JianhongOu, Jun Yu, HaiboLiu and LihuaJulie Zhu

April 2008

Jianhong Ou, Jun Yu, Haibo Liu and Lihua Julie Zhu

Integrated Analysis Of ChIP-seq/chip using ChIPpeakAnno, GeneNetworkBuilder and TrackViewer

Bioconductor Annual MeetingBostonJuly 28th 2017

• Introduction of ChIP-seq and ChIP-chip analysis workflow

• ChIPpeakAnno

• GeneNetworkBuilder

• TrackViewer

• Demo

Outline

HIGH-THROUGHPUT IDENTIFICATION OF DNA BINDINGSITES

• ChIP-seq– ChIP followed by high-throughput sequencing

• ChIP-chip– ChIP followed by genome tiling array analysis

Cross-link with formaldehyde

Fragment DNA

Add specific antibody to immunoprecipitate

Reverse cross-link, purify and amplify DNA

High- throughput sequencing

Hybridize to DNA microarray post DNA labeling

Fastq sequence file@HWI-ST570:42:D0PJJACXX:4:1101:1436:2323 1:N:0:CATGGATCGGAAGAGGGAANTCATCTTTGGCCCGGTGTTTCGTCCTTTCC+CCCFFFFFHHHHHHHIJGG#2AEGHIGJIJJJJJJ?FFHJGIGHIIIJIJ

Adapted from: Zhu LJ. Integrative analysis of ChIP-chip and ChIP-seq dataset. Methods Mol Biol. 2013; 1067:105-24.

ANALYSIS WORKFLOW

Adapted from: Zhu LJ. Integrative analysis of ChIP-chip and ChIP-seq dataset. Methods Mol Biol. 2013; 1067:105-24.

CHIPPEAKANNO

• Batch annotate enriched peaks– ChIP-seq– ChIP-chip– PAS-seq (Poly(A) Site Sequencing)– Cap Analysis of Gene Expression (CAGE)– Any experiments resulting in a large number of

enriched genomic regions

FUNCTIONALITY• Find the nearest genes for each set of peaks and graph the distribution around features.• Find all genes within a certain distance from the peaks • Identify enriched Gene Ontology (GO) terms and pathways associated with adjacent genes of the peaks.• Label peaks with any annotation of interest

• a dataset from the literature• CpG island• conserved element• histone modification marks

• Determine the significance of overlap and drawing Venn diagrams to visualize the extent of the overlap • binding sites among replicates• binding sites among transcription factors within a complex• binding sites among different experiments such as yours and the ones in literature

• Retrieve genomic sequences flanking putative binding sites for motif discovery, cloning or PCR amplification• Find the peaks with bi-directional promoters with summary statistics• Summarize motif occurrence in peaks• Irreproducibility Discovery Rate (IDR)

GENENETWORKBUILDER

DAF-12 EXAMPLE DATASET

• ChIP-chip peaks were downloaded from GEO at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28350 (Hochbaum, Zhang et al. 2011, PLoS Genet 7(7): e1002179)

• Expression Microarray results were downloaded from (Fisher and Lithgow 2006, Aging Cell 5(2): 127-138).

OVERLAP ANALYSIS AND DISTRIBUTION OF PEAKS AROUND TSS

Replicate1 Replicate2

Replicate3 156

932

12

323

4

148

1

1424

Distance To Nearest TSS

Freq

uenc

y

−10000 −5000 0 5000 10000

05

1015

2025

DISTRIBUTION OF DAF-12-BINDING SITES

downstream

includeFeature

insideoverlapEnd

overlapStart

upstream

Exon% Intron% 5UTR% 3UTR% Proximal Promoter% Immediate Downstream% Enhancer%

Chromosome Region

% b

indi

ng s

ites

010

2030

4050

6070

TOP MOTIFS

m1forward RC

EWSR1−FLI1−

2.2641e−03

IRF1−

2.33e−02

SPIB−

2.3803e−02

m2forward RC

NR2F1+

1.6075e−02

SOX10+

2.4823e−02

HNF4A−

2.7161e−02

m3forward RC

RREB1−

5.2626e−03

Egr1+

1.1045e−02

Foxd3+

1.9366e−02

m4forward RC

SPIB−

2.0655e−02

Tal1::Gata1−

6.646e−02

SOX10+

6.9122e−02

m5forward RC

Pax4−

4.2833e−03

Sox5−

1.6952e−02

NKX3−1−

2.2411e−02

Logo

DAF-12 REGULATORY NETWORK

C02F5.5

mi r -46C33D9.9

C01B10.4

mi r -52

mi r -53

C17A2.4

mi r -228

F01D5.1

srd-16

mb f - 1

mi r -74

mi r -229Y37D8A.5

lsy-2

mi r -75

C50F4.9

mi r -73

hpd -1

F32A5.4

C08E8.3

K10C2.3

Y69E1A.5

mi r -76

htas-1

Y59E9AL.3

dod-24

R10H10.3

sss-1

mi r -240

K09H11.7

pos-1

mi r -43

F55G11.7T01D3.6

cdh-5

T05E12.6

mi r -788

F57G8.7

C33F10.1

F01D5.3

K03H1.12

scl-27

m i r - 2

mi r -242

bre-1

mi r -63nhr -86

hpo-28

Y106G6A.4

F35H8.4

msp-51

clec-66

mi r -72

F44A2.5

ZK1248.17

msp-76

mir-239.1

mi r -87

B0507.10

T11B7.5

pho-1

clec-52

f l i - 1

T05C3.6

nspd-10

Y54G11A.14F21C10.11

Y49G5A.1

F36D1.4

K10D3.6

C27F2.7

nspd-1

ceh-45

meg-2

col-172

z ip -2

tbc -7

mi r -230

wago-9col -19

m i r - 1clec-4

g rd -5

gst -11

dyc-1

nhr -20

T22B7.7

F35E12.5

mi r -243

mi r -44

m ig -5

mi r -80

nhr -42

h lh-30

mi r -40

C24B9.3

i lys-2

mi r -58Y44A6D.1

Y75B8A.23

gst -27

cyp-14A5

col-20

C53A5.2

mi r -34far -3

msp-38

Y67A6A.1

W10C8.5

nhr -10

a t f -6

W01B6.4

ces-2mi r -237

l i n -4

nh r -1

daf -12

grd-10

C12D5.4

F09F7.4

mi r -84

sre-13lnp -1

msp-56

mi r -261

pho-12 T01B11.2

C36C9.1

mi r -42

msp-142le t -7

mi r -795

mi r -37

ug t -6

F11G11.4

ssq-2msp-63

C27D6.3

mi r -51

clec-76

msp-50

fu t - 1

mi r -39

Y40C5A.1

mi r -355

pd i -2

mi r -38

daf -3

mi r -41

l in -13mi r -241

mi r -35

mi r -36

scd-1

C32H11.4

K12H4.7

TRACKVIEWER

REFERENCES

• Zhu LJ, Gazin C, Lawson ND, Pagès H, Lin SM, Lapointe DS, Green MR. ChIPpeakAnno: a Bioconductor package to annotate ChIP-seq and ChIP-chip data. BMC Bioinformatics. 2010 May 11; 11:237. PMID: 20459804.

• Zhu LJ. Integrative analysis of ChIP-chip and ChIP-seqdataset. Methods Mol Biol. 2013; 1067:105-24. PMID: 23975789.