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RNA Transcription
Novel
Exon 1 exon Exon 2 AA(A)n Cell 1 AA(A)n
AA(A)n
TT(T)n
AA(A)n
TT(T)n
Cell 1
Epigenetics T C T C G
CEL-Seq
Genome
sequence Area of interest O O O TT(T)n AA(A)n O Bisulfite

CaptureSeq Index 2 Index 1 Base AA(A)n Cell 2 AA(A)n AA(A)n TT(T)n Cell 2
5’ OH 3’ P5 P7 HO P O P O P O TT(T)n
BS-Seq T U T C G

5’ o m
T7

Un apto r
— biased against detecting novel exons AA(A)n Cell 3 AA(A)n AA(A)n H3C O S O CH3

ad ot
pr
O

iq r
RNA capture sequenc- — can detect novel exons Purified RNA TruSeq stranded mRNA cDNA library PCR O O O AA(A)n
Hybridize capture probes Library enriched
Bisulfite-seq Bisulfite conversion of genomic DNA (bs-Seq)
TT(T)n PCR

ue
Cell 3
TT(T)n
ing (CaptureSeq) Design capture probes sample preparation

in
with targeted RNAs Pool Methylated DNA Shear DNA Bisulfite conversion T T T C G DNA

de
e
Cell expression by linear amplifica- Second strand Fragment, add PCR Separate cell sequences O

x
Index primer P5 primer Index primer P5 primer Index primer P5 primer N
N + tion and sequencing (CEL-Seq) RNA synthesis adapters and based on unique indices
Dimethyl sulfate (DMS) WGBS or whole-genome bisulfite sequencing (WGBS)
T C T C G
RASL-seq
-

AA (A)n 5’ OH AA(A)n 3’ 5’
P OH
AAAAA 5’ AAAAA 5’ AAAAA Barcode
N reverse transcribe Bisulfite
TTTTT TTTTT TTTTT
P7 P5
3’-azido-2’,3’-dideoxynucle-
RNA-mediated oligonucleotide Poly A+ RNA Add targeted primers Ligate Selection
Streptavidin

Elute fragment Add bar-coded Indexed cDNA otide triphosphate (AzNTP)

AA(A)n Cell 1 AA(A)n
TT(T) n CCC
AA(A)n
TT(T) n
GGG
CCC TT(T) n
Cell 1
EpiGnomeTM T U T C G

STRT GGG PCR

annealing, selection, and ligation P7 primer AA(A)n Cell 2 AA(A)
TT(T)nn CCC
AA(A)n
TT(T) n
GGG
CCC TT(T) n Cell 2
Bisulfite conversion HELP-Seq Bisulfite conversion of genomic Methylated DNA Bisulfite conversion Converted single-stranded Random priming 3’ tagging PCR DNA

5’
Un ap
T T T C G
sequencing (RASL-seq)

ad
DNA without shearing fragments DNA synthesis

iq tor
AA(A)n

ue
5’ cDNA cDNA
AA(A)n Cell 3 AA(A)n GGG NH2 NH2
N3 N3 TT(T) n

in
5’ CCC TT(T) n CCC
ClickSeq N3 TT(T) n Cell 3

de
5’ CH3
scBS-seq

x
5’ N3 N3 N3 O
Base
O
Base Single-cell tagged reverse Add oligo-dT primer cDNA Add 3 to 6 Template Introduce Pool Single-primer Separate cell sequences N N Random primer 1 Random primer 2
Generate RNAseq libraries from RNA Add semi-random primers and Purify single-stranded Click-ligate alkyne- PCR amplify cDNA library O O
transcription (STRT) synthesis cytosines switching primer unique index PCR and purify based on unique indices Adaptor Adaptor
stochastically terminated 3’-azi- AzNTPs - dNTP mixture cDNA modified adaptor N N
do-blocked cDNA fragments (ClickSeq)
O O Single-cell bisulfite Isolated Lyse Methylated DNA Bisulfite First random Repeat Extend Exo I and Second random PCR Align fragments from every Sequence
3Seq N3
Cu(I) N N
TCRα mRNA
Oil emulsion
Cytosine 5-Methyl Cytosine sequencing (scBSBS-seq) single cell conversion priming 4 times purify priming unique molecular tag
TCRα TCRα
3-Seq
NVTT(T) n
AA (A)n AA (A)n AA (A)n AA(A)n
P5 P7 Rt
N
TCR Chain AA(A)n TCRα TCRβ TCRα TCRβ Streptavidin
Random primer 2
3’-end-seq NVTT(T) n P7
Paring CDR3α CDR3β Biotin
TCRβ mRNA TCRβ TCRβ Adaptor
Adaptor
PAS-Seq
3’-end sequencing for expression quantifi-
cation (3SEQ) from FFPE samples. Poly(A)
Partially degraded
RNA from FFPE
poly A selected
fragments
P7 primer
N Random base
Anneal primer Reverse
transcribe
2nd strand
cDNA
Ligate P5
linker
cDNA
CDR3
AA(A)n
PBAT
O NH2 Post-bisulfite adaptor Methylated DNA Bisulfite First random Capture first strand on Streptavi- Second random Generate second strand Elution DNA with adaptors
SS3-Seq
O O
site sequencing (PAS-Seq). 3’-end regions sample V Any base, except T synthesis Identify T-cell Receptor Reverse Amplification Overlap extension Blocker Nested PCR amplification PCR supression of DNA
of transcripts (3’-end-seq). Strand-specific
O P O- O P O-
CH3 tagging (PBAT) conversion priming din coated magnetic beads priming
(TCR) alpha–beta chain transcription primers non-fused molecules
3’T-fill 3’-end RNA-seq (SS3-Seq and 3’T-fill)
O
Base
O
Base pairing in single cells N N
MmeI AluI MmeI
TT(T) n
Biotin O O
Repeat 2 Repeat 1 H1P1 P2 H2
N N
BSPP
O O
AA (A)n AA (A)n AA (A)n AA(A)n Whole-genome RNA Mutation
Repeat 1 Uracil 5-Methyl Cytosine
3P-Seq TT(T) n
DNA

Copper-catalysed cycloaddition
DNA
CirSeq AA(A)n
Repeat 2 Bisulfite sequencing with CpG island Bisulfite Bisufite-converted Padlock Hybridize Extension Exonuclease PCR End repair DNA with
Poly(A)-position profiling T1 RNase partial digest Biotinylated Ligate biotinylated Streptavidin Anneal primer and RNase H digestion cDNA Repeat 3 padlock probes (BSPP) conversion DNA probe and ligation digestion and adaptor adaptors
of 3’-azido blocked cDNA to Repeat 3 Error Corrected sequence
by sequencing (3P-Seq) splint primer primer purify reverse transcribe Identify low abundance Circularize RNA Circular RNA Random ligation
5’-hexynyl DNA adaptor
RNA viruses with circular with kinase and template primers NH2
Poly(A)-selected RNA NVTT(T) 20

2P-seq AA (A)n AA (A)n AA (A)n AA(A)n NVTT(T) 20

sequencing (CirSeq) RNA ligase 1
N
RRBS
cDNA Cell
NVTT(T) 20 Biotin
scRRBS Reduced representation bisulfite Methylated MspI Methylated Methylated End repair Bisulfite Converted fragments PCR DNA
TIVA Whole-genome RNA Cy3 UUUUUUUUUUUUUUUUUU Cy3 UUUUUUUUUUUUUUUUUU Cy3 UUUUUUUUUUUUUUUUUU
Poly(A)-primed sequencing (2P-seq) to N Random base Reverse Sequence with PL PL Cy3 UUUUUUUUUUUUUUUUUU Cy3 UUUUUUUUUUUUUUUUUU N O sequencing (RRBS-Seq). DNA digestion regions adapter and ligation conversion
AA(A)n S Cy5 AAAAAAA PL AAAAAAA AAAAAAAAAAAAAAAAAA Single cell RRBS (scRRBS)
quantify usage of known poly(A) sites T1 RNase partial digest V Any base, except T transcription Circularize and PCR primer ending in A 20 CPP S S Cy5 AAAAAAA PL AAAAAAA S Cy 5 PL
AAAAAAAAAAAAAAAAAA

