Re: [Useq-users] ORSS not counting reads properly?

[David N. <Dav...@hc...>]

Let me take a look, there are several filters that are applied to exclude
bad alignments before being counted by ODRSS.  Thus the alignments counted
will likely be a subset of the input file.

On 4/19/12 10:12 AM, "Oler, Andrew (NIH/NIAID) [C]" <and...@ni...>
wrote:

>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)
>OCICB/OSMO/OD/NIAID/NIH
>
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