Question: warning message after HTSeq
gravatar for BioDH
28 days ago by
BioDH0 wrote:

I have analyzed RNA-seq data with HTSeq.

My command that I used is

python -m HTSeq.scripts.count -f bam -r pos -s reverse -t ORF -i group -m union input.bam my.gff > output.txt

Warning message is

Warning: Mate records missing for 3752 records; first such record: <SAM_Alignment object: Paired-end read 'K00198:150:HGYHGBBXX:6:2219:20435:16049' aligned to chrII:[2341669,2341744)/+>.
11597356 SAM alignment pairs processed.

Do I have to concern the message? What does it mean?

rna-seq htseq software error • 160 views
ADD COMMENTlink modified 28 days ago • written 28 days ago by BioDH0

Is it only me that is confused, because DESeq2 is a BioC package. What command are you running there? Anyway, you are missing mate reads, where the read1 is probably flagged as paired. Probably you did any filtering (MAPQ maybe) where one of the two mates got removed. If that bothers you, you can use samtools fixmate to update the flags. Input would be a name-sorted bam file.

ADD REPLYlink written 28 days ago by ATPoint2.4k

Sorry. I have changed the title.

Yes, that is not DESeq2.

I just got the reads using HTSeq module of puthon.

ADD REPLYlink written 28 days ago by BioDH0

Hello BioDH,

Can you confirm that you are working with Pair-end data? This warning is telling that for 3752 reads of your BAM file, one of the two reads of the pair if missing. You should consider checking the program that gives you the BAM file. Or, you can skip this warning if you think that 7K reads of 11597k reads is negligible.

ADD REPLYlink written 28 days ago by glihm530

I'm sure that is paired-end data.

ADD REPLYlink written 28 days ago by BioDH0

What did you do prior to running htseq count?

ADD REPLYlink written 28 days ago by WouterDeCoster23k
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