PL
CPP S AAAAAAA AAAAAAA
R
3’-Seq BSAS
SS Disulfide bond Cytosine
AA(A)n AAAAAA(A)n AAAAAA(A)n NBAAAAAA(A)n NBAAAAAA(A)n NBAAAAAA(A)n NBAAAAAA(A)n
NVTTTUTT(T)n NVTTTUTT(T)n TT(T)n
NVTTTUTT(T)n NVTTTUTT(T)n NVTTTTTT(T)n NVTTTTTT(T)n Transcriptome in PL Photocleavable linker Load into CPP peptide Photoactivate Anneal to mRNA Capture on Streptavidin mRNA from
vivo analysis (TIVA) CPP Cell-penetrating peptide cells released coated magnetic beads single cell NH2 Bisulfite amplicon Methylated DNA Bisulfite Bisufite-con- PCR with primers specific for Amplicons Transposome with Tagmentation DNA
3’-Seq to detect Reverse transcribe Synthesize 2nd Introduce nick at U Translate nick 5’ to 3’ Blunt end at PCR and cDNA CH3 sequencing (BSAS) conversion verted DNA bisulfite converted DNA adaptor
3’ UTR isoforms strrand with RNase HII for 50-75 bases nick purify Oligo dT N
Single cell Molecular index
MspI
TIF-Seq 5’ P AA(A)n Biotin
CytoSeq AA(A)n Cell label
Universal N O
C
C
C
C
G
G
G
G
C
C
C
C
G
G
G
G
5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ P AA(A)n
TT(T) n Methyl-Seq
PEAT Gene expression cytometry Cell Each bead with Load cells and beads Cell lysis, mRNAs Pool all beads from cDNA synthesis Sequence Barcoded mRNA HpaII
R
MRE-Seq
C C G G C C G G
Transcript isoform sequencing Ligate ‘5oligocap’ Reverse Second Incorporate (CytoSeq) suspension unique oligos into microwells hybridize on bead microwells and amplification from single cells 5-Methyl Cytosine Methyl-seq and MRE-Seq use C C G G C C G G
Capped mRNA Tobacco acid oligonucleotide Purify cDNA
(TIF-Seq). Similar to paired-end pyrophosphatase transcription strand methyl-sensitive enzymes to Methylated sites Split sample Restriction Sequence Align sequences and Identified
biotinilated Circularize and identify methylation patterns
analysis of TSSs (PEAT) strategy (TAP) treatment synthesis primers fragment Oligo dT in the genome enzyme digest determine undigested sites methylation site
Single cell Molecular index NH2

5’ P Drop-Seq AA(A)n Cell label

Universal
HO
Displaced oligo
N
SMORE-Seq 5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ P AA(A)n 5’ P AA(A)n
AA(A)n Analyze mRNA transcripts Sequence Barcoded mRNA
T-WGBS
Cell Each bead with Load cells and beads Cell lysis, mRNAs Pool all beads from cDNA synthesis Tagmentation-based Oligo with Displace oligo Bisulfite PCR
from individual cells in suspension unique oligos into droplets hybridize on bead droplets and amplification from single cells N O Methylated DNA Transposome with Tagmentation DNA
Simultaneous mapping of RNA Capped mRNA Tobacco acid Ligate Fragmentation Ligate reverse Reverse transcription PCR and purify cDNA droplets (Drop-seq) whole-genome bisulfite methylated Hybridize methylated conversion
methylated adaptor
ends by sequencing (SMORE-seq) pyrophosphatase 5’ adapter 3’ adapter transcription R sequencing (T-WGBS) adaptor adaptor and gap repair
(TAP) treatment Single cell RNA 5-hydroxymethylcytosine (5hmc)
RNA On-bead transcriptome 5hmc residues
AA(A)n AA(A)n AAAAAAA
GPPP GPPP P
P OH OH OH
G&T-seq
AAAAAAA
TTTTTTTTTT
TTTTTTTTTT amplification with Smart-seq2
O JBP1-seq
TL-seq
NH2
5’ GPPP AA(A)n 5’ GPPP AA(A)n OH OH OH OH DNA DNA Whole genome amplification
PPP OH OH OH
H CH3 with MDA J-binding protein 1 sequencing Hydroxy-methyl- Transposomes Tagmentation T4-βGT Glucosylated 5-hmC JBP1-magnetic bead PCR DNA
TATL-seq Enzymatic capture of m7G-capped Capped mRNA Fragmentation Phosphatase Pyrophosphatase Ligate Reverse Gel purify Ligate Reverse cDNA N
Genome and transcriptome Cell Isolate single Lyse cell Streptavidin magnetic bead Separate the DNA and the RNA Sequence Align RNA and
H N (JBP1-seq), for genome-wide profiling ated DNA pull down
mRNA 5’ ends (TL-seq). Transla- and size selection 5’ adapter transcription 3’ adapter transcription of 5-hydroxy-methylcytosine (5hmC)
N sequencing from a single cell suspension cell with mRNA capture primer genome
tion-associated TL-seq (TATL-seq) N
(G&T-seq) N O
O
5hmc residues
rRNA-depleted RNA N N Aba-seq
TAIL-seq AA(A)n AAAA(A)n AAAA AAAA AAAA AAAA AAAA
O P O
O 5-formylcytosine (5fC)
R
O AbaSI coupled with sequencing Hydroxy-methyl- T4-βGT Glucosylated 5-hmC AbaSI Biotinylated Ligate Fragment Streptavidin magnetic DNA
Measure genome-wide poly(A)
tail lengths (TAIL-seq)
3’ adaptor ligation Partial digestion
with RNase T1
Pull down with
streptavidin
5’ end
phosphorylation
Gel purify 5’ adaptor
ligation
RT, PCR and purify cDNA
OH OH
RNA Modifications O NH2
(Aba-seq) to map high-resolution
hydroxymethylome (5hmC)
ated DNA primers bead pull down

N3 N3S-S Biotin
HO N 5mC 5hmC 5mC g5hmC 5cmC g5hmC g5hmC
AAAAAAAAAAAAA

AAAAAAAAAAAAA

Methylated RNA Methylated RNA

TTUTTTTUTTTTU

TTUTTTTUTTTTU

N6-Methyladenosine (m6A) 5mc residue βGT TET g5hmC

MeRIP-Seq
AAAAAAAAAA

AAAAAAAAAA
TTTUTTTUTT

TTTUTTTUTT

Biotin
N O TAmC-Seq C T C C G C T C C G C T C C G C T C C G C T C C G
AAAAAA

AAAAAA
TTTUTT

TTTUTT

TTTTT
PAL-seq AA(A)n AA(A)n AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA TTTTTTTTTT m6A-seq Tet-assisted 5-methylcytosine
sequencing (TAmC-Seq)
Glucosylation Oxidation βGT-catalyzed
UDP-6-N3 glucosylation
Biotinylation Streptavidin pulldown
and DTT cleavage
DNA

m6A-RIP
TTTTT O R
m6A-specific methylated RNA immunoprecipitation with RBP Extract RNA Fractionate RNA Immunoprecipitate Reverse transcription cDNA
NH nextgeneration sequencing (MeRIP-Seq), (m6A-seq) 5-carboxylcytosine (5caC)
C mC C Biotin
Poly(A)-tail length Total RNA Splint Ligate Partial Bind to streptavi- Ligate adaptor Reverse transcribe Anneal sequencing Sequence and flow in fluores- Scan 5fc residue 5m 5h 5fc 5ca Blocked 5hmC residue 5hmC residue N3 N3S-S

profiling by sequencing oligo splint digest with din beads and and release from primer and extend with cent streptavidin. Measure flowcell N O Methylated RNA fC-Seal C C C C C C C C C C C C C C C C C C C C C C C C C
(PAL-seq) oligo RNase T1 wash beads dTTP and biotin-dUTP fluorescence intensity HO
O miCLIP A 5-formylcytosine-selective βGT-catalyzed NaBH4 Convert 5fc to βGT-catalyzed Biotinylation Streptavidin pulldown DNA
P7 P7 P5 P7 chemical labeling (fC-Seal) approach glucosylation 5hmc UDP-6-N3 glucosylation and DTT cleavage
FRT-Seq AA (A)n 5’ OH AA(A)n 3’ 5’ OH OH 3’ 5’ OH ddC P ddC AmC6 ddC
HO HO
m6A individual-nucleotide-res-
olution cross-linking and
Sheared cellular RNA Add anti-m6A antibody UV 365 nm Immunoprecipitate Reverse transcription truncated
at binding sites
cDNA HO
HO
OH
O for genome-wide profiling of 5fC
C mC
P ddC AmC6 OH
C
5m 5h 5fc 5ca
RNA DNA DNA RNA
Flowcell reverse transcription Poly A RNA
+
Fragment and P7 primer Gel purify P5 primer Hybridize to flowcell immunoprecipitation (miCLIP) O NH

sequencing (FRT-seq) for dephosporylate Phosphorylate and reverse transcribe Uridine

Ψ-seq Control
HO O- O- C C C C C T C C T T
o-acylisourea
strand-specific RNA-Seq Pseudouridine residues
N O C mC C C mC C C mC
O P O P O O 5m 5h 5fc 5ca 5m 5h 5fc 5ca 5m 5h 5fc 5caC S-S Biotin 5caC S
PSI-seq
AA(A)n AA(A)n Nu S-S
CAB-Seq
O
OH OH 5caC residue C C C C C C C C C C C C C C C C C C C C T C C T C

