBS)
Methylated regions
Methylated adapter
End repair and ligation
Bisul�te conversion
Converted fragments PCRPCRMspI digestion
PBATPost-bisul�te adaptortagging (PBAT)
Methylated DNA Capture �rst strand on Streptavi-din coated magnetic beads
Second random priming
Streptavidin
BiotinAdaptor
Bisul�te conversion
First random priming
AdaptorRandom primer 2
Generate second strand DNA with adaptorsElution
BS-SeqBisulfite-seqWGBS
DNAShear DNAMethylated DNA Bisul�te conversionBisul�te conversion of genomic DNA (bs-Seq) or whole-genome bisul�te sequencing (WGBS)
C GTCT
C GTUT
Bisul�te
C GTTT
PCR
BSPPBisul�te sequencing with padlock probes (BSPP)
CpG island Bisul�te conversion
Bisu�te-converted DNA
HybridizePadlock probe
End repair and adaptor ligation
Extension and ligation
Exonuclease digestion
P1 P2H1 H2
AluI MmeIMmeI
PCR DNA with adaptors
EpiGnomeTM HELP-Seq DNAMethylated DNA Bisul�te conversionBisul�te conversion of genomic DNA without
shearing. HpaII tiny fragment enrichment by ligation-mediated PCR (HELP-Seq)
C GTCT
C GTUT
Bisul�te
C GTTT
PCR
Converted single-stranded fragments
Random primingDNA synthesis
3’ tagging PCR
Random primer 1
Methylated DNA Bisul�te conversion
Random primer 2scBS-seqSequenceSingle-cell bisul�te
sequencing (scBSBS-seq)Align fragments from every unique molecular tag
Isolated single cell
Lyse First random priming
Second random priming
Repeat 4 times
PCRExtend
Adaptor AdaptorExo I and purify
Protein-Protein Interactions
DNA-Protein Interactions
ATAC-Seq Fragmented and primed DNAAssay for transposase accessible
chromatin (ATAC-Seq)Tn5 TransposomeOpen DNA DNA puri�cation
Ampli�cationInsert in regions of open chromatin
MAINE-SeqMNase-SeqNucleo-Seq Open chromatin Isolate trimmed complexesMNase digestion DNA extraction DNAMNase-assisted isolation of nucleosomes (MAINE-Seq).
Also Micrococcal nuclease sequencing (MNase-Seq)
FAIRE-seqSono-Seq
DNAOpen DNA Formaldehyde-assisted isolation of regula-tory elements (FAIRE-Seq) and sonication of cross-linked chromatin (Sono-Seq)
Crosslink protein and DNA with formalin Sonicate Phenol extract and purify DNA from the aquous phase
NOMe-SeqOpen DNA Nucleosome Occupancy Methylome-
Sequencing (NOMe-Seq), a single-molecule nucleosome positioning assay
Bisul�te conver-sion BS-Seq
CpG dinucleotides Methylated CpG
CpG dinucleotides Methylated CpG
GpC methyltransferase (M.CviPI) and S-Adenosyl methionine (SAM)
Protected methylated
Protected unmethylated
Unprotected methylated
Unprotected unmethylated
Protected Protected
M.CviPI
Control
DNADNA extraction
Enrich DNA fragments
Add biotinylated compound
Crosslink
Chem-seq
Identify sites bound by small chemical molecules (Chem-seq)
DNA-protein complex with putative drug binding site
in vivo
in vitro
Streptavidin
Add biotinylated compound
Harvest cells and fragment DNA
HiTS-FLIPTarget sequence
Scan �owcellHybridizeHigh-throughput sequencing: �uorescent ligand interaction pro�ling (HiTS-FLIP)
Protein binding site
Prepare sequencing libraries and sequence �rst strandRemove second strand DNA
Flowcell
Hybridize primer
Synthesize second strand with unmodi-�ed nucleotides
Add �uorescent-ly labeled ligand
