Thanks Rory.

We find quite different results when using bamControls, with heatmaps looking much cleaner (no residual batch effects between replicates).

We will look with more care into differences. Luckily we have a few loci for which we know the true differences, and since this is a methyl-seq experiment we expect an enrichment of significantly differential peaks overlapping CpG islands.

Best,
Daniele and Thomas

On 2013-07-26, at 1:44 PM, Rory Stark wrote:

Hello Thomas-

This should work fine (using the same bam files as both ChIP and control). I don't think I've actually tested this scenario, so if there is a problem it would be a bug that would get fixed.

Cheers-
Rory



From: Thomas Nalpathamkalam <thomas.nalpathamkalam@sickkids.ca<mailto:thomas.nalpathamkalam@sickkids.ca>>
Date: Fri, 26 Jul 2013 17:32:48 +0000
To: Rory Stark <rory.stark@cruk.cam.ac.uk<mailto:rory.stark@cruk.cam.ac.uk>>
Cc: Daniele Merico <daniele.merico@sickkids.ca<mailto:daniele.merico@sickkids.ca>>
Subject: Diffbind : use of controlBam

Dear Rory Stark ,

we used DiffBind with MACS peaks.

In our experiment, we had 3 biological replicates x 2 experimental conditions (tumor, control). We did not have input samples, so when we did peak calling in MACS we paired samples as follows: rep1-ctrl vs rep1-tumor, rep2-ctrl vs rep2-tumor, rep3-ctrl vs rep3-tumor, rep1-tumor vs rep1-ctrl, rep2-ctrl vs rep2-tumorand rep3-ctrl vs rep3-tumor . This gave us the peaks for each control and tumor replicate. This is approved by the MACS protocol.

When we load the MACS peaks in DiffBind, we are not sure if we can use the same replicate twice: as a "bamReads" for its condition, and as a "controlBam" for the other condition. This follows the sample matching we have used in MACS, but it may not work right in DiffBind.

What is opinion  on this? Do you prefer to do analysis without "controlBam" in this scenario?

Thank you in advance,


Thomas Nalpathamkalam




Thomas Nalpathamkalam
The Centre for Applied Genomics (TCAG)
The Hospital for Sick Children
MaRS Building - East Tower
101 College St., Room 14-701
Toronto, ON  M5G 1L7
thomas.nalpathamkalam@sickkids.ca<mailto:thomas.nalpathamkalam@sickkids.ca>
(416)-813-7032
www.tcag.ca<http://www.tcag.ca>

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Daniele Merico
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PhD, Molecular And Cellular Biology
Informatics Core Facility Manager
The Centre for Applied Genomics (TCAG)
Toronto (ON), Canada

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