Output of HTseq-count?

[kasutubh]

Hi Everyone,
I'm trying my hands at HTseq-count and was able to run it on our university cluster. We're using version htseq/0.4.6p1. My input command was
htseq-count --stranded=no -o samout q1.sam GeneWithUTR.gff
At the end of the run I got following output message,
43033693 reads processed.
no_feature 39980188
ambiguous 0
too_low_aQual 0
not_aligned 0
alignment_not_unique 63473377.
Is this all that is of the output? I looked for a file with read counts but didn't end up having one. Is there some problem here. I tried to give -o as output string..but to an error message saying that there's no such option. What am I doing wrong?
Thanks in advance for the answer!
Best,

[Simon Anders]

As a first step, try to use a current version of HTSeq, please.

Then, remember that you need to specify the '-i' and '-t' options unless you use an Ensembl GTF file.

[kasutubh]

Hi Simon,
Thanks a lot for the reply. I'll try the latest version!
Best,

[kasutubh]

Hi Simon,
I tried the latest version and also added the attributes..it works now.
Thanks a lot..
Best,

[billstevens]

Hi,

I'm switching over to try HTSeq to use with DESeq, and I'm having a little trouble getting it to run. Specifically, I'm running it like this:

python -m HTSeq.scripts.count -o HT_Out accepted_hits.sam hg19_knowngene2.gtf

I got my accepted_hits.sam file by first sorting by output from Tophat (accepted_hits.bam) and then using samtools view to convert the sorted accepted_hits.bam to a sorted accepted_hits.sam.

However, every line outputted says the following:
Warning: Read HWI-1KL118:23:C0J57ACXX:8:1106:5750:192499 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)

What am I doing wrong?

[kopi-o]

You need to sort by read name before inputting to HTSeq. It seems you have sorted in the "regular way", by genomic coordinates.

[billstevens]

Thanks, that did the trick!

[billstevens]

Can I run more than one count table at a time? Sorry, I'm not familiar with Python, so I've been running my HTSeq like this:

python -m HTSeq.scripts.count -o HT_Out accepted_hits.sam hg19_knowngene2.gtf

And after I run that on one of my samples, when I go to run it on another, it seems like the first file stopped running.

[Simon Anders]

How do you start the multiple processes? Most likely, you have made a mistake that has little to do with Python or htseq.