Hi guys, I'm having some trouble running HTseq-count on paired end RNA-seq data. I use the following command to run HTse-count:
And the following command is used to sort the bam prior to running HTseq-count:
There seems to be a problem with matching paired reads. The following error message is reported and empty .count files are generated:
And the relevent section of the SAM file looks like this:
Has anyone else experienced this? Do you think its a problem with the fastq files, Tophat, Samtools, or HTseq-count?
Thanks
Code:
htseq-count -s no -a 10 Sorted.sam Homo_sapiens.GRCh37.74.gtf > Sorted.count
Code:
samtools sort -n bamfile.bam sorted_bamfile
Code:
Warning: Read HISEQ:309:C4UR9ACXX:2:1101:2467:41039 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Code:
HISEQ:309:C4UR9ACXX:2:1101:2467:41039 81 X 71494941 3 50M 19 22189602 0 GACCCTTGGTGTCATAGATCAGACGGAAATTCTCTCCCGTCTTGTCAATG FBIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFIIIIFFFFFFFFFFBBB AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:UU XS:A:- NH:i:2 CC:Z:= CP:i:71494941 HI:i:0 HISEQ:309:C4UR9ACXX:2:1101:2467:41039 337 X 71494941 3 50M 19 22394287 0 GACCCTTGGTGTCATAGATCAGACGGAAATTCTCTCCCGTCTTGTCAATG FBIIIIIIIIIIIIIIIIIIIIIIIIIIIIFFFIIIIFFFFFFFFFFBBB AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:50 YT:Z:UU XS:A:- NH:i:2 HI:i:1
Has anyone else experienced this? Do you think its a problem with the fastq files, Tophat, Samtools, or HTseq-count?
Thanks