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@dimitra-alexopoulou-6348
Last seen 7.4 years ago
Germany

Hi all (and Alejandro!)

I have come across a problem with the DEXSeq count script (DEXSeq_1.14.2), namely getting the following error while wanting to produce the counts per exon bin by running dex_seq_count.py:

I'm using gsnap for alignment and when working on paired end data the count script went through. It was only when working on single read that I got this type of error, which was corrected once I removed the unmapped reads from my bam file.

Looking at the script itself dexseq_count.py) lines 182-189, I think I know the reason:

if not is_PE:

for a in reader( sam_file ):
if a.optional_field("NH") > 1:
continue
if not a.aligned:
counts[ '_notaligned' ] += 1
continue

So initially, the first if clause looks for the NH flag and then there is the control whether or not the read is aligned. Reversing the order of these two, so first checking whether the read has been aligned has now worked for me. I just wanted to let people with the same problem know how they could work around this, but I guess this should be as well changed in the next version.

Best,

Dimitra

dexseq • 1.8k views
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Alejandro Reyes ★ 1.8k
@alejandro-reyes-5124
Last seen 6 days ago
Novartis Institutes for BioMedical Rese…

Thanks Dimitra for your report and for the fix! I have implemented it to the latest versions of DEXSeq.

Alejandro

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@julsjaeger-7621
Last seen 7.0 years ago
Germany

Hi,

I am using the 3.2 version but I got the same problem:

python /home/julian/R/x86_64-pc-linux-gnu-library/3.2/DEXSeq/python_scripts/dexseq_count.py -f bam dexseq/annotation/RefGene.fixed.gff 2monthsCTX/WT/WT1/mm10.bam dexseq/annotation/dexseq/WT1fb.txt

Traceback (most recent call last):
File "/home/julian/R/x86_64-pc-linux-gnu-library/3.2/DEXSeq/python_scripts/dexseq_count.py", line 187, in <module>
if a.optional_field("NH") > 1:
File "_HTSeq.pyx", line 1399, in HTSeq._HTSeq.SAM_Alignment.optional_field (src/_HTSeq.c:26483)

The alignment was done with the STAR aligner.

Any Ideas how it can be approached?

Thanks, Julian

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Hi, the problem was that I used the wrong bam files. works now. Sorry for the useless post.