Question: Counting Raw Reads with DiffBind
0
gravatar for jess.schust
4.0 years ago by
United States
jess.schust0 wrote:

Hello, 

I am trying to use diffbind on paired end read data. I have done the following to get our proper peaks of interest ( we have technical replicates: 

myDBA=dba(sampleSheet="All_samplesheet.csv", attributes=c(DBA_ID,DBA_CONDITION,DBA_REPLICATE))

myDBA=dba.peakset(myDBA, consensus=-DBA_REPLICATE, minOverlap=3)

I then used Rory's instructions to extract the raw read counts (this is what I need for our next step): 

myDBA <- dba.count(myDBA, peaks=NULL, score=DBA_SCORE_READS)

counts <- dba.peakset(myDBA, bRetrieve=TRUE)

However, when I look at the bam files (using IGV) there appear to be  way more reads than are reported in the count matrix.  How exactly are the reads counted? I have been using your raw read matrix to move forward in my analysis and I am concerned that I am using at the wrong counts.  

Any advice would be greatly appreciated.  

~Jessica 

diffbind • 1.1k views
ADD COMMENTlink modified 4.0 years ago by Gord Brown590 • written 4.0 years ago by jess.schust0
Answer: Counting Raw Reads with DiffBind
4
gravatar for Gord Brown
4.0 years ago by
Gord Brown590
United Kingdom
Gord Brown590 wrote:

Hi, Jessica,

Sorry for the slow response.  Rory passed along the issue (and your data) to me.  As far as I can tell the answer is that DiffBind by default removes reads that have a mapping quality below 15.  Particularly near centromeres and telomeres, quite a few reads tend to fall into this category.  We usually remove them prior to analysis, since we aren't confident that they're correctly mapped.

If you re-count with:

myDBA <- dba.count(myDBA, score=DBA_SCORE_READS, mapQCth=0, bRemoveDuplicates=FALSE)

you should get everything.  Let me know if you still don't see the numbers you're expecting.  Also, you can try adding the parameter "bUseSummarizedOverlaps=TRUE" which uses a slightly different counting algorithm.

Cheers,

 - Gord

ADD COMMENTlink written 4.0 years ago by Gord Brown590
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