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F DNA or all y ou seq... 2b-RAD Wang S et al. (2012) Nat Methods 9:808-810 3C Duan 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 Sun Z. et al. (2013) Cell Rep 3: 567-576 ATAC-Seq Buenrostro J. D. et al. (2013) Nat Methods 10: 1213-1218 BisChIP-Seq Statham A. L. et al. (2012) Genome Res 22: 1120-1127 Bisulfite-seq Berman B. P. et al. (2012) Nat Genet 44: 40-46 BLESS Crosetto, N. et al. Nat. Methods 10, 361–365 (2013). Break-seq Hoffman E. A. et al. (2015) Genome Res 25: 402-412 BSAS Masser D. R. et al. (2013) Epigenetics Chromatin 6: 33 BSPP Deng J. et al. (2009) Nat Biotechnol 27: 353-360 BS-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: 9315-9317 caMAB-seq Wu H et al. (2016) Nat Protoc 11:1081-100 CAP-seq Illingworth R. S. et al. (2010) PLoS Genet 6: e1001134 Capture-C Hughes J. R. et al. (2014) Nat Genet 46: 205-212 CATCH-IT Deal R. B. et al. (2010) Science 328: 1161-1164 Chem-Seq Anders L. et al. (2014) Nat Biotechnol 32: 92-96 Chia-PET Li G. et al. (2010) Genome Biol 11: R22 ChIP-BS-seq Brinkman A. B. et al. (2012) Genome Res 22: 1128-1138 ChIP-exo Yen K. et al. (2013) Cell 154: 1246-1256 ChIPmentation Schmidl C et al. (2015) Nat Methods 12:963-5 ChIP-Seq Barski A. et al. (2007) Cell 129: 823-837 CPT-seq Amini S. et al. (2014) Nat Genet 46: 1343-1349 DamID Vogel M. J. et al. (2007) Nat Protoc 2: 1467-1478 ddRADseq Peterson B. K. et al. (2012) PLoS One 7: e37135 Digenome-seq Kim D et al. (2015) Nat Methods 12:237-43 DIP-seq Shen L. et al. (2013) Cell 153: 692-706 Dnase I SIM Cumbie JS et al. (2015) Plant Methods 11:42 DNaseI-Seq Hesselberth J. R. et al. (2009) Nat Methods 6: 283-289 DNase-Seq Boyle A. P. et al. (2008) Cell 132: 311-322 Drop-ChIP Rotem A et al. (2015) Nat Biotechnol 33:1165-72 DR-Seq Dey SS et al. (2015) Nat Biotechnol 33:285-9 DSB-seq Baranello L et al. (2014) Int J Mol Sci 15:13111-22 Duplex-Seq Schmitt M. W. et al. (2012) Proc Natl Acad Sci U S A 109: 14508-14513 EC-seq Parkinson N. J. et al. (2015) Genome Res 25: 226-234 EpiRADSeq Schield DR et al. (2016) Methods in Ecology and Evolution 7:60-69 FAIRE-Seq Gaulton K. J. et al. (2010) Nat Genet 42: 255-259 fCAB-Seq Song C. X. et al. (2013) Cell 153: 678-691 fC-CET Xia B et al. (2015) Nat Methods 12:1047-50 fC-Seal Song C. X. et al. (2013) Cell 153: 678-691 FiT-Seq Cejas P et al. (2016) Nat Med G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522 G4-seq Chambers V. S. et al. (2015) Nat Biotechnol 33: 877-881 GUIDE-seq Tsai SQ et al. (2015) Nat Biotechnol 33:187-97 HELP-Seq Oda M. et al. (2009) Nucleic Acids Res 37: 3829-3839 Hi-C Lieberman-Aiden E. et al. (2009) Science 326: 289-293 Histone meth. Barski A. et al. (2007) Cell 129: 823-837 HiTS-Flip Nutiu R. et al. (2011) Nat Biotechnol 29: 659-664 hMeDIP-seq Xu Y et al. (2011) Mol Cell 42:451-64 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 HydEn-seq Clausen A. R. et al. (2015) Nat Struct Mol Biol 22: 185-191 hyRAD Suchan T et al. (2016) PLoS One 11:e0151651 Ig-Seq Vollmers C. et al. (2013) Proc Natl Acad Sci U S A 110: 13463-13468 IMS-MDA Seth-Smith H. M. et al. (2013) Nat Protoc 8: 2404-2412 INSeq Goodman AL et al. (2009) Cell Host Microbe 6:279-89 JBP1-seq Cui L. et al. (2014) Genomics LAM-HTGTS Hu J et al. (2016) Nat. Protocols 11:853-871 MAB-seq Wu H. et al. (2014) Nat Biotechnol MAF 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 MeDIP-Seq Down T. A. et al. (2008) Nat Biotechnol 26: 779-785 MethylCap-Seq Brinkman A. B. et al. (2010) Methods 52: 232-236 Methyl-seq Brunner A. L. et al. (2009) Genome Res 19: 1044-1056 MIDAS Gole J et al. (2013) Nat Biotechnol 31:1126-32 MiGS Serre D. et al. (2010) Nucleic Acids Res 38: 391-399 MINCE-seq Ramachandran S et al. (2016) Cell 165:580-92 Mint-ChIP van Galen P et al. (2016) Mol Cell 61:170-80 MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761 MIRA Rauch T. A. et al. (2010) Methods 52: 213-217 MNase-Seq Schones D. E. et al. (2008) Cell 132: 887-898 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 NS-seq Foulk MS et al. (2015) Genome Res 25:725-35 Nucleo-Seq Valouev A. et al. (2011) Nature 474: 516-520 nuc-seq Wang Y. et al. (2014) Nature 512: 155-160 ORGANIC Zentner G. E. et al. (2013) PLoS Genet 9: e1003317 OS-Seq Myllykangas S. et al. (2011) Nat Biotechnol 29: 1024-1027 oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937 PAT–ChIP Fanelli M et al. (2011) Nat Protoc 6:1905-19 PBAT Miura F. et al. (2012) Nucleic Acids Res 40: e136 PB-seq Guertin M. J. et al. (2012) PLoS Genet 8: e1002610 PD-Seq Arango D. et al. (2013) Proc Natl Acad Sci U S A 110: E2153-2162 PDZ-Seq Ernst A. et al. (2010) Mol Biosyst 6: 1782-1790 PE RAD-Seq Willing E. M. et al. (2011) Bioinformatics 27: 2187-2193 ProP-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: e3376 Rapture Ali OA et al. (2016) Genetics 202:389-400 RBBS Meissner A et al. (2008) Nature 454:766-70 RC-Seq Baillie J. K. et al. (2011) Nature 479: 534-537 RedBS-Seq Booth M. J. et al. (2014) Nat Chem 6: 435-440 Repli-Seq Hansen R. S. et al. (2010) Proc Natl Acad Sci U S A 107: 139-144 Rep-Seq Benichou J. et al. (2012) Immunology 135: 183-191 Ribose-seq Koh K. D. et al. (2015) Nat Methods 12: 251-257 RRBS Meissner A. et al. (2008) Nature 454: 766-770 RRMAB-seq Neri F. et al. (2015) Cell Rep 10: 674-683 Safe-SeqS Kinde I et al. (2011) Proc Natl Acad Sci U S A 108:9530-5 scABA-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: 817-820 scChIP-seq Rotem A et al. (2015) Nat Biotechnol 33:1165-72 scM&T-seq Angermueller C et al. (2016) Nat Methods advance online 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-Seq Klein I. A. et al. (2011) Cell 147: 95-106 THS-seq Sos BC et al. (2016) Genome Biol 17:20 TN-Seq van 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 advance online publication WGBS Lister R. et al. (2009) Nature 462: 315-322 X-ChIP-seq Skene P. J. et al. (2014) Elife 3: e02042 References TruSeq RNA AAAAA mRNA TTTTT AAAAA 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 Moleculo Sheared genomic DNA End repair Adapter ligation Prepared fragments ~10kb P5 Index 2 Index 1 P7 P5 Index 2 Index 1 P7 Transposase Tagmentation ~600bp P5 Index 2 Index 1 P7 P5 P7 P5 P7 P5 P7 Generate clonal pools Amplify Add indices Pool and purify Sequencing by Synthesis TruSeq PCR Free Double-stranded DNA Fractionate Size select A-overhang End repair Phosphorylate P P A A P P T P P5 P7 Index T P P5 P7 Index Adaptor ligation P5 P5 P7 Index P7 Index Add Adaptors Product ready for cluster generation TruSeq Nano Double-stranded DNA Fractionate Size select A-overhang End repair Phosphorylate P P A A P P T P P5 P7 Index 1 Index 2 Index 2 Index 1 T P P5 P7 Adaptor ligation Denature and amplify Add Adaptors P5 P7 Index 1 Index 2 Index 2 Index 1 P5 P7 P5 P7 Index 1 Index 2 Double-stranded DNA Product ready for cluster generation TruSeq Custom Amplicon Add custom probes Region of interest Double-stranded DNA Double-stranded DNA Add custom probes Extension and ligation Add sequencing primers Index 2 Index 1 P5 P7 Index 1 P7 Index 2 P5 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 P7 Index Product ready for cluster generation TruSeq RNA Stranded 5’ 3’ RNA Random primer cDNA Create cDNA Create second strand cDNA dUTP + dCTP + dATP + dGTP dTTP + dCTP + dATP + dGTP End repair Phosphorylate A-overhang Adaptor ligation Denature and amplify P5 P7 P5 P7 Index 1 Index 2 U UU U U U U U U U U Sense strand A A P P U UU U U U U U U U Sense strand P7 Index 1 P5 Index 2 U UU U U U U U U U Index 2 P5 Index 1 P7 Sense strand P5 P7 U UU U U U U U U U Sense strand Block polymerase Product ready for cluster generation Nextera Library Preparation Transposase DNA ~300bp Tagmentation Amplification P5 P7 Index 1 Index 2 P5 Index 2 Index 1 P7 Product ready for cluster generation Nextera Mate Pair Adaptor ligation Isolate biotinylated fragment Denature and amplify P5 P7 Transposase DNA Tagmentation Circularize R R R R Biotinylated junction adapter R R R R R R Fragment R R R R P5 P7 P5 P7 R R Product ready for cluster generation 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 A A T T C G C Synthesize second strand The second read is sequenced Sequence Index2 A A T T C G C Deblock P5 primer and add unlabeled bases Read 2 primer The forward- strand is cleaved and washed away 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 A A T T C G C A A T T C G C 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 C H 3 O CH 3 Nextera Rapid Capture Elute Target Target P5 P7 Index 1 Index 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 Target P5 P7 Index 1 Index 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 