diffReps: automated ChIP-seq differential analysis package

Li ShenAsst. Professor

Neuroscience, Mount Sinai06/28/2013

Slides adapted from previous presentation

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ChIP-seq differential analysisTreatment(coc i.p.)

Control(sal i.p.)

Rep1Rep2

Rep3

Rep1Rep2

Rep3

Differences

Venn diagram for peak lists

Treatment Control

False positive

False negativeTreatment Control

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Subtle changes of chromatin modifications

H3K4me3 from ENCODE

K562

ESC

? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?

ASUN: Asunder, Spermatogenesis Regulator

[0, 1.2]

[0, 1.2]

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Existing programs for differential analysis

• ChIPDiff(2008): HMM-based approach. NOT sensitive enough for brain data.

• Peak-based: DIME(2011), DBChIP(2012). Caveats.

• Read counts + DESeq(2010)/edgeR(2010): Not convenient to use.

K562

ESC

Peaks

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diffReps: a ChIP-seq differential analysis package

• Written in PERL, easy to use command line tool; Do everything in one command.

• Sliding window strategy.

Background modeling

Normalization

Differential test

Merge and re-test

Multiple testing

correction

Workflow

diffReps.pl -tr A.bed B.bed -co C.bed D.bed -gn mm9 -re report.txt

Google code:

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Differential analysis & tail behavior

Gaussian: p=1E-5

Empirical: p=1E-5

H3K4me3 from mouse brain; bin1kb counts normalized.

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Statistical tests for differential analysis

• Negative binomial test: models biological replicates, over-dispersion

• T-test: NOT recommended• X2 test: SUM((exp – emp)^2)

=> X2 distr (p-val).• G-test: SUM(ln(emp / exp))

=> X2 distr (p-val). A modification to X2 test, recommended.

diffReps on H3K4me3: cocaine vs. saline

Negative binomial test T-test6527

282

130

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Two additional tools1. Find hotspots - hotspots are regions where the differential sites or peaks occur significantly more often than random chance.

Hotspot

Differential sites

Greedy search algorithmLocal Poisson

Eval

2. Region analysis - any file with the first 3 columns to be: chromosome, start, end. Annotate gene and heterochromatic regions

Easy to use: region_analysis.pl -i input.txt

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Test data: ENCODE H3K4me3 between K562 and ESC

Target: H3K4me3 Mock: DNA Input

Identify differential chromatin modification sites

ESC K562

Rep1

Rep2

Rep1

Rep2

Estimate empirical false positive rate

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Sensitivity & SpecificityTarget

Mock

Negative binomial vs. G-test

eFDR < .05%

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Overlapped & specific sitesUp-regulated sites, do the same for down sites

“Specific”

“Overlapped”

Promoter

Genebody Promoter Genebody

Using default p<1E-4

RNA-seq

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Correlating differential sites with transcription

“Specific”“Overlapped”

K562, ESC RNA-seq TopHat-Cufflinks: gene exp change, alternative promoter/splicing

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diffReps “specific” sites - examples

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diffReps is used in many works

Big cocaine project:

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diffReps: current status & community feedback

diffRepspublished

Great to see diffreps has found a nice home in plos one. It is literally the program which has saved my sanity, my phD and probably the paper i'm writing!

- Michael Reschen, Oxford Univ., UK

http://dx.plos.org/10.1371/journal.pone.0065598

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Acknowledgement

Role Li Shen Ningyi Shao Xiaochuan Liu Eric NestlerDevelopmentTest & resultDocumentation

Google codeMoney$

diffReps: