HTSeq: Error at the end of HTSeq analysis and an output of .count file with 0 byte
At the beginning of the process I get this warning:
Warning: Read HS1_07961:1:1308:18104:63516#3 claims to have an aligned mate which could not be found in an adjacent line.
Then, at the end of the SAM file, I get:
10800000 SAM alignment record pairs processed.
10900000 SAM alignment record pairs processed.
Error occured when processing SAM input (record #11138334 in file 7961_1_3.bam):
reference_id -1 out of range 0<=tid<22
[Exception type: ValueError, raised in calignmentfile.pyx:640]
and my output .count file was empty, with 0 byte.
The code I entered for this process is:
htseq-count -f bam -s no -a 10 7961_1_3.bam Mus_musculus.GRCm38.82.gtf > 7961_1_3.count
note that my BAM files contain pair ended strands.
Any help will be appreciated!
Thank you in advance.
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What's the output of
samtools view 7961_1_3.bam | awk '{if(NR==11138334) print $0}'? It's likely that there's a malformed line.Is your bam sorted by name? htseq-count expects pairs to be sorted by name, thus they should be on adjacent lines.
GTF file wrong?
How was the BAM file generated? Does it pass Picard tools validation?