Expression Target ULSO DLSO Total RNA cDNA Hybridization P7 Index 1 P5 P5 P7 5’ P 5’ P Index 2 Target Index 1 Index 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 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. EDTA O O O - O O O - O - O - N N M Display methods on mobile device N N N N C H 3 O CH 3 Display methods on mobile device N N N N DNA Low-Level Detection DNA Rearrangements and Markers Duplex-Seq α β Very 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 variant Random error Ligate and PCR Rare variant Sequence Create single strand consensus sequence from every unique molecular tag Consensus Create duplex sequences based on molecular tags and sequencing primers Add adaptors Ig-seq Rep-Seq MAF CDR3 junction region V D J Constant DNA DNA sequencing of immunoglobulin genes (Ig-seq), repertoire sequencing (Rep-Seq), and molecular amplification fingerprinting (MAF) Extracted RNA Reverse transcription 8N UID 8N UID Second strand synthesis PCR Purify CpG island CAP-seq DNA CXXC affinity purification 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 CXXC X Elute unmethylated CpG enriched fragments TC-Seq AID-dependent rearrangement Infect I-Sel Purification I-SceI site +AID -AID A A A A DNA Genomic DNA TC-Seq: translocation capture sequencing Sonicate, blunt and A-tail Ligate linkers Cut I-Scel Semi-nested PCR Linker cleavage RC-Seq Genomic DNA Fractionate DNA fragments Hybridize Read1 Read2 Transposon 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-Seq INSeq Transposon Transposon 20bp MmeI MmeI MmeI digestion Add adapters 20bp PCR and sequence Transposon sequencing (TN-Seq) and insertion sequencing (INSeq) Inverted MmeI recognition site MmeI recognition site Transposon insertion sites MALBAC Genome Hybridize primers PCR 27-bp common sequence 8 random nucleotides Partial amplicons Template Denature Denature Hybridize primers Synthesis Multiple annealing and looping-based amplification cycles (MALBAC) DNA Cycles of quasilinear amplification Looped full amplicons Bst DNA polymerase OS-Seq Gene Target sequence Adaptor sequence Flow cell Sequencing Primers Target sequence Single adaptor library Hybridize Hybridize Sequence Oligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on the flow cell Create target-specific 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 DNA Indexed transpo- some reactions EDTA Pool Contiguity-preserving transposition sequenc- ing (CPT-seq) Genome Indexed PCR primers EC-seq V(D)J antigen receptor region Excision circle sequencing (EC-seq) Lymphocyte-specific recombination activat- ing gene (RAG) recombinase, bound genome V 1 V 2 V 3 J 5 J 4 J 3 J 2 J 1 V 1 V 2 V 3 J 5 J 4 J 3 J 2 J 1 J 5 J 4 J 3 J 2 J 1 V 1 V 2 V 3 Synapsis, cleavage and coding end hairpin formation Resolved genomic coding junction Liberated excision circle with resolved signal junction Align read pairs to reference genome V 1 V 2 J 3 J 2 J 1 V 3 J 5 J 4 V1 V2 V3 J5 J4 J3 J2 J1 HydEn-seq Genome Ribonucleotides Hydrolytic end sequencing (HydEn-seq) to reveal replicase- and strand-specific 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 R 5’HO 5’P-O 2’,3’-cyclic phosphate R R Genomic DNA Gene smMIP Copy target sequence Exonuclease Corrected sequence Align fragments from every unique molecular tag Sample index Read1 Read2 True variant Random error Single Molecule Molecular Inversion Probes (smMIPs) for detecting low frequency targets PCR amplification Degenerate molecular tag Targeted STR Short tandem repeat (STR) MIPSTR Copy target STR Amplify and sequence Targeted capture of STR loci by smMIPs (MIPSTR) Degenerate molecular tag Strain I Strain II Strain I Strain I Natural variation between individuals Somatic variation within an individual G4-seq Potential G-quadruplex Genome 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 flow cell Sequencing Primer Read stabilized squence Polymerase stalls nuc-seq SNES Cell 1 Cell 2 Cell 3 Cell sorting from G2/M distribution Lyse cell Nucleus Single G2/M nucleus sequencing of cells in S phase (nuc-seq). Single nucleus exome sequencing (SNES) Single cell genome Phi 29 Limited amplification S1 nuclease Synthesis DNA Ribose-seq Genome 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 by AtRNL Degradation of linear ssDNA Remove 2’-phos- phate and PCR R 5’ P P 5’ R 5’ P P 