for all you seq dna | ngs dna library prep methods€¦ · prepa red fragments ~10kb p5 index 2...
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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
Illumina, Inc. • 5200 Illumina Way, San Diego, CA 92122 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • techsupport@illumina.com • illumina.com
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
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
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
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
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