Bubble-Seq HN NH
Pseudouridine site identification PolyA selected CMC treatment Fragmentation Adaptor ligation Reverse Purification cDNA
HO HO
Chemical modification-assisted 1-ethyl-3-[3-dimethylamino-propyl]-car- Linker Streptavidin pulldown Bisulfite treatment Sequence
O sequencing (PSI-seq and Ψ-seq) DNase treated RNA and OH- treatment transcription Uridine diphosphate bisulfite sequencing (CAB-Seq) bodiimide hydrochloride (EDC) chemistry and DTT cleavage PCR amplification
HO glucose (UDP-Glu) for 5caC detection
Libraries of restriction fragments Bubble-containing Restriction Cast fragments in Recover bubble-con- Add sequencing O Pseudouridine residues C mC
that contain replication initiation fragment digest trapping gel
Run gel
taining plug
DNA extraction
primers
DNA
Pseudo-seq AA(A)n 5m 5h 5fc 5ca
C
Bisulfite treatment
sites (bubbles) in vivo C mC C T C C T T
HO HO 5hmc residue 5m 5h 5fc 5ca Control C C C C C PCR amplification
OH
ncRNA ncRNA Pseudouridine (Ψ)
Pseudo-seq, a method for
genome-wide pseudouri-
PolyA selected RNA Fragmentation and
size selection
CMC treatment
and OH- treatment
Adaptor ligation Reverse
transcription
Size select Circularize with
circLigaseTM
PCR and purify DNA
O oxBS-Seq C C C C C 5fc Bisulfite treatment T C T T T DNA
HO Oxidative bisulfite sequencing KRuO C C C C C PCR amplification
dine identification HO O NH2 (oxBS-Seq) to map 5-methylcytosine 4
ChIRP Control T C I C G Reverse transcription
PCR amplification
T C G C G
OH
and 5-hydroxymethylcytosine
C mC C
H3C + Inosine residue Inosine residue N 5m 5h 5fc 5ca
N c N ICE T C I C G
N1-cyanoethylinosine
cDNA
C mC C Bisulfite treatment
5m 5h 5fc 5ca
N C C C C C T C C T T
Chromatin Isolation by RNA Purification (ChIRP) RNA-binding protein Crosslink Biotinylated tiling oligos Capture on Streptavidin RNase H DNA DNA Acrylonitrile N O 5fc residue Control PCR amplification
Fragment magnetic beads Inosine chemical erasing (ICE) Reverse transcription
Hybridize extraction T C I C G
PCR amplification X RedBS-Seq C C C C C
5h
mC
R DNA
O O Reduced bisulfite sequencing NaBH Bisulfite treatment
Glucosylated 5hmc 4 C C C C C T C C C T
(redBS-Seq), to map 5-formylcyto- PCR amplification
H3C s O-
CHART RNA Structure
ncRNA ncRNA sine (5fC) in DNA
C mC C
O 5m 5h 5fc 5ca
C mC C Bisulfite treatment
5m 5h 5fc 5ca
T C C T T
Capture hybridization analysis of Crosslink and Hybridize biotinylated Capture, wash Extract DNA or Western DNA 1-cyclohexyl-(2-morpholino- 5fc residue Control
C C C C C
PCR amplification
ethyl)carbodiimide-
RNA targets (CHART) lyse cells probes and elute blot analysis of proteins
metho-p-toluene sulfonate
N3
fCAB-Seq C C C C C Blocked
DNA
O 5fC chemically assisted bisulfite Bisulfite treatment
(CMCT) HO sequencing (fCAB-seq) method for
O-ethylhydroxylamine (EtONH2) C C C C C
PCR amplification
T C C C T

lncRNA lncRNA
SHAPE-Seq HO
OH
O NH2
the base-resolution detection of 5fC
RAP Selective 2’-hydroxyl acylation RNA Barcoded RNA 1M7 reaction Reverse transcription RNA hydrolysis cDNA N
C mC
5m 5h 5fc 5ca
C
Bisulfite treatment
C mC C T C C T T
RNA antisense purification (RAP) Crosslink and Hybridize biotinylated 120 Capture, wash and elute PCR and reverse analyzed by primer extension 5fC/5caC residues 5m 5h 5fc 5ca Control
C C C C C
PCR amplification
cDNA sequencing (SHAPE-Seq) N O
lyse cells bp antisense probes transcribe mC
MAB-seq
C C C C C 5
Bisulfite treatment DNA
GRO-seq Nascent RNA Br-UTP O NH Cell NAI-N3
N3 biotin
N3-5GMC
R M.SssI methylase-assisted bisulfite
sequencing (MAB-seq) to map 5fC/5caC
M.SssI C C C C C
PCR amplification
C C C T T

BRIC-Seq HN N
icSHAPE N3 C mC
5m 5h 5fc 5ca
C
N
C mC Bisulfite treatment
Bru-Seq O
5fC/5caC residues 5m 5h 5fc 5ca
C
Control
C C C C C
PCR amplification
T C C T T

mC
BruChase Global run-on-sequencing (GRO-seq)
5’-Bromo-uridine immunoprecipitation
Run-on with analog Isolate and hydrolyze Bead coated with
α-BrdUTP antibody
Elute
Cap removal
Reverse
transcription
cDNA HO O
H3C
In vivo click selective
2’-hydroxyl acylation and pro-
In vivo folded RNA Add azide group DIBO-biotin ‘click’ Reverse transcription Capture on Streptavi-
din beads
Elute cDNA
RRMAB-seq Reduced representation M.SssI Digest Add methylated
C C C C C
M.SssI
5
Bisulfite treatment C C C T T DNA
C C C C C
-Seq chase (BRIC-Seq) End Repair amplification
O
N filing experiment (icSHAPE)
OH NH2 SO3 -
NH2
methylase-assisted bisulfite
sequencing (MAB-seq)
with MspI adapters
PCR amplification

Nascent RNA Br-UTP HO HO C mC C C

mC
5’ cap 5’ cap N N
5ca 5g 5ca 5ca
5’-GRO-seq
OH
OH
5’ cap
OH OH
Ψ-CMC adduct CIRS-seq NaHSO3
5hmc residue C mC
5m 5h 5fc 5ca
C βGT 5g TET Bisulfite treatment
OH
OH OH OH OH OH
DMS N O NaOH N O TAB-Seq C C C C C C C C C C
C C C C C
PCR amplification
T T C T T

TET-assisted bisulfite sequencing, (TAB-Seq) Glucosylation Oxidation DNA

Detect nascent RNA with a 5’ 7-methyl- Bead coated with Cap Reverse R R to map 5-hydroxymethylcytosine
Run-on with analog DNase 3’ dephosphorylate 5’ dephosphorylate Ligate adaptors with cDNA Chemical inference of RNA In vivo folded RNA Proteinase K RNA Reaction Reverse transcription RNA hydrolysis cDNA
guanylated cap (5’-GRO-seq) treatment and with polynucleotide α-BrdUTP antibody with calf intestinal- removal truncated mutant RNA transcription structures (CIRS-seq) treatment 5hmC CMS
hydrolyze kinase phosphatase ligase 2 and RNA ligase 1 amplification O C G C
C C
Br Adduct induced cytosine-5-methyl- 5mc CpG island 5m 5m C G C
NH SHAPE adducts sulfonate (CMS)
C G C

MIRA
Nascent RNA DRB Br-UTP
mutations C G C G C G C C G C G C G C G C G C G

BruDRB-seq RNAPII
HO P
O
O P
O
O P
O
O
N O Methylated-CpG island Fractionate MBD2B/MBD3L1 Isolate on glutathione-coated DNA purification DNA
Mn2+ Profile recovery assay (MIRA) protein complex beads PCR amplification
O O O O
SHAPE-MaP
BruDRB-seq— to reveal gene-specific Transient inhibition of Remove Synchronized run-on Isolate and hydrolyze Bead coated with α Elute Reverse cDNA
differences in elongation rates initiated RNAPII with analog Brd-UTP antibody Cap removal transcription
DRB
MeDIP-Seq
OH OH O
End Repair amplification
Br-UTP Selective 2’-hydroxyl acylation 1M7 reaction cDNA reaction Library prep and Align sequence and
DIP-seq
N NH
Nascent RNA DRB Biotin analyzed by primer extension and sequencing count mutations
mutational profiling (SHAPE-MaP) Methylated DNA Immunoprecipitation Methylated DNA Extract DNA Fractionate Immunoprecipitate DNA purification DNA
4sUDRB-seq RNAPII A C A C
CH2OH
N N (MeDIP-Seq), DNA immunoprecipitation followed
by high throughput sequencing (DIP-seq))
Denature
O +DMS O
Br A C
4sUDRB-seq— to reveal gene-specific
differences in elongation rates in vivo
Transient inhibition of
initiated RNAPII
Remove
DRB
Synchronized run-on
with analog
Biotinylated fragments Bead coated with
streptavidin
Elute Reverse
transcription
cDNA NH
Struc-
amplification
N O ture-Seq Control
A C A C OH OH hMeDIP-Seq
BrdU HO Inosine
Nascent RNA O DMS-Seq Hydroxymethylated DNA immunopre- Hydroxymethylated Extract DNA Fractionate Immunoprecipitate DNA purification DNA
Repli Seq Use dimethyl sulphate
methylation of unprotected
Structure
in vivo
In vivo
treatment
Use DMS to methylate the RNA
base-pairing faces of A and C
Random
hexamers
Reverse
transcription
Ligate linker PCR DNA N
cipitation combined with next genera- DNA Denature
adenines and cytosines in loops Acrylonitrile tion DNA sequencing (hMeDIP-seq)
OH (Structure-Seq)
Repli Seq—to map temporally ordered
replicating DNA
Run-on with analog Sort cells and lyse Bead coated with
anti-BrdU antibody
Elute
End Repair
Purify DNA 5’-Bromo-uridine (BrdU)
Base to loop
N MBDCap-seq
Nascent RNA
processing P5 P7 O Methyl- MBD Biotin

