ChIP Seq Presentation

8/6/2019 ChIP Seq Presentation

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PRACTICAL: CHIP-SEQ DATA

ANALYSISAndre Faure & Petra Schwalie

Paul Flicek Lab, Vertebrate Genomics, EMBL-EBI9. March 2010

Wednesday, 9 March 2011


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RESOURCES

http://www.bioconductor.org (R packages & workflows; help)

http://seqanswers.com (software overview; forum)

data repositories: ArrayExpress & GEO, ENA

ENCODE, modENCODE (collaborative efforts, ChIP-seq)

Reviews + Benchmarks (see last slide)

http://seqanswers.com/http://seqanswers.com/http://seqanswers.com/http://www.bioconductor.org/http://www.bioconductor.org/


3/23

WORKFLOW

Raw data (ENCODE CTCF, H3K36me3, Input in K562 & HepG2)

Quality check & align (not discussed here)

(1) Peak-calling

(2) Genomic context

Read profile plots

(3) Motif analysis (de novo & scanning)

(4) Differential enrichment



4/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots



Peak-calling



5/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots





6/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots



Genomic context



7/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots





8/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots



Read profile plots



9/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots





10/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots



Motif analysis

G

C

T

GT

A

GT

G

ACACGTAA

G

C

G

A

T

CG

TAGAGT

GAGACTGTACT

A

G

A

G

C

T

C

C

A

G



11/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots





12/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots


(4) Differential enrichmentDifferential enrichment



13/23

WORKFLOW



(1) Peak-calling

(2) Genomic context

Read profile plots





14/23

(1) PEAK-CALLING

chipseq, GenomicRanges (Bioconductor)

estimating fragment length

extending reads

islands of enrichment

modeling the background (e.g. Poisson, neg. binomial) calling peaks (manual, MACS, SWEMBL)

genomic overlaps: comparison of peak-calling results



15/23

(2) GENOMIC CONTEXT

biomart, GenomicRanges (Bioconductor)

obtaining annotation (Ensembl)

overlaps with annotation (e.g. promoters)

enrichment of peaks in genomic areas (e.g. promoters) (not discussed here)

functional term enrichment (not discussed here) (e.g. GREAT, McLean et al. Nat Biotechnol)

average profile plots on genomic feature/peak summit



16/23

(3) MOTIF ANALYSIS

BSgenome, seqLogo, GenomicRanges (Bioconductor)

MEME (de novo motif discovery)

obtaining the peak sequences

de novo motif discovery

motif scanning: motifs per peaks?

motif enrichment vs. background (not discussed here)

refining the PWM for a given factor

motif profile plot (distribution of motif around peak summit)



17/23

(4) DIFFERENTIAL ENRICHMENT

DESeq, GenomicRanges (Bioconductor)

defining regions of interest (ROI) obtaining counts per regions of interest (replicates & conditions)

estimating library sizes

estimating variation of counts per ROIs

calling differentially modified regions (negative binomial distribution)

overview of significantly modified regions



18/23

http://www.ebi.ac.uk/~schwalie/chipseqprac_0311/chipseq_practical.pdf

http://www.ebi.ac.uk/~schwalie/chipseqprac_0311/http://www.ebi.ac.uk/~schwalie/chipseqprac_0311/


19/23

(1) PEAK-CALLING



20/23

PEAK ANALYSIS



21/23

(3) MOTIF ANALYSIS

motif discovery

motif profile

motifs/peaks

MACS Swembl



22/23

(4) DIFFERENTIAL HISTONE

MODIFICATION



23/23

CHIP-SEQ REVIEWS +

BENCHMARKS ChIP-seq: advantages and challenges of a maturing technology (Park, Nat Rev Genet 2009)

Computation for ChIP-seq and RNA-seq studies (Peke et al, Nat Methods 2009)

Design and analysis of ChIP-seq experiments for DNA-binding proteins (Kharchenko et al, NatBiotechnol 2008)

Q&A: ChIP-seq technologies and the study of gene regulation (Liu et al, MBC Biol 2010)

Evaluation of algorithm performance in ChIP-seq peak detection (Wilbanks, PLos ONE 2010)

A practical comparison of methods for detecting transcription factor binding sites in ChIP-seqexperiments (Laajala et al, BMC Bioinformatics)

ChIP Seq Presentation

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