Elute with increas-ing stringency
Binding siteKlenow
ChIP-SeqChIP-exoHT-ChIPMint-ChIP
Exonuclease digestion Immunoprecipitate DNADNA-protein complex DNA extraction
Crosslink proteins and DNA Sample fragmentationChromatin immune precipitation (ChIP-Seq), High-throughput chromatin immunoprecipi-tation (HT-ChIP))
PB-seqDNADNA-protein complex DNA
extractionHybridize with DNA-binding protein
Isolate protein-bound DNA
Purify DNA Shear DNAProtein/DNA binding (PB–seq), to determine the binding energy landscape
ProP-PDPDZ-Seq
Hybridize
Proteomic peptide-phage display (ProP-PD) to identify short linear motif (SLiM) interac-tions or PDZ domains (PDZ-Seq)
Protein target
Identify C-terminal sequences
Create oligo library
Construct phage display library
Bait proteins immobi-lized on 96-well plate
Select phages against baits
Peptide counts
Isolate and sequence
21
CATCH-ITMNase digestion DNACovalent attachment of tags to
capture histones and identify turnover (CATCH-IT)
Isolate nucei
Cycloaddition reaction
Remove of H2A–H2B dimersand non-histone proteins
Cells starved ofmethionine
DNA extraction
Add methionine analogue l-azidohomoalanine (AHA)
Biotin
Streptavidin
ORGANICMNase digestion DNAOccupied regions of genomes from
a�nity-puri�ed naturally isolated chromatin (ORGANIC)
Isolate nuceiCell Isolated chromatin Soluble extract Immunoprecipitate and DNA extraction
X-ChIPMNase digestion DNAHigh resolution of mapping of in vivo
chromatin associated proteins (X-ChIP)Crosslink cells in vivo
Lyse cells Cross-linked chromatin Sonicate Soluble extract
Immunoprecipitate and DNA extraction
Chia-PET
Sample fragmentation Immunoprecipitate Ligation Restriction enzyme digestion DNAChromatin interaction analysis by paired-end tag sequencing (ChIA-PET)
4-C
LigationCrosslink proteins and DNA Sample fragmentation DNARestriction digest Self-circularization and Reverse PCR
Chromatin conformation capture circular (4-C)
5-C
LigationCrosslink proteins and DNA Sample fragmentation LMA: Ligation-mediated ampli�cation DNA
T7 T3
Chromatin conformation capture carbon copy (5-C)
DNA-protein complex Exonuclease digestion Immunoprecipitate DNADNA extractionCrosslink proteins and DNA
Sample fragmentation
Histone methylation
ChIP-Seq of methylated histones (Histone methylation)
DamID
DNA adenine methyltransferase interaction detection (DamID)
DNA-protein interaction
Create fusion protein
Align sequences and determine di�erentially digested sites
Split sample DpnI digestion Unmethylated GATCs are cut by DpnII
Adaptor ligation
Non-targeted methylation
Speci�c and non-targeted methylation
DNA adeninemethyltransferase (DAM)
Protein of interestDAM
Fusion protein DpnII PCRDpnI
SELEXSELEX-seqHT-SELEX
Binding site
High-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX)
Target sequence
Transcription factor binding siteDNA binding regionLuciferaseStreptavidin binding peptide
pD40htSELEX
Expression vectorLigand
Forward sequencing adaptorSequencing primerBarcode to identify sample14N with all possible combinationsReverse sequencing adaptor
Fusion protein immobilized in well
Matching ligand binds
Wash and elute
Recovered matching ligand
PCR and Sequence
LuciferaseFusion protein
Pu-seq ROrigin of replication
DNAPolymerase usage sequencing (Pu-seq)
Klenow reaction(+ random primer, dATP, dGTP,dCTP, dUTP)
Attach adaptors
Uracil DNA glycosylase-and DNA lyase (USER)
Alkali treatment
5’ 3’3’ 5’ U U U
PCR and purify
Index 1Index 2
Break-seq Breakage at replication fork
DNADouble-stranded breaklabeling to map chromosome breaks (Break-seq)
End-repair with dGTP, dCTP, dTTP, and biotinylated-dATP
Elution and fragmentation
Attach adaptors
DNA trapped in agarose gel
Break5’ 3’3’ 5’
PCR and purify
Index 1Index 2
Streptavidin magnetic bead pull down
5’ 3’3’ 5’
Yellow highlights indicate the target of the protocol
PD-SeqWash
Hybridize
PD-Seq identi�es candidate cellular targets for proteins
Protein target
Beads
Pyridine
Polyethylene glycol linker added
Immobilized protein
RNA from cells
NH2 NH2K2CO3
DMF
NH
Create phage display cDNA library
Repeat cycle three times
PCR phage insert
DNAProtein-protein interaction
NH2
NH2
MeDIP-SeqDIP-seq
Extract DNA FractionateDenature
ImmunoprecipitateMethylated DNAMethylated DNA Immunoprecipitation (MeDIP-Seq), DNA immunoprecipitation followed by high throughput sequencing (DIP-seq))
DNADNA puri�cation
hMeDIP-Seq
Extract DNA FractionateDenature
ImmunoprecipitateHydroxymethylated DNA
Hydroxymethylated DNA immunopre-cipitation combined with next genera-tion DNA sequencing (hMeDIP-seq)
DNADNA puri�cation
T-WGBSDNATagmentation-based
whole-genome bisul�te sequencing (T-WGBS)
Methylated DNA TagmentationTransposome withmethylated adaptor
Displaced oligo
Oligo withmethylated adaptor
Displace oligoHybridize methylated adaptor and gap repair
PCRBisul�te conversion
TAmC-Seq5cmC
Tet-assisted 5-methylcytosine sequencing (TAmC-Seq)
5mc residue βGT
Glucosylation OxidationGCC CT
5hmC 5mC
GCC CT
g5hmC 5mCTET
GCC CT
g5hmC
DNAβGT-catalyzed UDP-6-N3 glucosylation
Biotinylation Streptavidin pulldownand DTT cleavage
N3
GCC CT
g5hmC BiotinN3S-S
GCC CT
g5hmC
DNA
fC-Seal5fc residue
Blocked 5hmC residue
NaBH4
5hmC residue
A 5-formylcytosine-selective chemical labeling (fC-Seal) approach for genome-wide pro�ling of 5fC
βGT-catalyzed glucosylation
βGT-catalyzed UDP-6-N3 glucosylation
BiotinylationConvert 5fc to 5hmc
Streptavidin pulldownand DTT cleavage
5hmC
CC C
5caC
CC
5fc5mC
CC C CC CC C CC
N3
CC C CC
BiotinN3S-S
CC C CC
oxBS-SeqOxidative bisul�te sequencing (oxBS-Seq) to map 5-methylcytosine and 5-hydroxymethylcytosine
DNAKRuO4
5hmc residue ControlBisul�te treatmentPCR ampli�cation
Bisul�te treatmentPCR ampli�cation
C TCT T
TCT TT
5hmC
CC C
5caC
CC
5fc5mC
5fc
5hmC
CC C
5caC
CC
5fc5mC
C C CCC
MAB-seqM.SssI methylase-assisted bisul�te sequencing (MAB-seq) to map 5fC/5caC
DNAM.SssI
5fC/5caC residues ControlC TCT T
C TCC T
Bisul�te treatmentPCR ampli�cation
Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC5hmC
CC C
5caC
CC
5fc5mC
CCC CC
5mC
RRMAB-seq Reduced representation M.SssI methylase-assisted bisul�te sequencing (RRMAB-seq)
DNAM.SssI Digest with MspI
Add methylated adapters
5fC/5caC residues ControlC TCT T
C TCC T
Bisul�te treatmentPCR ampli�cation
Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC
CCC CC
5hmC
CC C
5caC
CC
5fc5mC
5mC
C TCT T
RedBS-SeqcaMAB-seq Reduced bisul�te sequencing
(redBS-Seq), to map 5-formylcyto-sine (5fC) in DNA
DNANaBH4
5fc residue Control
CCT TC
Bisul�te treatmentPCR ampli�cation
Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC5hmC
CC C
5caC
CC
5fc5mC
C CC C C
5hmC
C TCT T
fCAB-Seq5fC chemically assisted bisul�te sequencing (fCAB-seq) method for the base-resolution detection of 5fC
DNAO-ethylhydroxylamine (EtONH2)
5fc residue ControlBlocked
CCT TC
Bisul�te treatmentPCR ampli�cation
Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC5hmC
CC C
5caC
CC
5fc5mC
C CC C C
C TT T TTAB-SeqTET-assisted bisul�te sequencing, (TAB-Seq) to map 5-hydroxymethylcytosine
DNA
5hmc residue βGT
Glucosylation Oxidation
TET Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC 5gmC
CC C CC
5gmC
CC C
5caC
C
5caC
C
5caC
JBP1-seqDNAJ-binding protein 1 sequencing
(JBP1-seq), for genome-wide pro�ling of 5-hydroxy-methylcytosine (5hmC)
TagmentationTransposomes Glucosylated 5-hmC PCRJBP1-magnetic bead pull down
5hmc residues
T4-βGTHydroxy-methyl-ated DNA
Aba-seqDNAAbaSI coupled with sequencing
(Aba-seq) to map high-resolution hydroxymethylome (5hmC)
Glucosylated 5-hmC Streptavidin magnetic bead pull down
5hmc residues
T4-βGTHydroxy-methyl-ated DNA
AbaSI Biotinylated primers
Ligate Fragment
CAB-SeqSequence
5caC residue
Chemical modi�cation-assisted bisul�te sequencing (CAB-Seq) for 5caC detection
1-ethyl-3-[3-dimethylamino-propyl]-car-bodiimide hydrochloride (EDC) chemistry
Streptavidin pulldownand DTT cleavage
Nu S-S
Linker Bisul�te treatmentPCR ampli�cation
5hmC
CC C
5caC
CC
5fc5mC o-acylisourea5hmC
CC C
5caC
CC
5fc5mC BiotinS-S5hmC
CC C5caC
CC
5fc5mCCCT T T
C CC TT
Control5hmC
CC C
5caC
CC
5fc5mC
CCC CCS5caC
Extract DNA Fractionate Elute with increasingsalt concentration
Methylated DNA
Methyl-CpG binding domain-based capture and sequencing (MBDCap-seq). Capture of methylated DNA using the MBD domain of MeCP2 (Methyl-Cap-Seq). MBD-isolated Genome Sequencing (MiGS)
DNADNA puri�cation
Capture biotinylated MBD on Streptavidin coated magnetic beads
MBDStreptavidinBiotin
MBDCap-seqMethyl-Cap-seqMBD-SeqMiGS
MIRAMethylated-CpG island recovery assay (MIRA)
DNA
5mc CpG island
DNA puri�cationPCR ampli�cation
CGCGC
5mC
GC
5mC
G GC
CGC
GC G GCGC G
MBD2B/MBD3L1 protein complex
Isolate on glutathione-coated beads
Fractionate
CGCGCGC G
CGC
Methyl-SeqMRE-Seq Methyl-seq and MRE-Seq use
methyl-sensitive enzymes to identify methylation patterns
Methylated sites in the genome
MspI
HpaII
GGC CGGC C
GGC CGGC C
GGC CGGC C
GGC CGGC C
Sequence Align sequences and determine undigested sites
Split sample Identi�ed methylation site
Restriction enzyme digest
BSASDNABisul�te amplicon
sequencing (BSAS)Methylated DNA TagmentationTransposome with
adaptorAmpliconsPCR with primers speci�c for
bisul�te converted DNABisul�te conversion
Bisu�te-con-verted DNA
Key
5-Methyl Cytosine
Cytosine
R
NH2
HO
N
N
O
5-formylcytosine (5fC)R
NH2
H
N
N
O
O
5-carboxylcytosine (5caC)R
NH2
HO
N
N
O
O
R
NH2
N
N
O
CH3
R
NH2
N
N
O
5-hydroxymethylcytosine (5hmc)
N
NH2
NO
N
NH2
NO
CH3
Cytosine 5-Methyl Cytosine
N
NH2
NO
CH3