Cap-seq
N Streptavidin
or N
PRO-seq Cl SPARE P5 P7
MBD-Seq
N
Loop to base N N Methyl-CpG binding domain-based capture and Methylated Extract DNA Fractionate Capture biotinylated MBD on Elute with increasing DNA DNA
NSCR processing P5 P7 CH2OH sequencing (MBDCap-seq). Capture of methylated DNA Streptavidin coated magnetic beads salt concentration purification
Precision nuclear run-on and sequencing (PRO-seq) Single biotin-NTP run-on Isolate and Hydrolyze Bead coated with Purify Reverse transcrip- cDNA OH
N
Cl
O MiGS DNA using the MBD domain of MeCP2 (Methyl-
Cap-Seq). MBD-isolated Genome Sequencing (MiGS)
Nascent strand capture and release (NSCR) streptavidin tion amplification O Specific parallel amplification of 5’ Processing RNA-adapter Processing Reverse Precursor-specific PCR Sequence
RNA Ends (SPARE) to identify intermediates ligation intermediates transcription primer
Nascent RNA microRNA processing intermediates OH OH
OH OH
N1-cyanoethyl inosine
NS-seq 5,6-dichlorobenzimidazole 5’ P 3’ OH
5’ P
5’ OH
3’ OH
3’ OH
T C
Bisulfite
T C G

BisChIP-Seq
BisChIP-Seq
RNAse S1
1-β-D-ribofuranoside (DRB) 5’ OH 3’ OH
T U T C G
5’ P 3’ OH 5’ P 3’ OH PCR
Nascent strand sequencing (NS-seq) to discover
DNA replication origins and G4 structures
DNA replication origin Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo) Digestion Purify Amplify DNA ChIP-BS-seq T T T C G

EcoP151 site
PARS 5’ P 3’ OH
5’ P
5’ OH
3’ OH
3’ OH
Bisulfite-treated chromatin immuno- DNA-protein complex with Sonicate Immunoprecipitate Purify DNA Bisulfite conversion DNA
precipitated DNA (BisChIP-seq) and methylated histones and and shear
CAGE
RNA Cap
AA(A)n AA(A)n
O dsRNA-Seq RNAse V1
5’ P
5’ OH 3’ OH
3’ OH
ChIP-BS-seq), to correlate protein methylated DNA
5’ P 3’ OH modifications with DNA methylation
N
Cap-analysis gene expression (CAGE) Capped mRNA N15 random Reverse Biotinylate RNase digestion Capture only Purify Add linker and cDNA
RNA + N
Parallel analysis of RNA structure (PARS) PolyA RNA RNAse digestion RNA fragments with 5’ phosphate ends Random fragmentation Reverse transcription cDNA
to map initiation sites of both primer with transcription complete 5’ ends cDNA digest wth EcoP151 N also double-stranded RNA (dsRNA-Seq)
capped coding and noncoding RNAs
RNA
EcoP151 site
MmeI
3’ adapter
Acylation
N3
5’ P 3’ OH
5’ P
5’ OH
3’ OH
3’ OH
DNA-Protein Interactions
DeepCAGE Cap
AA(A)n AA(A)n
75ºC RNAse V1
5’ P 3’ OH 5’ P
5’ OH 3’ OH
3’ OH DNA adenine
O DNA-protein interaction methyltransferase (DAM) Specific and non-targeted
RNA methylation DpnI DpnII PCR
High throughput Cap-analysis Capped mRNA Random Reverse Biotinylate Capture only MmeI digestion Purify Ligate PCR cDNA Fusion protein
gene expression (CAGE) primer transcription RNase digestion complete 5’ ends O PARTE 5’ P 3’ OH
5’ P
5’ OH
3’ OH
3’ OH DamID Protein of interest
RNA RNA 25ºC RNAse V1 5’ P 5’ OH 3’ OH
3’NT method
3’ OH Non-targeted methylation
Cap Cap Cap N 5’ P 3’ OH DAM
5’ P 3’ OH
N3 DNA adenine methyltransferase Create fusion Split sample DpnI digestion Adaptor ligation Unmethylated GATCs Align sequences and determine
Parallel analysis of RNA RNAse V1 digestion at different N interaction detection (DamID) protein are cut by DpnII differentially digested sites
3’ End of nascent transcripts Transcriptome complex isolated in PolyA RNA RNA fragments with 5’ phosphate ends Random fragmentation Reverse transcription cDNA
Insoluble chromatin complex RNA extraction Reverse transcription cDNA structures with temperature temperatures N N N
(3’NT) hypotonic buffers and nonionic detergents DIBO-biotin “click” elevation (PARTE)
RNA RNA O DNase-Seq
NET-Seq Cap Cap Cap
RNA P1 endonuclease
CH3
O 3’ OH 5’ P 5’ P
O DNaseI-Seq
H3C DNase I hypersensitive sites sequencing
Native elongating transcript
sequencing (NET-Seq)
Transcriptome complex Immunoprecipitate complex RNA extraction Reverse transcription cDNA Frag-seq Endogenous 5’P control
(DNase-Seq, DNaseI-Seq)
Active chromatin DNase I digestion Isolate trimmed complexes DNA extraction DNA
N 3’ OH 5’ P 5’ P
N
RNA RNA OH P N N
Cap m7G OH 3’ OH 5’ OH Display methods on your
mNET-seq
Endogenous 5’OH control
T4 kinase
cDNA
mobile device MAINE-Seq
Native elongating transcript Pol II Transcriptome MNase digestion Soluble Immunoprecipitate 5’ phosphorylation Purify and Reverse cDNA Fragmentation sequenc- In vitro folded P1 endonuclease digestion
3’ OH 5’ P 5’ P
reverse transcription cDNA
www.illumina.com/method-selector
MNase-Seq
ing (Frag-seq) polyA RNA MNase-assisted isolation of nucleosomes (MAINE-Seq).
sequencing technology for
mammalian chromatin (mNET-seq)
complex chromatin with Pol II Ab with PNK size select transcription Nucleo-Seq Also Micrococcal nuclease sequencing (MNase-Seq)
Open chromatin MNase digestion Isolate trimmed complexes DNA extraction DNA

5’ P 5’ P
miRNA directed cleavage Biotin
PARE-Seq 5’ GPPP AA(A)n
5’ GPPP AA(A)n
5’ GPPP 3’ OH
5’ P AA(A)n MmeI
3’ adapter
Preparation of acylated CapSeq 5’ OH 5’ OH 5’ OH 5’ OH 5’ OH 5’ OH 5’ OH
TT(T) 21 5’ PPP 5’ PPP 5’ PPP
5’ P AA(A)n RNA for biotin–streptavidin
Parallel analysis of RNA ends (PARE) Capped mRNA Fragment Poly(A) RNA Ligate Reverse Second strand MmeI digestion Purify Ligate PCR cDNA purification. 5’ GPPP 5’ GPPP 5’ GPPP 5’ GPPP 5’ P 5’ P 5’ P X-ChIP
RNA extraction adapter transcription synthesis DIBO, dibenzocyclooxtyne High resolution of mapping of in vivo Crosslink Lyse cells Cross-linked chromatin MNase digestion Sonicate Soluble Immunoprecipitate DNA
5’ anchored profiling of Pol II Total RNA Terminator CIP TAP Primer Ligation Random Primer Reverse Purification cDNA chromatin associated proteins cells in vivo extract and DNA extraction
transcripts (CapSeq) transcription
5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n 3’ adapter
5’ GPPP 5’ GPPP AA(A)n AA(A)n
TT(T)n TT(T)n index 5’ P 5’ P
GMUCT 1.0 AA(A)n AA(A)n AA(A)n
TT
TT(T)n
AA(A)n
TT(T)n 5’ adapter 5’ OH 5’ OH
5’ OH 5’ OH 5’ OH 5’ OH

Genome-wide mapping of Degraded RNA Poly(A) RNA Ligate 5’ RNA Add Reverse PCR Fragmen- Ligate adapters PCR Indexed cDNA CIP-TAP 5’ PPP 5’ PPP ORGANIC
uncapped transcripts (GMUCT) selection adapter oligo-dT transcription tation O
NH H O 5’ GPPP 5’ GPPP 5’ P 5’ P Occupied regions of genomes from Cell Isolate nucei Isolated chromatin MNase digestion Soluble extract Immunoprecipitate DNA
5’ GPPP 5’ GPPP
affinity-purified naturally isolated and DNA extraction
5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n 5’ GPPP AA(A)n HN OH CIP-TAP for identifying capped csRNA CIP 3’ primer ligation and gel purify TAP 5’ primer ligation and gel purify cDNA synthesis PCR Purification cDNA chromatin (ORGANIC)
5’ GPPP 5’ GPPP AA(A)n AA(A)n small RNA (csRNA) species Biotin
GMUCT 2.0 H S
AA(A)n AA(A)n AA(A)n