5’ A A R A A mA T T Am A mA T A Am R T A mA T A Am R T R T R T A mA T A Am 2’P 2’P R T Epigenetics Methylated DNA DNA RRBS scRRBS Reduced representation bisulfite sequencing (RRBS-Seq). Single cell RRBS (scRRBS) Methylated regions Methylated adapter End repair and ligation Bisulfite conversion Converted fragments PCR PCR MspI digestion PBAT Post-bisulfite adaptor tagging (PBAT) Methylated DNA Capture first strand on Streptavi- din coated magnetic beads Second random priming Streptavidin Biotin Adaptor Bisulfite conversion First random priming Adaptor Random primer 2 Generate second strand DNA with adaptors Elution BS-Seq Bisulfite-seq WGBS DNA Shear DNA Methylated DNA Bisulfite conversion Bisulfite conversion of genomic DNA (bs-Seq) or whole-genome bisulfite sequencing (WGBS) C G T C T C G T U T Bisulfite C G T T T PCR BSPP Bisulfite sequencing with padlock probes (BSPP) CpG island Bisulfite conversion Bisufite-converted DNA Hybridize Padlock probe End repair and adaptor ligation Extension and ligation Exonuclease digestion P1 P2 H1 H2 AluI MmeI MmeI PCR DNA with adaptors EpiGnome TM HELP-Seq DNA Methylated DNA Bisulfite conversion Bisulfite conversion of genomic DNA without shearing. HpaII tiny fragment enrichment by ligation-mediated PCR (HELP-Seq) C G T C T C G T U T Bisulfite C G T T T PCR Converted single-stranded fragments Random priming DNA synthesis 3’ tagging PCR Random primer 1 Methylated DNA Bisulfite conversion Random primer 2 scBS-seq Sequence Single-cell bisulfite sequencing (scBSBS-seq) Align fragments from every unique molecular tag Isolated single cell Lyse First random priming Second random priming Repeat 4 times PCR Extend Adaptor Adaptor Exo I and purify Protein-Protein Interactions DNA-Protein Interactions ATAC-Seq Fragmented and primed DNA Assay for transposase accessible chromatin (ATAC-Seq) Tn5 Transposome Open DNA DNA purification Amplification Insert in regions of open chromatin MAINE-Seq MNase-Seq Nucleo-Seq Open chromatin Isolate trimmed complexes MNase digestion DNA extraction DNA MNase-assisted isolation of nucleosomes (MAINE-Seq). Also Micrococcal nuclease sequencing (MNase-Seq) FAIRE-seq Sono-Seq DNA Open 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-Seq Open DNA Nucleosome Occupancy Methylome- Sequencing (NOMe-Seq), a single-molecule nucleosome positioning assay Bisulfite 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 DNA DNA 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-FLIP Target sequence Scan flowcell Hybridize High-throughput sequencing: fluorescent ligand interaction profiling (HiTS-FLIP) Protein binding site Prepare sequencing libraries and sequence first strand Remove second strand DNA Flowcell Hybridize primer Synthesize second strand with unmodi- fied nucleotides Add fluorescent- ly labeled ligand Elute with increas- ing stringency Binding site Klenow ChIP-Seq ChIP-exo HT-ChIP Mint-ChIP Exonuclease digestion Immunoprecipitate DNA DNA-protein complex DNA extraction Crosslink proteins and DNA Sample fragmentation Chromatin immune precipitation (ChIP-Seq), High-throughput chromatin immunoprecipi- tation (HT-ChIP)) PB-seq DNA DNA-protein complex DNA extraction Hybridize with DNA-binding protein Isolate protein-bound DNA Purify DNA Shear DNA Protein/DNA binding (PB–seq), to determine the binding energy landscape ProP-PD PDZ-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 2 1 CATCH-IT MNase digestion DNA Covalent attachment of tags to capture histones and identify turnover (CATCH-IT) Isolate nucei Cycloaddition reaction Remove of H2A–H2B dimers and non-histone proteins Cells starved of methionine DNA extraction Add methionine analogue l-azidohomoalanine (AHA) Biotin Streptavidin ORGANIC MNase digestion DNA Occupied regions of genomes from affinity-purified naturally isolated chromatin (ORGANIC) Isolate nucei Cell Isolated chromatin Soluble extract Immunoprecipitate and DNA extraction X-ChIP MNase digestion DNA High 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 DNA Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) 4-C Ligation Crosslink proteins and DNA Sample fragmentation DNA Restriction digest Self-circularization and Reverse PCR Chromatin conformation capture circular (4-C) 5-C Ligation Crosslink proteins and DNA Sample fragmentation LMA: Ligation-mediated amplification