for all you seq dna | ngs dna library prep methods€¦ · prepa red fragments ~10kb p5 index 2...

FDNA

or all you seq...

Aba-seq Sun Z. et al. (2013) Cell Rep 3: 567-576BisChIP-Seq Statham A. L. et al. (2012) Genome Res 22: 1120-1127Bisulfite-seq Berman B. P. et al. (2012) Nat Genet 44: 40-46BSAS 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

CAB-Seq Lu X. et al. (2013) J Am Chem Soc 135: 9315-9317CAP-seq Illingworth R. S. et al. (2010) PLoS Genet 6: e1001134ChIP-BS-seq Brinkman A. B. et al. (2012) Genome Res 22: 1128-1138CPT-seq Amini S. et al. (2014) Nat Genet 46: 1343-1349ddRADseq Peterson B. K. et al. (2012) PLoS One 7: e37135DIP-seq Shen L. et al. (2013) Cell 153: 692-706

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: 226-234fCAB-Seq Song C. X. et al. (2013) Cell 153: 678-691fC-Seal Song C. X. et al. (2013) Cell 153: 678-691G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522G4-seq Chambers V. S. et al. (2015) Nat Biotechnol 33: 877-881

HELP-Seq Oda M. et al. (2009) Nucleic Acids Res 37: 3829-3839hMeDIP-seq Jin C. et al. (2014) Nucleic Acids Res 42: 6956-6971HydEn-seq Clausen A. R. et al. (2015) Nat Struct Mol Biol 22: 185-191Ig-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 van Opijnen T. et al. (2013) Nat Rev Microbiol 11: 435-442

JBP1-seq Cui L. et al. (2014) Genomics MAB-seq Wu H. et al. (2014) Nat Biotechnol MALBAC Zong C. et al. (2012) Science 338: 1622-1626MBD-Seq Nair S. S. et al. (2011) Epigenetics 6: 34-44MDA Dean F. B. et al. (2001) Genome Res 11: 1095-1099MDBCap-Seq de Assis S. et al. (2012) Nat Commun 3: 1053

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: 1044-1056MiGS Serre D. et al. (2010) Nucleic Acids Res 38: 391-399MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761MIRA Rauch T. A. et al. (2010) Methods 52: 213-217

MRE-Seq Maunakea A. K. et al. (2010) Nature 466: 253-257Nuc-Seq Wang Y. et al. (2014) Nature 512: 155-160OS-Seq Myllykangas S. et al. (2011) Nat Biotechnol 29: 1024-1027oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937PBAT Miura F. et al. (2012) Nucleic Acids Res 40: e136RAD Baird N. A. et al. (2008) PLoS One 3: e3376

RAD-Seq (PE) Willing E. M. et al. (2011) Bioinformatics 27: 2187-2193RC-Seq Baillie J. K. et al. (2011) Nature 479: 534-537RedBS-Seq Booth M. J. et al. (2014) Nat Chem 6: 435-440Rep-Seq Benichou J. et al. (2012) Immunology 135: 183-191Ribo-Seq Ingolia N. T. et al. (2009) Science 324: 218-223RRBS Meissner A. et al. (2008) Nature 454: 766-770

RRMAB-seq Neri F. et al. (2015) Cell Rep 10: 674-683scBS-seq Smallwood S. A. et al. (2014) Nat Methods 11: 817-820scRRBS Guo H. et al. (2015) Nat Protoc 10: 645-659smMIP Hiatt J. B. et al. (2013) Genome Res 23: 843-854SNES Leung M. L. et al. (2015) Genome Biol 16: 55TAB-Seq Yu M. et al. (2012) Cell 149: 1368-1380

TAmC-Seq Zhang L. et al. (2013) Nat Commun 4: 1517TC-Seq Klein I. A. et al. (2011) Cell 147: 95-106TN-Seq van Opijnen T. et al. (2013) Nat Rev Microbiol 11: 435-442T-WGBS Wang Q. et al. (2013) Nat Protoc 8: 2022-2032WGBS See BS-Seq

References

TruSeq RNA

AAAAA mRNA

T T T T TAAAAA polyA select

Fragment

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FOR RESEARCH USE ONLY

© 2015 Illumina, Inc. All rights reserved.Illumina, HiSeq, MiSeq, 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.