Genome-wide mapping of Degraded RNA Poly(A) RNA Ligate 5’ RNA Random hexamer Reverse transcrip- PCR to Indexed cDNA Biotin Streptavidin
uncapped transcripts (GMUCT) selection adapter fused to 3’ primer tion add index CATCH-IT
DNA Rearrangements and Markers
H2N O
Covalent attachment of tags to Cells starved of Add methionine analogue Isolate Cycloaddition MNase digestion Remove of H2A–H2B dimers DNA DNA
capture histones and identify methionine l-azidohomoalanine (AHA) nucei reaction and non-histone proteins extraction
Photoactivatable Nucleosides H2N
O O H2N turnover (CATCH-IT)
RAD
RNA-Protein Interactions
N
S
Genome Restriction sites
ChIP-Seq
NH NH
Paired-end- O O

High affinity binding site

NH
RAD-Seq Restriction-site associated DNA Restriction Add barcoded adapters Shear (Second restriction Add P2 Amplify DNA ChIP-exo
RBNS HO
N O
ddRADseq marker generation (RAD) digestion digest in ddRADseq) adapter
HT-ChIP
O
Chromatin immune precipitation (ChIP-Seq), DNA-protein complex Crosslink proteins and DNA Sample fragmentation Exonuclease digestion Immunoprecipitate DNA DNA
Ribonucleotides High-throughput chromatin immunoprecipi- extraction
Genome 2’,3’-cyclic phosphate Pyridostatin (PDS)
RNA Bind-n-Seq (RBNS) Random RNA RNA binding protein with
streptavidin binding peptide tag
Incubate with various
protein concentrations
Bead coated with
streptavidin
Purify Reverse transcrip-
tion amplification
cDNA
OH OH
HydEn-seq 5’
3’
R R
3’
5’ 5’HO 5’P-O
R R tation (HT-ChIP))

4-thiouridine (4SU) Hydrolytic end sequencing (HydEn-seq) to reveal replicase- and Alkaline Phosphorylation (T4 PNK Ligate oligo with 5’-amino- Add sequencing primers, Map locations
RNA RNA strand-specific patterns of ribonucleotides in the genome hydrolysis (KOH) 3-phosphatase minus) terminated C6 spacer PCR and sequence on the genome Histone
Ribo-Seq RNA RNA
rRNA O R methylation
GTI-Seq rRNA I Genome with ribonucleotides R mA R Am
T R
2’P

T
NH R R R R R
Ribose-seq
T

2’P

A Am A Am A R ChIP-Seq of methylated histones DNA-protein complex Crosslink proteins Sample Exonuclease digestion Immunoprecipitate DNA extraction DNA
ARTseqTM Ribo-Seq: Ribosome profiling. Global Ribosome RNase digestion RNA extraction rRNA depletion RNA extraction Reverse transcription cDNA 5’ P 5’ P A
T

T A Am
T

A T
translation initiation sequencing (GTI-Seq) P 5’ A P 5’ mA A T mA A T mA A T (Histone methylation) and DNA fragmentation
N O 5’
OH Detect ribonucleotides Fragmented dA tailing Adaptor ligation Alkali treatment Self-ligation by Degradation of Remove 2’-phos- DNA for O
Base Streptavidin
phate and PCR
RIP-Seq O embedded in DNA
(Ribose-seq)
genomic DNA AtRNL linear ssDNA sequencing O P O
O
O

Sequencing Primer Add biotinylated Harvest cells and

OH OH
O P O
O OH
Base
Chem-seq in vivo
compound fragment DNA
RNA immunoprecipitation (RIP) RNA-protein complex Immunoprecipitate RNase digestion RNA extraction Reverse transcription cDNA
O

RNA-protein complex 5-iodouridine (5IU) Genome Potential G-quadruplex K+ or PDS Polymerase stalls O

G4-seq O OH
Base
Crosslink
CLIP O
O P O
O
O
Chem-seq: to identify the sites DNA-protein complex with
in vitro
Add biotinylated Enrich DNA
fragments
DNA
extraction DNA
Br bound by small chemical putative drug binding site compound
HITS-CLIP NH
Determine the location of Add K+ or PDS to
O OH
Base molecules
Target sequence Fragment and Hibridize Read reference Denature and remove Hibridize Read stabilized Compare O P O O

PTB-Seq Crosslinking and immunoprecipita-

tion sequencing (CLIP-Seq) or
RNA-protein complex UV 254 nm RNase T1 digestion Proteinase K RNA extraction Reverse transcription cDNA N O potential G-quadruplexes in create library on sequencing sequence read fragment sequencing stabilize G4 regions squence sequence reads S
DNA (G4-seq) flow cell primer primer 1 and 2
FAIRE-seq
OH O OH

PIP-Seq high-throughput sequencing of CLIP Base

O O P O O
cDNA library (HITS-CLIP) S
Sono-Seq
XC
CX

CXXC CXXC CXXC

X
XC
CX
XC

CpG island OH OH
CX

OH OH Methylated CpG CXXC CXXC FAIRE-Seq: formaldehyde-assisted Isolation Open DNA Crosslink protein and DNA with formalin Sonicate Phenol extract and purify DNA DNA
3’
Pol II CLIP CAP-seq CXXC CXXC of regulatory elements and Sonication of from the aquous phase
4-bromo uridine (5BrU) Unmethylated CpG Phosphorothioate modifica- cross-linked chromatin, (Sono-Seq)
Crosslinking and immunoprecipita- RNA-protein complex UV 254 nm Immunoprecipitate Proteinase K RNA extraction Reverse transcription cDNA CXXC affinity purification plus CXXC bound to nickel-charged Hybridize to sepharose column Elute unmethylated CpG DNA
tion of Pol II (Pol II CLIP) RNA-protein complex deep sequencing (CAP-seq) sepharose beads enriched fragments
tion of the two 3’-terminal CpG dinucleotides CpG dinucleotides
Protected Protected

NOMe-Seq
S Control
nucleotides Methylated CpG Methylated CpG Protected Unprotected
N methylated methylated
NH (5’-NpNpNpNpSNpSN-3’)
Nucleosome Occupancy Methylome-
PAR-CLIP GpC methyltransferase (M.CviPI) and Bisulfite conver- M.CviPI
N NH2 Open DNA Protected Unprotected
N Sequencing (NOMe-Seq), a single-molecule S-Adenosyl methionine (SAM) sion BS-Seq unmethylated unmethylated
OH Genome nucleosome positioning assay
Photoactivatable-Ribonucleo- RNA-protein Incorporate 4-thiouridine (4SU) UV 365 nm RNase T1 digestion Proteinase K RNA Reverse cDNA O
side-Enhanced Crosslinking and complex into transcripts of cultured cells extraction transcription CPT-Seq
Immunoprecipitation (PAR-CLIP) OH OH

iCLIP 6-Thioguanosine (6SG) ATAC-Seq

cleavable Contiguity-preserving Divide sample Indexed transpo- EDTA Dilute and divide SDS Indexed PCR DNA
adapter transposition sequenc- into 96 reactions some reactions Pool into 96 reactions PCR primers Assay for transposase accessible Open DNA Tn5 Transposome Insert in regions of open chromatin Fragmented and primed DNA purification DNA
Individual nucleotide RNA-protein complex Immunoprecipitate cross-linked Proteinase K Cross-linked peptides remain cDNA truncates at Circularize Linearize and PCR cDNA ing (CPT-seq) chromatin (ATAC-Seq) Amplification
resolution CLIP (iCLIP) RNA-protein complex after proteinase K digestion binding site
Novel retrotrans- Retrotransposon
miRNA O O O position events binding sites Sequenced fragment Known
RC-Seq Read1 retrotrans-poson
Chia-PET
AGO-CLIP N Reference sequence
Read2 Align
Map miRNA binding to RNA-protein complex Incorporate 4-thiouridine (4SU) into UV 365 nm RNase T1 T4-RNA Proteinase K RNA extraction Reverse cDNA RC-Seq: Retrotransposon Genomic DNA Fractionate DNA Hybridize Microarray with transposon Transposon sites Novel retrotransposition
events
AGO sites in C. elegans transcripts of cultured cells digestion ligase transcription capture sequencing fragments binding sites Chromatin interaction analysis by Sample fragmentation Immunoprecipitate Ligation Restriction enzyme digestion DNA
Same RNA Pyridine paired-end tag sequencing (ChIA-PET)
Transposon Transposon 20bp MmeI
Same RNA RNA 1
Adaptor A NO2
TN-Seq
Hi-C
20bp
hi-CLIP Different RNA Different RNA RNA 1 1-methyl-7-nitroisatoic
INSeq Transposon sequencing (TN-Seq) Inverted MmeI
recognition site
MmeI
recognition site
MmeI
MmeI digestion Add adapters PCR and sequence Transposon
anhydride (1M7)
Adaptor B RNA 2 and insertion sequencing (INSeq) insertion sites
3-C
RNA hybrid and individu-
al-nucleotide resolution
Partial Adaptor ligation Sequence
AID-dependent rearrangement
I-SceI site
+AID A A Capture-C
ultraviolet crosslinking RNA-protein UV Immunoprecipitate Remove 3’ block Second RNA Proteinase K Map RNA sequences to Chromatin conformation capture Crosslink proteins and DNA Sample fragmentation Ligation PCR amplify ligated junctions DNA
RNase
and immunoprecipitation
(hiCLIP)
complex 254 nm
digestion
RBP-RNA complex from adaptor B ligation RNA extraction the genome
TC-Seq -AID A A
(3-C, Hi-C and Capture-C)
32-bp Ter sequence Tus Tus Tus
TC-Seq: translocation Genomic DNA Infect I-Sel Sonicate, blunt Ligate linkers Purification Semi-nested PCR DNA
capture sequencing and A-tail Cut I-Scel Linker cleavage
Klenow 4-C
HiTS-RAP Target sequence
CDR3 junction region V D J Constant 8N UID