5-Methyl Cytosine
N
NO
O
Uracil
Bisul�te conversion
R
NH2HO
N
N
O
OHO
O
OH
OH
Glucosylated 5hmc
HO HO
O O
HOHO
O
HO
OH
OH
P
O-
O
OH
P
O-
ON O
NH
O
Uridine diphosphate glucose (UDP-Glu)
R
NH2HO
N
N
O
OHO
O
OH
N3
N3-5GMC
Pyridostatin (PDS)
O
H2N
NH NHO
N
H2N
H2N
O
O O
Pyridine
Bubble-Seq
Libraries of restriction fragments that contain replication initiation sites (bubbles) in vivo
Bubble-containing fragment
Restriction digest
Run gelCast fragments in trapping gel
Recover bubble-con-taining plug
DNA extraction Add sequencing primers
DNA
2b-RADGenome BsaXI restriction sites
Restriction-site associated DNA marker generation (RAD) with type IIB restriction endonucleases (2b-RAD)
Restriction digestion with type IIB restriction endonucleases e.g. BsaXI
Add restriction-site-speci�c adapters
Amplify Amplify DNAAdd sample-speci�c adapters
RADPE RAD-Seq
Genome Restriction sites
Restriction-site associated DNA marker generation (RAD)
Restriction digestion
Shear and size select
Add barcoded adapters Add P2 adapter
Amplify DNA
Genome DNA and mRNA sequencing (DR-Seq)
DR-SeqAA(A)n
Single cell RNA
DNA
AA(A)n
RNA
DNA
Single cell
RT with barcoded primerLyse cell Ad-2 primer
Split samples
Quasilinear ampli�cation
SequencegDNA ampli�cation
cDNA ampli�cationT T T T T T T T T TAAAAAAA
PCR and Remove adaptors
2nd strand synthesis
SLAF-seq Genome Restriction sites
Speci�c locus ampli�ed fragment sequenc-ing (SLAF-seq) for large scale genotyping
Digest with MseI Add MseI adaptors
PCRPCR with barcoded MseI primer
DNAAdd sequencing adaptors
Digest with AluI Purify and pool
Genome and transcriptome sequencing from a single cell (G&T-seq)
G&T-seq
Align RNA and genome
AA(A)n
Single cell RNA
DNA
AA(A)n
RNA
DNA
Cell suspension
Isolate single cell
Separate the DNA and the RNALyse cell Sequence
T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primer
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-seq2Whole genome ampli�cation with MDA
Methylome and transcrip-tome sequencing from a single cell (scM&T-seq)
scM&T-seq
Align RNA and methylome
AA(A)n
Single cell RNA
DNA
AA(A)n
RNA
DNA
Cell suspension
Isolate single cell
Separate the DNA and the RNALyse cell Sequence
T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primerStreptavidin magnetic bead with mRNA capture primer
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-seq2Whole genome bisul�te sequencing with scBS-seq
Methylome and transcrip-tome sequencing from a single cell (scMT-seq)
scMT-seq
Align RNA and methylome
AA(A)n
Single cell RNA
DNA
AA(A)n
RNA
DNA
Cell suspension
Isolate single cell
Separate the DNA and the RNALyse cell Sequence
T T T T T T T T T TAAAAAAA
Streptavidin magnetic bead with mRNA capture primerStreptavidin magnetic bead with mRNA capture primer
T T T T T T T T T TAAAAAAA On-bead transcriptome
ampli�cation with Smart-seq2Whole genome ampli�cation with RRBS
Rapture Genome Restriction sites
Restriction-site associated DNA marker generation (RAD) capture (Rapture)
Digest with restriction enzyme
Restriction enzyme digest
Add well-speci�c barcodes
Pool
Plate 1
DNAHybridize Biotinylatedcapture bait
Pool wells and shear
Library prep with plate barcodes
