[Useq-users] ORSS not counting reads properly?

[Oler, A. (NIH/N. [C] <and...@ni...>]

Hi David,

I have two questions about ORSS.  I'm using ORSS in USeq 8.2.0.  I was checking on a few genes (randomly chosen) that had no read counts and noticed that there are some reads for those exons of those genes.  Is there a threshold number of reads that must be passed before the counts are reported?  (e.g., 5 in at least one dataset?)  Is there a way to get ORSS to report all of the read counts regardless of threshold (and only skip the differential testing if below a threshold)? (e.g., Dnajb7, Apol6, Dnahc5).

What I'm more concerned about is a few genes that I noticed with large differences between replicates.  ORSS reports high counts, but when I look at the raw reads, there is actually no expression for these genes at all.  See the screenshots for Cav3 and Oxtr.  In the attached spreadsheet, you can see that the read counts for the genes are 572 and 574, respectively for T1 (aka KO1 bam file in IGB).  But there are no reads for these exons in the KO1 bam file.  I repeated with just a single replicate and I got the same result (KO1 as treatment and WT1 as control).  If you look at the wide shot of Cav3, you see that there are some spliced reads that flank these genes.  I think these are being counted, even though their aligned bases are not overlapping any exons; there are about 500-600 reads that are hidden that probably have the same profile, which is probably why the counts are ~572/4.  I also just tried this with USeq 8.2.2 and it gives the same result.

Thanks,

Andrew

Andrew Oler, Ph.D.
High-Throughput Sequencing Bioinformatics Specialist
Contractor – Medical Science & Computing, Inc.
Computational Biology Section
Bioinformatics and Computational Biosciences Branch (BCBB)
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