Flowcell Ig-seq 8N UID

Chromatin conformation Crosslink proteins and DNA Sample fragmentation Ligation Restriction digest Self-circularization DNA
DNA sequencing of immunoglobulin genes Extracted RNA Reverse transcription Second strand synthesis PCR Purify
High-throughput Prepare sequencing libraries and Hybridize Synthesize second Add Escherichia coli Transcribe Bind labeled Scan flowcell Rep-Seq (Ig-seq) for antibody repertoire determination
DNA capture circular (4-C) and Reverse PCR
sequencing–RNA affinity sequence first strand primer strand with unmodi- replication termina- and halt protein
profiling (HiTS-RAP) Remove second strand DNA fied nucleotides tor protein (Tus) V(D)J antigen receptor region T7 T3
EC-seq 5-C
V3 J4 J5
RNA HO OH V1 V2 V3 J5 J4 J3 J2 J1 V1 V2 V3 J5 J4 J3 J2 J1 V1 V2 J3 J2 J1 V1 V2 V3 J5 J4 J3 J2 J1

TRAP-Seq V2 J3 J5
V1 J2 J1
V3 J4
Cap 1,6-Hexanediol Excision circle sequencing Lymphocyte-specific recombination activat- Synapsis, cleavage and coding Resolved genomic Liberated excision circle with
Targeted purification of Polysomes RNA Reverse transcription cDNA Align read pairs to
polysomal mRNA (TRAP-Seq) Bead (EC-seq) ing gene (RAG) recombinase, bound genome end hairpin formation coding junction resolved signal junction reference genome Chromatin conformation capture Crosslink proteins and DNA Sample fragmentation Ligation LMA: Ligation-mediated amplification DNA
U U NNNNN
Left adaptor carbon copy (5-C)
NNNNN U U Right adaptor U U NNNNN NNNNN U U
DLAF AA(A)n AA(A)n AA(A)n

Directly ligate sequencing adaptors mRNA Random Reverse RNase cDNA 3’ Hexanediol
5’ Phosphate
Ligation in presence of Uracil-specific PCR and cDNA PB-seq
DNA Low-Level Detection
to the first-strand cDNA (DLAF) primer transcription digestion PEG and DMSO excision (USER) purify
Protein/DNA binding (PB–seq), to DNA-protein complex Purify DNA Shear DNA Hybridize with Isolate protein-bound DNA DNA
miRNA
miRNA miRNA True variant determine the binding energy landscape DNA-binding protein DNA extraction
Read1 Sample index
miTRAP Gene Degenerate
Pu-seq
Index 2 Index 1

Bind in vitro transcribed Incubate with Extract RNA-protein Isolate miRNA

O smMIP molecular tag
Read2
Random error
Origin of replication 5’
3’ R
3’
5’ U U U
miRNA trapping by RNA in vitro RNA-protein Amylose resin cDNA
affinity purification (miTRAP) complex bait RNA cell extract complex Single Molecule Molecular Genomic DNA Copy target sequence Exonuclease PCR amplification Align fragments from every Corrected Polymerase usage sequencing Alkali Klenow reaction(+ random Attach Uracil DNA glycosylase- PCR and DNA
O- (Pu-seq) treatment primer, dATP, dGTP,dCTP, dUTP) adaptors and DNA lyase (USER) purify
O Inversion Probes (smMIPs) for unique molecular tag sequence
A A A detecting low frequency targets
N O- Degenerate
CLASH
B B B
Strain I Strain I Breakage at replication fork
Short tandem repeat (STR)
Break-seq
Index 2 Index 1
N
M
O- MIPSTR molecular tag
Strain II Strain I
5’
3’
Break 3’
5’
5’
3’
3’
5’
Crosslinking, ligation, and RNA duplex UV crosslinking Crosslinked Affinity Ligate ends Reverse transcription cDNA Targeted capture of STR Targeted STR Copy target STR Amplify and sequence Natural variation between individuals Somatic variation
sequencing of hybrids (CLASH) complex purification Uses double-stranded break DNA trapped in End-repair with dGTP, dCTP, Elution and Streptavidin magnetic Attach PCR and DNA
O
loci by smMIPs (MIPSTR) within an individual labeling to map chromosome agarose gel dTTP, and biotinylated-dATP fragmentation bead pull down adaptors purify
O- breaks (Break-seq)
O Forward sequencing adaptor

RNA Low-Level Detection

3’ blocked random hexamer primers Nascent replication fork Sequencing primer pD40htSELEX
Genome Fusion
EDTA MDA SELEX
Transcription factor binding site Barcode to identify sample
14N with all possible combinations DNA binding region
protein Luciferase
Reverse sequencing adaptor Luciferase
Digital RNA cDNA1 cDNA1
cDNA1
IMS-MDA Multiple displacement amplification (MDA).
Immunomagnetic separation for targeted
Hybridize primers Phi 29 Synthesis Phi 29 Synthesis S1 nuclease Amplified DNA
SELEX-seq Streptavidin binding peptide
cDNA bacterial enrichment for MDA (IMS-MDA) High-throughput systematic Target Ligand Expression vector Fusion protein Matching ligand Wash and Recovered PCR and Binding
HiRes-Seq cDNA2 cDNA2
cDNA2
Hybridize primers Synthesis
HT-SELEX evolution of ligands by exponential
enrichment (HT-SELEX)
sequence immobilized in well binds elute matching ligand Sequence site

FREQ-Seq 27-bp common sequence

8 random nucleotides
Unique molecular barcodes are added after cDNA Adapters with Some fragments Align sequences and determine True RNA
RNAtag-Seq synthesis for quantitative allele frequency detection unique barcodes
Amplify
amplify preferentially
Sequence
actual ratio based on barcodes abundance
MALBAC
Genome
Cycles of quasilinear
amplification Protein binding site Klenow
TTTTT T7 PCR
PCR
TTTTT
Blocking primer with LNA Multiple annealing and looping-based Hybridize primers Bst DNA Denature
Partial amplicons
Looped full PCR DNA HiTS-FLIP Target sequence
Binding site

Quartz-Seq
PCR
AA(A)n AAAAA AAAAA AAAAA TTTTT AAAAA amplification cycles (MALBAC) polymerase amplicons
T T T T T T7 PCR T T T T T T7 PCR AAAAA T T T T T T7 PCR AAAAA TTTTT Template
Flowcell
T7 PCR
Denature High-throughput sequencing: Prepare sequencing libraries and Hybridize Synthesize second Add fluorescent- Hybridize Elute with increas-
Whole-transcript amplifi- Add polyA primer Reverse transcription Poly A addition and Generate Add blocking Enrich with suppres- Single cell genome Scan flowcell
cDNA fluorescent ligand interaction sequence first strand primer strand with unmodi- ly labeled ligand ing stringency
cation for single-cells with T7 promoter and Primer digestion oligo dT primer with second strand primer sion PCR Cell 1
(Quartz-Seq) and PCR target PCR target Nuc-seq Cell 2
profiling (HiTS-FLIP) Remove second strand DNA fied nucleotides