Well 1Well 2
Streptavidin pull down
Streptavidin pull down
Plate 2
Hi-C3-CCapture-C
LigationCrosslink proteins and DNA Sample fragmentation PCR amplify ligated junctions DNAChromatin conformation capture (3-C, Hi-C and Capture-C)
NG Cap-ture-C
LigationFormaldehyde �xation Restriction enzyme digestion
De-crosslink and extract DNA
Sonicate to 200 bp fragments
DNAPCRNext-generation Capture-C (NG Capture-C)
Add indexed sequencing adaptors
Hybridize biotinylatedcapture bait
Streptavidin pull down
LAM-HTGTSDNALinear ampli�cation-mediated
high-throughput genome-wide sequencing (LAM-HTGTS)
Fragmentation by sonication
Tagged PCREnzyme blocking
Nested PCR
Purify
Breakage in genome
Streptavidin magnetic bead pull down
Adaptor ligationLAM-PCR with biotinilated primer
NNNNNNNN
HTGTSDNALinear ampli�cation-mediated
high-throughput genome-wide sequencing (HTGTS)
Fragmentation by sonication
Tagged PCRNested PCR
Purify
Breakage in genome
Streptavidin magnetic bead pull down
End repair 3’ A addition Adaptor ligation and PCR
A
A
A
A
A
A
ChIPmenta-tion
Fragmented and primed
DNAChromatin immunoprecipitation with sequencing library preparation by Tn5 transposase (ChIPmentation)
Chromatin immunoprecipitationChromatin DNA puri�cationAmpli�cation
Adaptor insertionTn5 Transposome
DNaseI-SIM
DNase I digestion Terminate DNase I digestion
DNA extraction
DNADnase I simpli�ed in-nucleus method (Dnase I SIM) for plants
Cells Lyse and centrifuge
Sort nuclei
Supernatant
Nuclei
Nucleus
Nucleus
Polish ends
DNA
fC-CET5fc residue
5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR
5hmC
CC C
5caC
CC
5fc5mC N3
CC C CC
BiotinN3
S-S
CC C CC
N3SH
CC C CC CC C CT
DBCO-S-S-PEG3-biotinAzido 1,3-indandione (AI)
Pulldown, NaOH and DTT
Adaptor ligation and PCR
Purify
DNase-SeqDNaseI-Seq
Active chromatin Isolate trimmed complexesDNase I digestion DNA extraction DNADNase I hypersensitive sites sequencing (DNase-Seq, DNaseI-Seq)
MPE-seq
Active chromatin Isolated nuclei Add bathophenanthrolineMPE digestion DNA extraction DNAMethidiumpropyl-EDTA sequencing (MPE-seq)
Methidiumpro-pyl-EDTA (MPE)
O
O- O
O
O-
O-
O-
N
N
N
NH
Fe
H
NH2H2N
O
N
Me
Single-cell triple omics sequencing (scTrio-seq)
scTrio-seqAA(A)n
Single cell RNA
DNA
DNA methylation
Cell suspension
Isolate single cell
Lyse and centrifuge
Supernatant
Nucleus
AA(A)nRNA
Add carrier RNA
AA(A)nT T (T)ncDNA synthesis PCR and sequenceAdd poly A with TDTHybridize oligo
AA(A)n
DNAAdd sequencing adaptors PCR and sequence
Align sequencesMethylated regions
Methylated adapter
End repair and ligation
Bisul�te conversion
Converted fragments
MspI digestion
PCR and sequence
Methylated DNA
GUIDE-seqOligo insert
DNAGenome-wide, unbiased identi�cation of DSBs enabled by sequencing (GUIDE-seq)
Genome DNA dsOligo tag integrat-ed in live cells
DNA isolationRandom shearing
End repairAdaptor ligation
dsOligo-speci�c ampli�cation
dsOligo insert
PCR
Oligo tagUMI
UMIP7
P5 Index 2
Index 1
Preparation of acylated RNA for biotin–streptavidin puri�cation. DIBO, dibenzocyclooxtyne
N
O
N3
ODNA
N
O
ODNA
NN
N
Biotin
N
NN
O
N3
DNA +
Acylation
DIBO-biotin “click”
MINCE-seqSonication and MNase digestion
