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Originally posted by TEFA
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Actually no, right now I'm trying to avoid using '-' as an htseq-count argument without using it in a pipeline with samtools view, I'll let you know what happens... -
Was this by any chance a TopHat output? Somebody alerted me recently to a bug that causes mishandling of SAM lines that contain the "NH" flag, and that seems to appear in newer tophat output. I'll try to fix this ASAP.Originally posted by EGrassi View Post
SimonComment
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Yep, I can confirm you that removing the NH flags from a subset of my sam files I get some counts different from 0. This could be a temporary workaroud.Comment
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Ok, on the whole data it falls back to all 0:
samtools view S1.sorted.bam.bam | sed 's/NM:i:\d//g' | htseq-count --stranded=no - /rogue/bioinfotree/prj/ewing-rnaseq/local/share/data/Homo_sapiens/UCSC/hg19/Annotation/Genes/genes.gtf > counts_S1_htseqq
I'm really a little surprised by the amount of errors and other glitches in rna seq software (this is a generic rant, not focused on htseq!)...it's a really complicated and new area, that's true, but right now using any of the existing tool seems a bet and to tell the truth I never feel confident on the results, as long as with every version results changes, and when something runs without errors I never know if that means that it worked or that an exception just had not screamed enough :/Comment
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The bug with the NH flags, which was introduced during some refactoring in the version of a week ago or so is now fixed in version 0.5.3p8. Sorry that the recent changes turned out to be so volatile.Comment
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Thanks, 0.5.3p9 has produced counts different from zero. Let's see what deSeq thinks of this data!
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Hello, I have performed the mapping of miRNA reads to the mirbase precursor file by SHRiMP. But I'm facing a probem in HTSeq while taking the read counts from the output.sam file.Originally posted by Simon Anders View Post
The error which I'm trying to solve is;
Error occured when processing SAM input (line 2305 of file HBI_10.sam): ("'seq' and 'qualstr' do not have the same length.", 'line 2305 of file HBI_10.sam') [Exception type: ValueError, raised in _HTSeq.pyx:808]
Has anyone of you come across this error? What might be the solution?Comment
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Sushant - what does line 2305 of your SAM file look like? Unless the quality entry is the special value '*' only then it is a likely an unrelated issue.Comment
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Actually, I did see that SAM file. The problem is that my sequence string & its respective sequence quality string is not of equal length.Originally posted by maubp View Post
However, I converted my fastq files to fasta and then performed the alignment, which solved this problem.
But the mapping percentage I'm getting with SHRiMP is too low(2.77%), for the mirna aligning to mature.
What parameters should I consider?Comment
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The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
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