[BioC] Diffbind : use of controlBam

[Rory Stark]

Hi Daniele and Thomas-

Given that the controls are actually ChIPs with signal, you are certainly
correct to take them into account, and not surprising that this has an
effect on clustering.

One thing I'd suggest to get a better handle on this effect is to compare
different counting/normalization scores. You can try these by using the
"score" parameter to dba.plotHeatmap (or simply plot) and dba.plotPCA (I'd
definitely be looking at PCA plots in this case, esp. if you are seeing
batch effects in some cases).

For example, you can compare scores that do or don;t take the control into
account:

* DBA_SCORE_READS vs. DBA_SCORE_READS_MINUS or DBA_SCORE_READS_FOLD
* DBA_SCORE_RPKM vs DBA_SCORE_RPKM_FOLD
* DBA_SCORE_TMM_READS_FULL or DBA_SCORE_TMM_READS_EFFECTIVE vs
DBA_SCORE_TMM_MINUS_FULL or DBA_SCORE_TMM_MINUS_EFFECTIVE

The DBA_SCORE_RPKM scores would be a good place to start!

Cheers-
Rory

From:  Daniele Merico <daniele.merico at sickkids.ca>
Date:  Fri, 26 Jul 2013 17:51:49 +0000
To:  Rory Stark <rory.stark at cruk.cam.ac.uk>
Cc:  Thomas Nalpathamkalam <thomas.nalpathamkalam at sickkids.ca>,
"bioconductor at r-project.org" <bioconductor at r-project.org>
Subject:  Re: Diffbind : use of controlBam


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 at sickkids.ca>
Date: Fri, 26 Jul 2013 17:32:48 +0000
To: Rory Stark <rory.stark at cruk.cam.ac.uk>
Cc: Daniele Merico <daniele.merico at 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 at sickkids.ca
(416)-813-7032
www.tcag.ca <http://www.tcag.ca>





________________________________________

This e-mail may contain confidential, personal and/or health
information(information which may be subject to legal restrictions on use,
retention and/or disclosure) for the sole use of the intended recipient.
Any review or distribution by anyone other than
 the person for whom it was originally intended is strictly prohibited. If
you have received this e-mail in error, please contact the sender and
delete all copies.







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

daniele.merico at sickkids.ca
daniele.merico at gmail.com

1===0
 0===1
  1=0
   0
  0=1
 0===1
 1===0
  0=0
   1
  0=1
 1===0







________________________________________

This e-mail may contain confidential, personal and/or health
information(information which may be subject to legal restrictions on use,
retention and/or disclosure) for the sole use of the intended recipient.
Any review or distribution by anyone other than
 the person for whom it was originally intended is strictly prohibited. If
you have received this e-mail in error, please contact the sender and
delete all copies.