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


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


TruSeq Small RNA

3’5’ Small RNA fragment

Ligate adaptors

Add primer

Reverse transcription


5’ Adapter 3’ Adapter

P7

Index 1

P5

P5 P7Index


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


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


Nextera Library Preparation

Transposase

DNA

~300bp

Tagmentation

P5 P7Index 1Index 2

P5

Index 2

Index 1

P7


Nextera Mate Pair

Adaptor ligation

Isolate biotinylatedfragment


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


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


Extension-Ligation

O O

OH

NH

HN

H

H S

Biotin

EDTAO

O

O-O

O

O-

O-

O-

N

NM

Display methods on mobile device

N

NN

N

DNA Low-Level Detection

DNA Rearrangements and Markers

GenomeMDAIMS-MDA Hybridize primers

Nascent replication fork

Phi 29 Phi 29 S1 nuclease

3’ blocked random hexamer primers

Synthesis SynthesisImmunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA)

Duplex-Seq α βVery rare mutation

Duplex sequencing detects very 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

Consensus

Create duplex sequences based on molecular tags and sequencing primers

Add adaptors

Ig-seqRep-Seq

CDR3 junction region V D J Constant

DNADNA sequencing of immunoglobulin genes (Ig-seq) for antibody repertoire determination

Extracted RNA Reverse transcription8N UID

8N UID

Second strand synthesis PCR Purify

CpG island

CAP-seqDNA

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

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

RADPE RAD-SeqddRADseq

Genome Restriction sites

Restriction-site associated DNA marker generation (RAD) Double digest restric-tion-site associated DNA marker genera-tion (ddRADseq)

Restriction digestion

Shear (Second restriction digest in ddRADseq)

Add barcoded adapters Add P2 adapter

Amplify DNA

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 DNA

Cycles of quasilinear

Looped full amplicons

Bst DNA polymerase

OS-Seq Gene

Target sequence

Adaptor sequence

Flow cell

Sequencing PrimersTarget sequence Single adaptor library

Hybridize Hybridize

SequenceOligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on

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)

-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 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

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

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 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

sequencing (RRBS-Seq). Single cell RRBS (scRRBS)

Methylated regions

Methylated adapter

End repair and ligation conversion

Converted fragments PCRPCRMspI digestion

PBATtagging (PBAT)

Methylated DNA -din coated magnetic beads

Second random priming

Streptavidin

BiotinAdaptor

conversionFirst random priming

AdaptorRandom primer 2

Generate second strand DNA with adaptorsElution

BS-SeqBisulfite-seqWGBS

DNAShear DNAMethylated DNA

C GTCT

C GTUT

C GTTT

PCR

BSPPpadlock probes (BSPP)

CpG islandconversion 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

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

C GTCT

C GTUT

C GTTT

PCR

Converted single-stranded fragments

Random primingDNA synthesis

3’ tagging PCR

Random primer 1

Methylated DNAconversion

Random primer 2scBS-seqSequence

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 Insert in regions of open chromatin

DNase-SeqDNaseI-Seq

Active chromatin Isolate trimmed complexesDNase I digestion DNA extraction DNADNase I hypersensitive sites sequencing (DNase-Seq, DNaseI-Seq)

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 FAIRE-Seq: formaldehyde-assisted Isolation of regulatory elements 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

-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

Chem-seq: to identify the sites bound by small chemical molecules

DNA-protein complex with putative drug binding site

in vivo

in vitro

Streptavidin

Add biotinylated compound

Harvest cells and fragment DNA

HiTS-FLIPTarget sequence

HybridizeHigh-throughput sequencing:

Protein binding site

Prepare sequencing libraries and

Remove second strand DNA

Flowcell

Hybridize primer

Synthesize second strand with unmodi-

-ly labeled ligand

Elute with increas-ing stringency

Binding siteKlenow

ChIP-SeqChIP-exoHT-ChIP Exonuclease digestion Immunoprecipitate DNADNA-protein complex DNA

extractionCrosslink 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

chromatin (ORGANIC)

Isolate nuceiCell Isolated chromatin Soluble extract Immunoprecipitate and DNA extraction

X-ChIPMNase digestion DNAHigh resolution of mapping of in vivo

chromatin associated proteinsCrosslink 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 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)