No secondary
structure
SNES Cell 3

AA(A)n TT(T)n TT(T)n Single G2/M nucleus sequencing of Cell sorting from Lyse cell Nucleus
Protein-Protein Interactions
Phi 29 Limited amplification Synthesis S1 nuclease DNA
DP-Seq AA(A)n AA(A) cells in S phase (Nuc-seq). Single G2/M distribution
Unique sequence n nucleus exome sequencing (SNES)
Designed Primer-based RNA-se- Define set of PolyA selection First strand cDNA Hybridize primers PCR DNA
quencing strategy (DP-seq) heptamer primers cDNA synthesis Fragment and add single adaptors NH2
Sequencing Primers Pyridine
Protein target NH2 NH2 K2CO3 NH Hybridize NH2
OS-Seq Gene
Target sequence Single adaptor library
PD-Seq
Smart-seq mRNA AAAAAAA
Adaptor DMF
Wash
NanoCAGE AAAAAAA AAAAAAA CCC TTTTTTT Target sequence
CCC TTTTTTT Oligonucleotide-selective Sequence
Adaptor PD-Seq identifies candidate Beads Polyethylene glycol Immobilized RNA from cells Create phage display Repeat cycle three Protein-protein PCR phage DNA
CAGEscan Switch mechanism at the 5’ mRNA fragment First strand synthesis with Moloney murine Second strand synthesis PCR amplification Purify DNA
sequencing (OS-Seq) capture
and sequence gene targets on
Adaptor sequence N cellular targets for proteins linker added protein cDNA library times interaction insert
end of RNA templates (Smart) leukemia virus reverse transcriptase O
the flow cell N N N
N
NH
mRNA AAAAAA
Locked nucleic acid (LNA) Index 2 Index 1
Flow cell Create target-specific oligos Extend and
Denature
Hybridize Extend and
Denature
Hybridize Extend and
Denature
Sequence reads
1 and 2 ProP-PD Protein target Hybridize
2
Smart-seq2 AAAAAA CCC TTTTTT GGG AAAAAA N N NH 2
PDZ-Seq
AAAAAAA CCC TTTTTT GGG P5 P7 1
Tem- CCC OH CH3
Adaptor
Duplex O
Very rare mutation P5 P7 Random error True variant
plate-switch- O α β
ing oligo H3C Proteomic peptide-phage Identify C-terminal Create oligo Construct phage Bait proteins immobi- Select phages against Isolate and Peptide
Switch mechanism at mRNA fragment First strand synthesis with cDNA synthesis PCR Tagmentation Gap repair, enrich- Enrichment-ready fragment
the 5’ end of RNA Moloney murine leukemia ment PCR and PCR OH O
Sequencing P7 12 random
base index
12 random
base index
P5 display (ProP-PD) to identify
short linear motif (SLiM) interac-
sequences library display library lized on 96-well plate baits sequence counts
templates (Smart) virus reverse transcriptase purification
Consensus tions or PDZ domains (PDZ-Seq)
P5
True variant Locked nucleic acid (LNA) Display methods on your
Degenerate molecular tag (N10) Duplex sequencing detects A mutation occurs on Add Ligate and PCR Sequence Create single strand Create duplex sequences Rare variant
mRNA very rare mutations by both strands adaptors consensus sequence from based on molecular tags
mobile device
UMI Method
Index
AAAAAAA AAAAAAA AAAAAAA www.illumina.com/method-selector
CCC TTTTTTT CCC TTTTTTT sequencing and aligning both every unique molecular tag and sequencing primers
Random error strands of the DNA
Key
P7
Unique molecular identifiers mRNA fragment First strand synthesis Second strand synthesis PCR amplification Align fragments from every DNA Yellow highlights indicate the target of the protocol
(UMIs) uniquely identify copies unique molecular tag
derived from each molecule

