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diffReps: automated ChIP-seq
differential analysis package
Li Shen
Asst. Professor
Neuroscience, Mount Sinai
06/28/2013
Sl...
ChIP-seq differential analysis
Treatment
(coc i.p.)
Control
(sal i.p.)
Rep1
Rep2
Rep3
Rep1
Rep2
Rep3
Differences
Venn diag...
Subtle changes of chromatin
modifications
H3K4me3 from ENCODE
K562
ESC
? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?...
Existing programs for differential
analysis
• ChIPDiff(2008): HMM-based
approach. NOT sensitive
enough for brain data.
• P...
diffReps: a ChIP-seq differential analysis package
• Written in PERL, easy
to use command line
tool; Do everything in
one ...
Differential analysis & tail behavior
Gaussian: p=1E-5
Empirical: p=1E-5
 H3K4me3 from mouse
brain; bin1kb counts
normali...
Statistical tests for differential analysis
• Negative binomial test:
models biological replicates,
over-dispersion
• T-te...
Two additional tools
1. Find hotspots - hotspots are regions where the differential
sites or peaks occur significantly mor...
Test data: ENCODE H3K4me3 between
K562 and ESC
Target: H3K4me3 Mock: DNA Input
Identify differential chromatin
modificatio...
Sensitivity & Specificity
Target
Mock
Negative binomial vs. G-test
eFDR < .05%
10
Overlapped & specific sites
Up-regulated sites, do the same for down sites
“Specific”
“Overlapped”
Promoter
Genebody Promo...
Correlating differential sites with transcription
“Specific”“Overlapped”
K562, ESC RNA-seq TopHat-Cufflinks: gene exp chan...
diffReps “specific” sites - examples
13
diffReps is used in many works
Big cocaine project:
14
diffReps: current status & community
feedback
diffReps
published
Great to see diffreps has found a nice home in plos one. ...
Acknowledgement
Role Li Shen Ningyi Shao Xiaochuan Liu Eric Nestler
Development
Test & result
Documentation
Google code
Mo...
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diffReps: automated ChIP-seq differential analysis package

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diffReps is published in PLoS ONE. Link: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0065598

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  • I think your question is about the normalization method used. Briefly, diffReps calculates a normalization factor for each sample on each window, and then take the median across all windows on the genome. You may find more details in the diffReps paper which is published on PLoS One. Thx!
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  • Hi,

    Good tool. will consider using it. A question about it.

    How does DiffReps handle difference in total number of reads in Control and Treatment sample?

    Thanks.
       Reply 
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diffReps: automated ChIP-seq differential analysis package

  1. 1. diffReps: automated ChIP-seq differential analysis package Li Shen Asst. Professor Neuroscience, Mount Sinai 06/28/2013 Slides adapted from previous presentation
  2. 2. ChIP-seq differential analysis Treatment (coc i.p.) Control (sal i.p.) Rep1 Rep2 Rep3 Rep1 Rep2 Rep3 Differences Venn diagram for peak lists Treatment Control False positive False negativeTreatment Control 2
  3. 3. Subtle changes of chromatin modifications H3K4me3 from ENCODE K562 ESC ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ASUN: Asunder, Spermatogenesis Regulator [0, 1.2] [0, 1.2] 3
  4. 4. 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 4
  5. 5. 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: 5
  6. 6. Differential analysis & tail behavior Gaussian: p=1E-5 Empirical: p=1E-5  H3K4me3 from mouse brain; bin1kb counts normalized. 6
  7. 7. 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 7
  8. 8. Two additional tools 1. Find hotspots - hotspots are regions where the differential sites or peaks occur significantly more often than random chance. Hotspot Differential sites Greedy search algorithm Local 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 8
  9. 9. 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 9
  10. 10. Sensitivity & Specificity Target Mock Negative binomial vs. G-test eFDR < .05% 10
  11. 11. Overlapped & specific sites Up-regulated sites, do the same for down sites “Specific” “Overlapped” Promoter Genebody Promoter Genebody Using default p<1E-4 RNA-seq 11
  12. 12. Correlating differential sites with transcription “Specific”“Overlapped” K562, ESC RNA-seq TopHat-Cufflinks: gene exp change, alternative promoter/splicing 12
  13. 13. diffReps “specific” sites - examples 13
  14. 14. diffReps is used in many works Big cocaine project: 14
  15. 15. diffReps: current status & community feedback diffReps published 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 15 http://dx.plos.org/10.1371/journal.pone.0065598
  16. 16. Acknowledgement Role Li Shen Ningyi Shao Xiaochuan Liu Eric Nestler Development Test & result Documentation Google code Money$ diffReps: 16

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