Streptavidin capture
DNAMapping in vivo nascent chromatin with EdU and sequencing (MINCE-seq)
Crosslink with formaldehyde Click reactionto attach biotin
Label with ethynyl deoxyuridine (EdU)
DNA extraction
Chase with Thymidine
Polymerase
Newly-formed chromatin Streptavidin
SSB-SeqSingle-strand break
DNAMap DNA single-strand breaks (SSB-Seq)
Genome DNA DNA isolation and random shearing Immunoprecipitate with anti-digoxigenin antibody
DNA Pol I, dU-digoxigenin, and dNTPs
DSB-SeqDouble-strand break
DNAMap DNA double-strand breaks (DSB-Seq)
Genome DNA DNA isolation and random shearing Capture on streptavidin beadsTdT and biotinylated-dUTP
scAba-seqDNADetect 5hmC marks in single cells
with AbaSI nuclease (scAba-seq)Glucosylated 5-hmC
5hmc residues
T4-βGTHydroxy-methyl-ated DNA
AbaSI Ligate Pool T7 ampli�cationPrimer
Illumina 5’ adaptorT7 promoter
Adaptor with cell-speci�c barcodeSingle cell
Droplet-based single-cell ChIP-seq (Drop-ChIP)
Drop-ChIPscChIP-seq
Single cell
Barcoded sequences from single cells
Cell suspension
Droplet with unique oligos
Load single cells into droplets with lysis bu�er and MNase
Fuse droplets Pool all droplets SequenceChromatin immuno-precipitation
Single cell
scATAC-Seq(Microfluidics)
Fragmented and primed DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)
Lyse and introduce Tn5 transposase
Pool libraries from all cells
Amplify with cell-speci�c barcodes
Insert in regions of open chromatinCell suspension
Micro�uidics device
Isolate single cell
Very rare mutationSafe-SeqS
DNA Shear
Mutation
Amplify and solid phase capture
SequenceSafe-sequencing system is a unique molecular identi�er (UMI) approach to detect rare variants (Safe-SeqS)
Adaptor ligation Randomly sheared ends serve as UMIs
Align sequences and determine actual ratio
True mutant
FiT-seqHeat 40°C in SDS DNAPuri�cationFixed-tissue chromatin immuno-
precipitation sequencing (FiT-Seq)Depara�nation and rehydration
FFPE-�xed chromatin
Proteinase K digestion
Enzyme inactivation
Sonicate Soluble extract
Immunoprecipitate Cromatin elution
PAT-ChIPMNase digestion DNAPuri�cationPathology tissue chromatin
immunoprecipitation (PAT-ChIP)Depara�nation and rehydration
FFPE-�xed chromatin
Sonicate Soluble extract
Immunoprecipitate and chromatin elution
Reverse crosslink and proteinase K digestion
UMI-4C
Ligation Sonicate Single end adaptor ligation
Reverse crosslink and proteinase K digestion
HC DpnII digestion
Nested PCR ampli�cation DNATargeted chromosome conformation capture (4C) with unique molecular identi�ers (UMI-4C)
Crosslink proteins and DNA
Adaptor Universal primerDS
primerUS primer
Pad
BLESSDouble-strand break
DNABreaks labeling and enrichment on streptavidin and sequencing (BLESS)
Genome DNA DNA isolation and random shearing
Capture on streptavidin beads
Biotinylated proximal primer
Distal primer
Digest with I-SceI and PCR
Ligate primer
TTTTTTTT
TTTTTTTT
TTTTTTTT
TTTTTTTT
TTTTTTTT
TTTTTTTT
TTTTTTTT
Dige-nome-seq
Genome Cas9 target
In vitro Cas9-digested whole-ge-nome sequencing (Digenome-seq)
Sequence Align and determine sequence breaks
Target site digestion
Wild typeDeletion