DNA T7 T3 Chromatin conformation capture carbon copy (5-C) DNA-protein complex Exonuclease digestion Immunoprecipitate DNA DNA extraction Crosslink 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 differentially digested sites Split sample DpnI digestion Unmethylated GATCs are cut by DpnII Adaptor ligation Non-targeted methylation Specific and non-targeted methylation DNA adenine methyltransferase (DAM) Protein of interest DAM Fusion protein DpnII PCR DpnI SELEX SELEX-seq HT-SELEX Binding site High-throughput systematic evolution of ligands by exponential enrichment (HT-SELEX) Target sequence Transcription factor binding site DNA binding region Luciferase Streptavidin binding peptide pD40htSELEX Expression vector Ligand Forward sequencing adaptor Sequencing primer Barcode to identify sample 14N with all possible combinations Reverse sequencing adaptor Fusion protein immobilized in well Matching ligand binds Wash and elute Recovered matching ligand PCR and Sequence Luciferase Fusion protein Pu-seq R Origin of replication DNA Polymerase 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 1 Index 2 Break-seq Breakage at replication fork DNA Double-stranded break labeling 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 Break 5’ 3’ 3’ 5’ PCR and purify Index 1 Index 2 Streptavidin magnetic bead pull down 5’ 3’ 3’ 5’ Yellow highlights indicate the target of the protocol PD-Seq Wash Hybridize PD-Seq identifies candidate cellular targets for proteins Protein target Beads Pyridine Polyethylene glycol linker added Immobilized protein RNA from cells NH 2 NH 2 K 2 CO 3 DMF NH Create phage display cDNA library Repeat cycle three times PCR phage insert DNA Protein-protein interaction NH 2 NH 2 MeDIP-Seq DIP-seq Extract DNA Fractionate Denature Immunoprecipitate Methylated DNA Methylated DNA Immunoprecipitation (MeDIP-Seq), DNA immunoprecipitation followed by high throughput sequencing (DIP-seq)) DNA DNA purification hMeDIP-Seq Extract DNA Fractionate Denature Immunoprecipitate Hydroxymethylated DNA Hydroxymethylated DNA immunopre- cipitation combined with next genera- tion DNA sequencing (hMeDIP-seq) DNA DNA purification T-WGBS DNA Tagmentation-based whole-genome bisulfite sequencing (T-WGBS) Methylated DNA Tagmentation Transposome with methylated adaptor Displaced oligo Oligo with methylated adaptor Displace oligo Hybridize methylated adaptor and gap repair PCR Bisulfite conversion TAmC-Seq 5cmC Tet-assisted 5-methylcytosine sequencing (TAmC-Seq) 5mc residue βGT Glucosylation Oxidation G C C C T 5hmC 5mC G C C C T g5hmC 5mC TET G C C C T g5hmC DNA βGT-catalyzed UDP-6-N 3 glucosylation Biotinylation Streptavidin pulldown and DTT cleavage N 3 G C C C T g5hmC Biotin N 3 S-S G C C C T g5hmC DNA fC-Seal 5fc residue Blocked 5hmC residue NaBH4 5hmC residue A 5-formylcytosine-selective chemical labeling (fC-Seal) approach for genome-wide profiling of 5fC βGT-catalyzed glucosylation βGT-catalyzed UDP-6-N 3 glucosylation Biotinylation Convert 5fc to 5hmc Streptavidin pulldown and DTT cleavage 5hmC C C C 5caC C C 5fc 5mC C C C C C C C C C C N 3 C C C C C Biotin N 3 S-S C C C C C oxBS-Seq Oxidative bisulfite sequencing (oxBS-Seq) to map 5-methylcytosine and 5-hydroxymethylcytosine DNA KRuO 4 5hmc residue Control Bisulfite treatment PCR amplification Bisulfite treatment PCR amplification C T C T T T C T T T 5hmC C C C 5caC C C 5fc 5mC 5fc 5hmC C C C 5caC C C 5fc 5mC C C C C C MAB-seq M.SssI methylase-assisted bisulfite sequencing (MAB-seq) to map 5fC/5caC DNA M.SssI 5fC/5caC residues Control C T C T T C T C C T Bisulfite treatment PCR amplification Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC 5hmC C C C 5caC C C 5fc 5mC C C C C C 5mC RRMAB-seq Reduced representation M.SssI methylase-assisted bisulfite sequencing (RRMAB-seq) DNA M.SssI Digest with MspI Add methylated adapters 5fC/5caC residues Control C T C T T C T C C T Bisulfite treatment PCR amplification Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC C C C C C 5hmC C C C 5caC C C 5fc 5mC 5mC C T C T T RedBS-Seq caMAB-seq Reduced bisulfite sequencing (redBS-Seq), to map 5-formylcyto- sine (5fC) in DNA DNA NaBH 4 5fc residue Control C C T T C Bisulfite treatment PCR amplification Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC 5hmC C C C 5caC C C 5fc 5mC C C C C C 5hmC C T C T T