the dexseq_count.py is just yelling at me "claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)"! I sorted the bam files using "samtools sort" and then converted it to sam file using samtools view but Is still have these messages!
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Have you checke your free space. Out of quota maybe? Or maybe 'sort' puts its temporary files to /tmp (see option -T), an at least on our big server, this one is on a small partition which is always full. Also, use '-S 100G' to tell 'sort' that you have lots of memory. BTW, why '-s -c'? We don't need a stable sort.
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I have the same question as below.
Can anyone answer those questions?
very appreciate
Originally posted by glados View PostDear all.
I'm trying to find information about how HTSeq counts reads. I understood that one pair (properly paired) is counted as 1 count.
What about pairs that are not flagged as 'properly paired'?
What about the reads that lost their mate and became single reads?
Are they counted as 1 count as well? Or not counted at all?
Additionally I'm loosing quite many reads that have multiple mappings. Anyone figured out a way to deal with this in HTSeq, instead of just throwing them all out?
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A read that lost its mate will be counted once and a warning will be produced if the unmapped mate isn't actually in the file (tophat does this). I don't recall htseq-count caring about the properly paired flag.
Regarding multimappers, you don't know with certainty where they align, so the proper solution for downstream analyses toward which htseq-count is oriented would be to discard them.Last edited by dpryan; 09-09-2013, 08:11 AM.
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Originally posted by jkbonfield View PostThis task is a related one to the bamtofastq conversion in that collation by name is necessary, but not necessarily a full sort.
Collation is often fast, while sorting is very slow. I don't know if there are dedicated collation tools out there (but I'd be suprised if there aren't).
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Originally posted by Gonza View PostDear All,
Why not sort them BAM files instead of the SAM? SAM takes to much space. After running tophat2, I am thinking something like this:
$samtools sort -n accepted_hits.bam output.bam
then count using htseq-count.
Thoughts????
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Dear all,
Originally posted by dpryan View PostThere's no need to name sort anymore, htseq-count can handle coordinate sorted BAM files.
Code:-r ORDER, --order=ORDER 'pos' or 'name'. Sorting order of <alignment_file> (default: name). Paired-end sequencing data must be sorted either by [B]position[/B] or by read name, and the sorting order must be specified. Ignored for single- end data.
Many thanks
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