Hi-C3-CCapture-C

LigationCrosslink proteins and DNA Sample fragmentation PCR amplify ligated junctions DNAChromatin conformation capture (3-C, Hi-C and Capture-C)

DamID

DNA adenine methyltransferase interaction detection (DamID)

DNA-protein interaction

Create fusion protein

Align sequences and determine Split sample DpnI digestion Unmethylated GATCs are cut by DpnII

Adaptor ligation

Non-targeted methylation

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

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))

DNA

hMeDIP-Seq

Extract DNA FractionateDenature

ImmunoprecipitateHydroxymethylated DNA

Hydroxymethylated DNA immunopre-cipitation combined with next genera-tion DNA sequencing (hMeDIP-seq)

DNA

T-WGBSDNATagmentation-based

sequencing (T-WGBS)

Methylated DNA TagmentationTransposome withmethylated adaptor

Displaced oligo

Oligo withmethylated adaptor

Displace oligoHybridize methylated adaptor and gap repair

PCRconversion

BisChIP-SeqChIP-BS-seq

ImmunoprecipitateSonicate DNA

C GTCT

C GTUT

C GTTT

PCR

-precipitated DNA (BisChIP-seq) and ChIP-BS-seq), to correlate protein

DNA-protein complex with methylated histones and methylated DNA

Purify DNA and shear

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

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-Seq(oxBS-Seq) to map 5-methylcytosine and 5-hydroxymethylcytosine

DNAKRuO4

5hmc residue ControlC TCT T

TCT TT

5hmC

CC C

5caC

CC

5fc5mC

5fc

5hmC

CC C

5caC

CC

5fc5mC

C C CCC

MAB-seqsequencing (MAB-seq) to map 5fC/5caC

DNAM.SssI

5fC/5caC residues ControlC TCT T

C TCC T

5hmC

CC C

5caC

CC

5fc5mC5hmC

CC C

5caC

CC

5fc5mC

CCC CC

5mC

RRMAB-seq Reduced representation M.SssI

sequencing (MAB-seq)

DNAM.SssI Digest with MspI

Add methylated adapters

5fC/5caC residues ControlC TCT T

C TCC T

5hmC

CC C

5caC

CC

5fc5mC

CCC CC

5hmC

CC C

5caC

CC

5fc5mC

5mC

C TCT T

RedBS-Seq(redBS-Seq), to map 5-formylcyto-sine (5fC) in DNA

DNANaBH4

5fc residue Control

CCT TC

5hmC

CC C

5caC

CC

5fc5mC5hmC

CC C

5caC

CC

5fc5mC

C CC C C

5hmC

C TCT T

fCAB-Seqsequencing (fCAB-seq) method for the base-resolution detection of 5fC

DNAO-ethylhydroxylamine (EtONH2)

5fc residue ControlBlocked

CCT TC

5hmC

CC C

5caC

CC

5fc5mC5hmC

CC C

5caC

CC

5fc5mC

C CC C C

C TT T TTAB-Seqto map 5-hydroxymethylcytosine

DNA

5hmc residue βGT

Glucosylation Oxidation

TET5hmC

CC C

5caC

CC

5fc5mC 5gmC

CC C CC

5gmC

CC C

5caC

C

5caC

C

5caC

JBP1-seqDNAJ-binding protein 1 sequencing

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

for 5caC detection

1-ethyl-3-[3-dimethylamino-propyl]-car-bodiimide hydrochloride (EDC) chemistry

Streptavidin pulldownand DTT cleavage

Nu S-S

Linker

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 Capture biotinylated MBD on Streptavidin coated magnetic beads

MBDStreptavidinBiotin

MBDCap-seqMethyl-Cap-seqMBD-SeqMiGS

MIRAMethylated-CpG island recovery assay (MIRA)

DNA

5mc CpG islandCGCGC

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 samplemethylation site

Restriction enzyme digest

BSASDNA

sequencing (BSAS)Methylated DNA TagmentationTransposome with

adaptorAmplicons

conversion-

verted DNA

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 primerStreptavidin magnetic bead with mRNA capture primer

T T T T T T T T T TAAAAAAA On-bead transcriptome

with MDA

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

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

for all you seq dna | ngs dna library prep methods€¦ · prepa red fragments ~10kb p5 index 2...

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