fCAB-Seq 5fC chemically assisted bisulfite sequencing (fCAB-seq) method for the base-resolution detection of 5fC DNA O-ethylhydroxylamine (EtONH 2 ) 5fc residue Control Blocked C C T T C Bisulfite treatment PCR amplification Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC 5hmC C C C 5caC C C 5fc 5mC C C C C C C T T T T TAB-Seq TET-assisted bisulfite sequencing, (TAB-Seq) to map 5-hydroxymethylcytosine DNA 5hmc residue βGT Glucosylation Oxidation TET Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC 5gmC C C C C C 5gmC C C C 5caC C 5caC C 5caC JBP1-seq DNA J-binding protein 1 sequencing (JBP1-seq), for genome-wide profiling of 5-hydroxy-methylcytosine (5hmC) Tagmentation Transposomes Glucosylated 5-hmC PCR JBP1-magnetic bead pull down 5hmc residues T4-βGT Hydroxy-methyl- ated DNA Aba-seq DNA AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome (5hmC) Glucosylated 5-hmC Streptavidin magnetic bead pull down 5hmc residues T4-βGT Hydroxy-methyl- ated DNA AbaSI Biotinylated primers Ligate Fragment CAB-Seq Sequence 5caC residue Chemical modification-assisted bisulfite sequencing (CAB-Seq) for 5caC detection 1-ethyl-3-[3-dimethylamino-propyl]-car- bodiimide hydrochloride (EDC) chemistry Streptavidin pulldown and DTT cleavage Nu S-S Linker Bisulfite treatment PCR amplification 5hmC C C C 5caC C C 5fc 5mC o-acylisourea 5hmC C C C 5caC C C 5fc 5mC Biotin S-S 5hmC C C C 5caC C C 5fc 5mC C C T T T C C C T T Control 5hmC C C C 5caC C C 5fc 5mC C C C C C S 5caC Extract DNA Fractionate Elute with increasing salt 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) DNA DNA purification Capture biotinylated MBD on Streptavidin coated magnetic beads MBD Streptavidin Biotin MBDCap-seq Methyl- Cap-seq MBD-Seq MiGS MIRA Methylated-CpG island recovery assay (MIRA) DNA 5mc CpG island DNA purification PCR amplification C G C G C 5mC G C 5mC G G C C G C G C G G C G C G MBD2B/MBD3L1 protein complex Isolate on glutathione-coated beads Fractionate C G C G C G C G C G C Methyl-Seq MRE-Seq Methyl-seq and MRE-Seq use methyl-sensitive enzymes to identify methylation patterns Methylated sites in the genome MspI HpaII G G C C G G C C G G C C G G C C G G C C G G C C G G C C G G C C Sequence Align sequences and determine undigested sites Split sample Identified methylation site Restriction enzyme digest BSAS DNA Bisulfite amplicon sequencing (BSAS) Methylated DNA Tagmentation Transposome with adaptor Amplicons PCR with primers specific for bisulfite converted DNA Bisulfite conversion Bisufite-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 NH 2 N O N NH 2 N O CH 3 Cytosine 5-Methyl Cytosine N NH 2 N O CH 3 5-Methyl Cytosine N N O O Uracil Bisulfite conversion R NH2 HO N N O O HO O OH OH Glucosylated 5hmc HO HO O O HO HO O HO OH OH P O - O OH P O - O N O NH O Uridine diphosphate glucose (UDP-Glu) R NH2 HO N N O O HO O OH N3 N3-5GMC Pyridostatin (PDS) O H 2 N NH NH O N H 2 N H 2 N O O O Pyridine Bubble-Seq Libraries of restriction fragments that contain replication initiation sites (bubbles) in vivo Bubble-containing fragment Restriction digest Run gel Cast fragments in trapping gel Recover bubble-con- taining plug DNA extraction Add sequencing primers DNA 2b-RAD Genome 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-specific adapters Amplify Amplify DNA Add sample-specific adapters RAD PE 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-Seq AA(A) n Single cell RNA DNA AA(A) n RNA DNA Single cell RT with barcoded primer Lyse cell Ad-2 primer Split samples Quasilinear amplification Sequence gDNA amplification cDNA amplification TTTTTTTTTT AAAAAAA PCR and Remove adaptors 2nd strand synthesis SLAF-seq Genome Restriction sites Specific locus amplified fragment sequenc- ing (SLAF-seq) for large scale genotyping Digest with MseI Add MseI adaptors PCR PCR with barcoded MseI primer DNA Add 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 RNA Lyse cell Sequence TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-seq2 Whole genome amplification 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 RNA Lyse cell Sequence TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer Streptavidin magnetic bead with mRNA capture primer TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-seq2 Whole genome bisulfite 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 