References
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3’-end-seq Haenni S. et al. (2012) Nucleic Acids Res 40: 6304-6318 2240-2245 ChIRP Chu C. et al. (2011) Mol Cell 44: 667-678 Drop-seq Macosko E. Z. et al. (2015) Cell 161: 1202-1214 HITS-CLIP Chi S. W. et al. (2009) Nature 460: 479-486 MDA Dean F. B. et al. (2001) Genome Res 11: 1095-1099 oxBS-Seq Booth M. J. et al. (2012) Science 336: 934-937 Quartz-Seq Sasagawa Y. et al. (2013) Genome Biol 14: R31 scRRBS Guo H. et al. (2015) Nat Protoc 10: 645-659 TATL-seq Arribere J. A. et al. (2013) Genome Res 23: 977-987
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3′-Seq Lianoglou S. et al. (2013) Genes Dev 27: 2380-2396 BS-Seq Lister R. et al. (2009) Nature 462: 315-322 CirSeq Acevedo A. et al. (2014) Nature 505: 686-690 A 109: 14508-14513 hmeDIP-seq Jin C. et al. (2014) Nucleic Acids Res 42: 6956-6971 MethylCap-Seq Brinkman A. B. et al. (2010) Methods 52: 232-236 PARE-Seq German M. A. et al. (2008) Nat Biotechnol 26: 941-946 RASL-Seq Li H. et al. (2012) Curr Protoc Mol Biol Chapter 4: Unit 4 13 SHAPE-MaP Siegfried N. A. et al. (2014) Nat Methods 11: 959-965 TC-Seq Klein I. A. et al. (2011) Cell 147: 95-106
3-C Duan Z. et al. (2012) Methods 58: 277-288 Bubble-Seq Mesner L. D. et al. (2013) Genome Res 23: 1774-1788 CIRS-seq Incarnato D. et al. (2014) Genome Biol 15: 491 EC-seq Parkinson N. J. et al. (2015) Genome Res 25: 226-234 HT-ChIP Blecher-Gonen R. et al. (2013) Nat Protoc 8: 539-554 Methyl-seq Brunner A. L. et al. (2009) Genome Res 19: 1044-1056 PARS Wan Y. et al. (2013) Nat Protoc 8: 849-869 11-19 SHAPE-Seq Lucks J. B. et al. (2011) Proc Natl Acad Sci U S A 108: TIF-Seq Pelechano V. et al. (2013) Nature 497: 127-131
3P-Seq Jan C. H. et al. (2011) Nature 469: 97-101 CAB-Seq Lu X. et al. (2013) J Am Chem Soc 135: 9315-9317 CLASH Kudla G. et al. (2011) Proc Natl Acad Sci U S A 108: FAIRE-Seq Gaulton K. J. et al. (2010) Nat Genet 42: 255-259 HT-SELEX Jolma A. et al. (2010) Genome Res 20: 861-873 miCLIP Linder B. et al. (2015) Nat Methods 12: 767-772 PARTE Wan Y. et al. (2012) Mol Cell 48: 169-181 RBBS Smith Z. D. et al. (2009) Methods 48: 226-232 11063-11068 TIVA Lovatt D. et al. (2014) Nat Methods 11: 190-196
3Seq or 3-Seq Beck A. H. et al. (2010) PLoS One 5: e8768 CAGE Takahashi H. et al. (2012) Nat Protoc 7: 542-561 10010-10015 fCAB-Seq Song C. X. et al. (2013) Cell 153: 678-691 HydEn-seq Clausen A. R. et al. (2015) Nat Struct Mol Biol 22: 185-191 MiGS Serre D. et al. (2010) Nucleic Acids Res 38: 391-399 PAS-Seq Shepard P. J. et al. (2011) RNA 17: 761-772 RBNS Lambert N. et al. (2014) Mol Cell 54: 887-900 Smart-Seq Ramskold D. et al. (2012) Nat Biotechnol 30: 777-782 TL-seq Arribere J. A. et al. (2013) Genome Res 23: 977-987
4-C Zhao Z. et al. (2006) Nat Genet 38: 1341-1347 CAGEscan Plessy C. et al. (2010) Nat Methods 7: 528-534 ClickSeq Routh A. et al. (2015) J Mol Biol 427: 2610-2616 fC-Seal Song C. X. et al. (2013) Cell 153: 678-691 ICE-seq Sakurai M. et al. (2010) Nat Chem Biol 6: 733-740 MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761 PBAT Miura F. et al. (2012) Nucleic Acids Res 40: e136 RBNS Lambert N. et al. (2014) Mol Cell 54: 887-900 Smart-seq2 Picelli S. et al. (2013) Nat Methods 10: 1096-1098v TN-Seq van Opijnen T. et al. (2013) Nat Rev Microbiol 11: 435-442
4sUDRB-seq Fuchs G. et al. (2014) Genome Biol 15: R69 Capp-Seq Newman A. M. et al. (2014) Nat Med 20: 548-554 See CLIP Johnson D. S. et al. (2007) Science 316: 1497-1502 Frag-Seq Underwood J. G. et al. (2010) Nat Methods 7: 995-1001 iCLIP Konig J. et al. (2010) Nat Struct Mol Biol 17: 909-915 MIRA Rauch T. A. et al. (2010) Methods 52: 213-217 PB-seq Guertin M. J. et al. (2012) PLoS Genet 8: e1002610 RC-Seq Baillie J. K. et al. (2011) Nature 479: 534-537 smMIP Hiatt J. B. et al. (2013) Genome Res 23: 843-854 TRAP-Seq Jiao Y. et al. (2010) Mol Syst Biol 6: 419
targeted sequencing. CPT-seq Amini S. et al. (2014) Nat Genet 46: 1343-1349 FREQ-Seq Chubiz L. M. et al. (2012) PLoS One 7: e47959 RedBS-Seq Booth M. J. et al. (2014) Nat Chem 6: 435-440 SMORE-seq Park D. et al. (2014) Nucleic Acids Res 42: 3736-3749 T-WGBS Wang Q. et al. (2013) Nat Protoc 8: 2022-2032
5’-GROseq Lam M. T. et al. (2013) Nature 498: 511-515 icSHAPE Spitale R. C. et al. (2015) Nature 519: 486-490 miTRAP Braun J. et al. (2014) Nucleic Acids Res 42: e66 PD-Seq Arango D. et al. (2013) Proc Natl Acad Sci U S A 110:
CapSeq Gu W. et al. (2012) Cell 151: 1488-1500 CytoSeq Fan H. C. et al. (2015) Science 347: 1258367 FRT-Seq Mamanova L. et al. (2011) Nat Protoc 6: 1736-1747 E2153-2162 Repli-Seq Hansen R. S. et al. (2010) Proc Natl Acad Sci U S A 107: SNES Leung M. L. et al. (2015) Genome Biol 16: 55 UMI Method Kivioja T. et al. (2012) Nat Methods 9: 72-74
5-C Dostie J. et al. (2007) Nat Protoc 2: 988-1002 Ig-Seq Vollmers C. et al. (2013) Proc Natl Acad Sci U S A 110: MNase-Seq Schones D. E. et al. (2008) Cell 132: 887-898
CAP-seq Illingworth R. S. et al. (2010) PLoS Genet 6: e1001134 DamID-Seq Vogel M. J. et al. (2007) Nat Protoc 2: 1467-1478 G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522 13463-13468 PDZ-Seq Ernst A. et al. (2010) Mol Biosyst 6: 1782-1790 139-144 Sono-Seq Auerbach R. K. et al. (2009) Proc Natl Acad Sci U S A 106: WGBS See BS-Seq
Aba-seq Sun Z. et al. (2013) Cell Rep 3: 567-576 mNET-seq Nojima T. et al. (2015) Cell 161: 526-540
Capture-C Hughes J. R. et al. (2014) Nat Genet 46: 205-212 ddRADseq Peterson B. K. et al. (2012) PLoS One 7: e37135 G4-seq Chambers V. S. et al. (2015) Nat Biotechnol 33: 877-881 IMS-MDA Seth-Smith H. M. et al. (2013) Nat Protoc 8: 2404-2412 PE RAD-Seq Willing E. M. et al. (2011) Bioinformatics 27: 2187-2193 Rep-Seq Benichou J. et al. (2012) Immunology 135: 183-191 14926-14931 X-ChIP Skene P. J. et al. (2014) Elife 3: e02042
AGO-CLIP Grosswendt S. et al. (2014) Mol Cell 54: 1042-1054 MRE-Seq Maunakea A. K. et al. (2010) Nature 466: 253-257
CaptureSeq Mercer T. R. et al. (2014) Nat Protoc 9: 989-1009 deepCAGE Valen E. et al. (2009) Genome Res 19: 255-265 GMUCT 1.0 Gregory B. D. et al. (2008) Dev Cell 14: 854-866 INSeq van Opijnen T. et al. (2013) Nat Rev Microbiol 11: 435-442 PEAT Ni T. et al. (2010) Nat Methods 7: 521-527 Ribo-Seq Ingolia N. T. et al. (2009) Science 324: 218-223 SPARE Schapire A. L. et al. (2013) Methods 64: 283-291 Ψ-seq Schwartz S. et al. (2014) Cell 159: 148-162
ATAC-Seq Buenrostro J. D. et al. (2013) Nat Methods 10: 1213-1218 nanoCAGE Plessy C. et al. (2010) Nat Methods 7: 528-534
CATCH-IT Deal R. B. et al. (2010) Science 328: 1161-1164 Digital RNA Shiroguchi K. et al. (2012) Proc Natl Acad Sci U S A 109: GMUCT 2.0 Willmann M. R. et al. (2014) Methods 67: 64-73 JBP1-seq Cui L. et al. (2014) Genomics 104: 368-375 PIP-Seq Silverman I. M. et al. (2014) Genome Biol 15: R3 Ribose-seq Koh K. D. et al. (2015) Nat Methods 12: 251-257, 253 p sRNA-Seq small RNA sequencing. See RNA-Seq
BisChIP-Seq Statham A. L. et al. (2012) Genome Res 22: 1120-1127 NET-Seq Churchman L. S. et al. (2011) Nature 469: 368-373 following 257
CEL-Seq Hashimshony T. et al. (2012) Cell Rep 2: 666-673 1347-1352 GRO-Seq Core L. J. et al. (2008) Science 322: 1845-1848 M6A-RIP Batista P. J. et al. (2014) Cell Stem Cell 15: 707-719 Pol II CLIP Li Z. et al. (2015) Nat Struct Mol Biol 22: 256-264 SS3-Seq Yoon O. K. et al. (2010) RNA 16: 1256-1267
Bisulfite-seq Berman B. P. et al. (2012) Nat Genet 44: 40-46 NOMe-Seq Han H. et al. (2011) Hum Mol Genet 20: 4299-4310 RIP-Seq Zhao J. et al. (2010) Mol Cell 40: 939-953
CHART Simon M. D. et al. (2011) Proc Natl Acad Sci U S A 108: DIP-seq Shen L. et al. (2013) Cell 153: 692-706 GTI-Seq Wan J. et al. (2014) Nucleic Acids Res 42: D845-850 M6A-seq Dominissini D. et al. (2012) Nature 485: 201-206 ProP-PD Ivarsson Y. et al. (2014) Proc Natl Acad Sci U S A 111: STRT Islam S. et al. (2011) Genome Res 21: 1160-1167
Break-seq Hoffman E. A. et al. (2015) Genome Res 25: 402-412 20497-20502 NSCR Kunnev D. et al. (2015) Genome Res 25: 558-569 RNA-Seq Mortazavi A. et al. (2008) Nat Methods 5: 621-628. See
DLAF Agarwal S. et al. (2015) Nat Commun 6: 6002 HELP-Seq Oda M. et al. (2009) Nucleic Acids Res 37: 3829-3839 MAB-seq Wu H. et al. (2014) Nat Biotechnol 32: 1231-1240 2542-2547 Structure-Seq Ding Y. et al. (2014) Nature 505: 696-700. See also
BRIC-seq Tani H. et al. (2012) Genome Res 22: 947-956 Chem-Seq Anders L. et al. (2014) Nat Biotechnol 32: 92-96 NS-seq Foulk M. S. et al. (2015) Genome Res 25: 725-735 TruSeq RNA. DMS-Seq
DMS-Seq Rouskin S. et al. (2014) Nature 505: 701-705 See also Hi-C Lieberman-Aiden E. et al. (2009) Science 326: 289-293 MAINE-Seq Ponts N. et al. (2010) Genome Res 20: 228-238 PRO-Seq Kwak H. et al. (2013) Science 339: 950-953
BruChase-Seq Paulsen M. T. et al. (2013) Proc Natl Acad Sci U S A 110: Chia-PET Li G. et al. (2010) Genome Biol 11: R22 Structure-Seq Nucleo-Seq Valouev A. et al. (2011) Nature 474: 516-520 RNAtag-Seq Shishkin A. A. et al. (2015) Nat Methods 12: 323-325 TAB-Seq Yu M. et al. (2012) Cell 149: 1368-1380
2240-2245 hiCLIP Sugimoto Y. et al. (2015) Nature 519: 491-494 MALBAC Zong C. et al. (2012) Science 338: 1622-1626 Pseudo-seq Carlile T. M. et al. (2014) Nature 515: 143-146
ChIP-BS-seq Brinkman A. B. et al. (2012) Genome Res 22: 1128-1138 DNaseI-Seq Hesselberth J. R. et al. (2009) Nat Methods 6: 283-289 Nuc-Seq Wang Y. et al. (2014) Nature 512: 155-160 RRBS-Seq Xi Y. et al. (2012) Bioinformatics 28: 430-432 TAB-Seq Yu M. et al. (2012) Cell 149: 1368-1380
BruDRB-seq Veloso A. et al. (2014) Genome Res 24: 896-905 HiRes-Seq Imashimizu M. et al. (2013) Nucleic Acids Res 41: MBDCap-Seq de Assis S. et al. (2012) Nat Commun 3: 1053 PSI-seq Lovejoy A. F. et al. (2014) PLoS One 9: e110799
ChIP-exo Yen K. et al. (2013) Cell 154: 1246-1256 DNase-Seq Boyle A. P. et al. (2008) Cell 132: 311-322 ORGANIC Zentner G. E. et al. (2013) PLoS Genet 9: e1003317 RRMAB Neri F. et al. (2015) Cell Rep 10: 674-683 TAIL-Seq Chang H. et al. (2014) Mol Cell 53: 1044-1052
9090-9104 PTB-Seq Xue Y. et al. (2009) Mol Cell 36: 996-1006

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

Add Adaptors Extension-Ligation Fragment

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

Moleculo
Sequencing by Synthesis
~10kb Sheared genomic DNA

End repair

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

strand strand

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

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

FOR RESEARCH USE ONLY

This poster was compiled by the Illumina Scientific Affairs. Additional information, the latest version of the poster, and a comprehensive list of *seq methods, are available at http://www.illumina.com/libraryprepmethods. Please contact Scientific Affairs with any questions, comments, or suggestions.
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