Insert scRC-SeqGenomic DNA Enriched library
Novel retrotrans-position events
Retrotransposon binding site
Single cell retrotransposon capture sequencing (scRC-Seq)
Cell suspension
FACS isolation
Pick nuclei
Whole genome ampli�cation
Create sequencing library
Sequence captureNucleus
EpiRADseqGenome Restriction sites
Double digest restriction-site associated DNA marker generation (ddRADseq) with a methyla-tion-sensitive restriction enzyme (EpiRADseq)
Restriction digestion
Second restriction digest with methylation-sensitive HpaII
Add barcoded adapters Add P2 adapter
Amplify DNA
ddRADseqGenome Restriction sites
Double digest restriction-site associated DNA marker generation (ddRADseq)
Restriction digestion
Second restriction digest Add barcoded adapters Add P2 adapter
Amplify DNA
hyRADGenome DNA to be capturedHigh quality DNA
Hybridization RAD (hyRAD) for degraded DNA
Shotgun library Hybridize to probesPrepare RAD-seq library and size select
DNAAdaptor removal and labeling
Biotinylated probes
Streptavidin pull down
GenomeMDAIMS-MDAMIDAS
Hybridize primers
Nascent replication fork
Phi 29 Phi 29 S1 nuclease Ampli�ed DNA
3’ blocked random hexamer primers
Synthesis SynthesisMultiple displacement ampli�cation (MDA). Immunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA) Microwell displacement ampli�cation system (MIDAS)
T7 promoterRead primer
THS-seqEnd �ll-in and IVT ampli�cation
Add barcodes and sequencing adaptorsTransposome hypersensitive site
sequencing (THS-seq)Tn5 TransposomeOpen DNA dsDNA synthesisInsert in regions of open chromatin Purify
Single cellscATAC-Seq(Cell index)
DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)
Barcode each well with Tn5 transposase
Cell suspension Isolate Nuclei Split sample
Pool and dilute
Split sample PCR-barcode every well
Pool for library prep
SMDBSingle-molecule droplet barcoding (SMDB)
DNA templates Single template encapsulation
Template ampli�cation Template fragmentation Barcode every droplet Pool for library prep
DNA
Digoxigenin
O
HN
HN
O-
O-
NH
O
O
OH
HOCH3
O
CH3
O
O
O N O
O
O
O
O-
O-
O
O-
O-
O OSNS
RNAprimer
gDNA
SNS
gDNA
NSCRNascent strand capture and release (NSCR)
DNA replication bubble Denaturation and size-selection
5’-biotinylation Streptavidin pull-down RNase I digestion
Isolate and amplify SNS
DNA
Biotin
5fC-AI-SH
N
N
DNA
O
NH2O
5fC-AI
N
NN
N
OHN
O
N
N
N
DNA
O
O
HS
N3
N
N
N
DNA
O
O
OO
N3 5fC
AI-mediated cyclization labeling in fC-CET
BisChIP-SeqChIP-BS-seq
ImmunoprecipitateSonicate DNABisul�te conversion
C GTCT
C GTUT
Bisul�te
C GTTT
PCR
Bisul�te-treated chromatin immuno-precipitated DNA (BisChIP-seq) and ChIP-BS-seq), to correlate protein modi�cations with DNA methylation
DNA-protein complex with methylated histones and methylated DNA
Purify DNA and shear
Repli SeqNascent DNA
BrdU
Run-on with analog Sort cells and lyse DNARepli Seq—to map temporally ordered replicating DNA
PurifyBead coated with anti-BrdU antibody
EluteEnd Repair
NS-seqNascent DNA
DNA replication origin Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo) DNANascent strand sequencing (NS-seq) to discover DNA replication origins and G4 structures
AmplifyPurifyDigestion