RNA Lyse cell Sequence TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer Streptavidin magnetic bead with mRNA capture primer TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-seq2 Whole genome amplification with RRBS Rapture Genome Restriction sites Restriction-site associated DNA marker generation (RAD) capture (Rapture) Digest with restriction enzyme Restriction enzyme digest Add well-specific barcodes Pool Plate 1 DNA Hybridize Biotinylated capture bait Pool wells and shear Library prep with plate barcodes Well 1 Well 2 Streptavidin pull down Streptavidin pull down Plate 2 Hi-C 3-C Capture-C Ligation Crosslink proteins and DNA Sample fragmentation PCR amplify ligated junctions DNA Chromatin conformation capture (3-C, Hi-C and Capture-C) NG Cap- ture-C Ligation Formaldehyde fixation Restriction enzyme digestion De-crosslink and extract DNA Sonicate to 200 bp fragments DNA PCR Next-generation Capture-C (NG Capture-C) Add indexed sequencing adaptors Hybridize biotinylated capture bait Streptavidin pull down LAM-HTGTS DNA Linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) Fragmentation by sonication Tagged PCR Enzyme blocking Nested PCR Purify Breakage in genome Streptavidin magnetic bead pull down Adaptor ligation LAM-PCR with biotinilated primer NNNN NNNN HTGTS DNA Linear amplification-mediated high-throughput genome-wide sequencing (HTGTS) Fragmentation by sonication Tagged PCR Nested 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 DNA Chromatin immunoprecipitation with sequencing library preparation by Tn5 transposase (ChIPmentation) Chromatin immunoprecipitation Chromatin DNA purification Amplification Adaptor insertion Tn5 Transposome DNaseI-SIM DNase I digestion Terminate DNase I digestion DNA extraction DNA Dnase I simplified in-nucleus method (Dnase I SIM) for plants Cells Lyse and centrifuge Sort nuclei Supernatant Nuclei Nucleus Nucleus Polish ends DNA fC-CET 5fc residue 5fC based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR 5hmC C C C 5caC C C 5fc 5mC N 3 C C C C C Biotin N 3 S-S C C C C C N 3 SH C C C C C C C C C T DBCO-S-S-PEG3-biotin Azido 1,3-indandione (AI) Pulldown, NaOH and DTT Adaptor ligation and PCR Purify DNase-Seq DNaseI-Seq Active chromatin Isolate trimmed complexes DNase I digestion DNA extraction DNA DNase I hypersensitive sites sequencing (DNase-Seq, DNaseI-Seq) MPE-seq Active chromatin Isolated nuclei Add bathophenanthroline MPE digestion DNA extraction DNA Methidiumpropyl-EDTA sequencing (MPE-seq) Methidiumpro- pyl-EDTA (MPE) O O - O O O - O - O - N N N NH Fe H NH 2 H 2 N O N Me Single-cell triple omics sequencing (scTrio-seq) scTrio-seq AA(A) n Single cell RNA DNA DNA methylation Cell suspension Isolate single cell Lyse and centrifuge Supernatant Nucleus AA(A) n RNA Add carrier RNA AA(A) n T T (T) n cDNA synthesis PCR and sequence Add poly A with TDT Hybridize oligo AA(A) n DNA Add sequencing adaptors PCR and sequence Align sequences Methylated regions Methylated adapter End repair and ligation Bisulfite conversion Converted fragments MspI digestion PCR and sequence Methylated DNA GUIDE-seq Oligo insert DNA Genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq) Genome DNA dsOligo tag integrat- ed in live cells DNA isolation Random shearing End repair Adaptor ligation dsOligo-specific amplification dsOligo insert PCR Oligo tag UMI UMI P7 P5 Index 2 Index 1 Preparation of acylated RNA for biotin–streptavidin purification. DIBO, dibenzocyclooxtyne N O N 3 O DNA N O O DNA N N N Biotin N N N O N 3 DNA + Acylation DIBO-biotin “click” MINCE-seq Sonication and MNase digestion Streptavidin capture DNA Mapping in vivo nascent chromatin with EdU and sequencing (MINCE-seq) Crosslink with formaldehyde Click reaction to attach biotin Label with ethynyl deoxyuridine (EdU) DNA extraction Chase with Thymidine Polymerase Newly-formed chromatin Streptavidin SSB-Seq Single-strand break DNA Map 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-Seq Double-strand break DNA Map DNA double-strand breaks (DSB-Seq) Genome DNA DNA isolation and random shearing Capture on streptavidin beads TdT and biotinylated-dUTP scAba-seq DNA Detect 5hmC marks in single cells with AbaSI nuclease (scAba-seq) Glucosylated 5-hmC 5hmc residues T4-βGT Hydroxy-methyl- ated DNA AbaSI Ligate Pool T7 amplification Primer Illumina 5’ adaptor T7 promoter Adaptor with cell-specific barcode Single cell Droplet-based single-cell ChIP-seq (Drop-ChIP) Drop-ChIP scChIP-seq Single cell Barcoded sequences from single cells Cell suspension Droplet with unique oligos Load single cells into droplets with lysis buffer and MNase Fuse droplets Pool all droplets Sequence Chromatin immuno- precipitation Single cell scATAC-Seq (Microfluidics) Fragmented and primed DNA Single-cell assay for transposase accessible chromatin (scATAC-Seq) Lyse and introduce Tn5 transposase Pool libraries from all cells Amplify with cell-specific barcodes Insert in regions of open chromatin Cell suspension Microfluidics device Isolate single cell Very rare mutation Safe-SeqS DNA Shear Mutation Amplify and solid phase capture Sequence Safe-sequencing system is a unique molecular identifier (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-seq Heat 40 ° C in SDS DNA Purification Fixed-tissue chromatin immuno- precipitation sequencing (FiT-Seq) Deparaffination and rehydration FFPE-fixed chromatin Proteinase K digestion Enzyme inactivation Sonicate Soluble extract Immunoprecipitate Cromatin elution PAT-ChIP MNase digestion DNA Purification Pathology tissue chromatin immunoprecipitation (PAT-ChIP) Deparaffination and rehydration FFPE-fixed 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 amplification DNA Targeted chromosome conformation capture (4C) with unique molecular identifiers (UMI-4C) Crosslink proteins and DNA Adaptor Universal primer DS primer US primer Pad BLESS Double-strand break DNA Breaks 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 T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T 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 type Deletion Insert scRC-Seq Genomic 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 amplification Create sequencing library Sequence capture Nucleus EpiRADseq Genome 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 ddRADseq Genome Restriction sites Double digest restriction-site associated DNA marker generation (ddRADseq) Restriction digestion Second restriction digest Add barcoded adapters Add P2 adapter Amplify DNA hyRAD Genome DNA to be captured High quality DNA Hybridization RAD (hyRAD) for degraded DNA Shotgun library Hybridize to probes Prepare RAD-seq library and size select DNA Adaptor removal and labeling Biotinylated probes Streptavidin pull down Genome MDA IMS-MDA MIDAS Hybridize primers Nascent replication fork Phi 29 Phi 29 S1 nuclease Amplified DNA 3’ blocked random hexamer primers Synthesis Synthesis Multiple displacement amplification (MDA). Immunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA) Microwell displacement amplification system (MIDAS) T7 promoter Read primer THS-seq End fill-in and IVT amplification Add barcodes and sequencing adaptors Transposome hypersensitive site sequencing (THS-seq) Tn5 Transposome Open DNA dsDNA synthesis Insert in regions of open chromatin Purify Single cell scATAC-Seq (Cell index) DNA Single-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 SMDB Single-molecule droplet barcoding (SMDB) DNA templates Single template encapsulation Template amplification Template fragmentation Barcode every droplet Pool for library prep DNA Digoxigenin O HN HN O - O - N H O O OH HO CH 3 O CH 3 O O O N O O O O O - O - O O - O - O O SNS RNA primer gDNA SNS gDNA NSCR Nascent 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 NH 2 O 5fC-AI N N N N O H N O N N N DNA O O H S N 3 N N N DNA O O O O N 3 5fC AI-mediated cyclization labeling in fC-CET BisChIP-Seq ChIP-BS-seq Immunoprecipitate Sonicate DNA Bisulfite conversion C G T C T C G T U T Bisulfite C G T T T PCR Bisulfite-treated chromatin immuno- precipitated DNA (BisChIP-seq) and ChIP-BS-seq), to correlate protein modifications with DNA methylation DNA-protein complex with methylated histones and methylated DNA Purify DNA and shear Repli Seq Nascent DNA BrdU Run-on with analog Sort cells and lyse DNA Repli Seq—to map temporally ordered replicating DNA Purify Bead coated with anti-BrdU antibody Elute End Repair NS-seq Nascent DNA DNA replication origin Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo) DNA Nascent strand sequencing (NS-seq) to discover DNA replication origins and G4 structures Amplify Purify Digestion
