Hi all,
I am having a bit of a problem with htseq-count. I have single-end stranded RNA-seq experiment, aligned with tophat-2.0.14. SAM file is names sorted and using HTSeq-0.6.1p1 on Mac 10.7.5 with python 2.7. I also tried a few other versions of HTSeq, but it comes out with the same result.
When trying to run htseq-count:
htseq-count --stranded=reverse -t exon --idattr=gene_id --mode=intersection-nonempty ./test.sam ./gtf.gtf > ./htseq_count.txt
I get this error message:
Error:
[Exception type: AssertionError, raised in __init__.py:599]
Here are a few lines of my sam file:
HWI-ST1146:240:C5HD2ACXX:1:1101:1040:33602 16 X 13759664 50 100M * 0 0 GGATGCCTCCAATGACCGACTGAAGCCGACCAAGTGGCTGACCGCCACCGAGCTGGAGAACGTGCCCTNCCTCAACGACATCACCTGGGAGCGTTTGGAG
C<C@BBCCCCECB>9BCCDCCC<<5BBC>CCCCCBCC?@>9CDA<HGGIFIIGHFF<HHIIIIGGB:0#IGIHGFHFEHEDHEIIIIHHH@HFFFFF@@@ AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:68C31 YT:Z:UU NH:i:1 XS:A:+
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:2544 0 2L 450124 50 68M1I30M * 0 0 GATCCATTCGCCCACCGGCTTGGTTACGTACNACTTTCCGCCTTCATGGTTCAGCATAAAGTTTATGATTTTTTTTGACGATCTTGCGCTGGTCTCGCA
CCCFFFFFHFHHHJJJIIIJJHIBBFEHHHI#08?FHIIJJJGIJEHIJ=@AEA?AHFBE@7;@CCCCEEEDCB@@>?CBB@BDDCDDDBBBCDCDC<@ AS:i:-9 XN:i:0 XM:i:1 XO:i:1 XG:i:1 NM:i:2 MD:Z:31A66 YT:Z:UU NH:i:1 XS:A:-
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:10782 0 2L 2191942 50 100M * 0 0 CGCTCATTCACATAGGTCACCTTGGTGGCCGNTTGACCCTGGCCCTGGCTCTTGTCCTTCTCCTTTTCGGATATCGTCCTCTCGATGGAGCAGGAGGCCG CC@FFFFFGDFHHJJJEGGJI@HIJHJJJJJ#1?DFHIGIJIIIGHIIGIJJJJIJIJJJJJIJEEHDE6BFCCE=CBDDDDDDCDDDCBDDDDD?BBB5 AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:31C68 YT:Z:UU NH:i:1 XS:A:-
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:30995 16 3R 7900087 50 100M * 0 0 AAGGCAAGGATCCACCTTTCCTGCTAGAAGTCAGCGCACAAATCACTTTTGTTCTGTTCCTGCTGTTCNTGACCATCATTCTGATGAACCTGCTCGTGGG @:?CA>ACA><B@:9DDDBADDCA@CDDCEADFFHCHCIHG@@@7FHDHJIJIJIIHIGFIHEGB?11#IHGIIIIHFCHJJHGFEBHHHFFDDFFFCC@ AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:68G31 YT:Z:UU NH:i:1 XS:A:+
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:48103 16 2L 14617161 50 100M * 0 0 AACCAGAACGGAGCCATCTGGAAACTGGACTTGGGCGCCCTGGAGGCCATCCAGTGGACCAAGCACTGNGACTCCGGCATCTAAGAAGTGATACTCCCAA
>
BBBABBBBBBA4>BAA>;A@;5>@:BB?B?695(ECECEE@FAC@CCFEFFFDEFIEF??;F?:0#IIFGFCBAIHHEBEEIIIFFFA:=DDDD@@@ AS:i:-3 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:36A31G31 YT:Z:UU NH:i:1 XS:A:+
If I use another experiment with paired-end reads it works without a problem. As far as I can get it is an error related to "paired-end handling", although my reads are single-end.
It would appear that htseq-count recognises my reads as paired-end rather than single-end. It also does not matter if I have selected stranded= yes,no or reverse, same error.
Would really appreciate any help.
I am having a bit of a problem with htseq-count. I have single-end stranded RNA-seq experiment, aligned with tophat-2.0.14. SAM file is names sorted and using HTSeq-0.6.1p1 on Mac 10.7.5 with python 2.7. I also tried a few other versions of HTSeq, but it comes out with the same result.
When trying to run htseq-count:
htseq-count --stranded=reverse -t exon --idattr=gene_id --mode=intersection-nonempty ./test.sam ./gtf.gtf > ./htseq_count.txt
I get this error message:
Error:
[Exception type: AssertionError, raised in __init__.py:599]
Here are a few lines of my sam file:
HWI-ST1146:240:C5HD2ACXX:1:1101:1040:33602 16 X 13759664 50 100M * 0 0 GGATGCCTCCAATGACCGACTGAAGCCGACCAAGTGGCTGACCGCCACCGAGCTGGAGAACGTGCCCTNCCTCAACGACATCACCTGGGAGCGTTTGGAG
C<C@BBCCCCECB>9BCCDCCC<<5BBC>CCCCCBCC?@>9CDA<HGGIFIIGHFF<HHIIIIGGB:0#IGIHGFHFEHEDHEIIIIHHH@HFFFFF@@@ AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:68C31 YT:Z:UU NH:i:1 XS:A:+
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:2544 0 2L 450124 50 68M1I30M * 0 0 GATCCATTCGCCCACCGGCTTGGTTACGTACNACTTTCCGCCTTCATGGTTCAGCATAAAGTTTATGATTTTTTTTGACGATCTTGCGCTGGTCTCGCA
CCCFFFFFHFHHHJJJIIIJJHIBBFEHHHI#08?FHIIJJJGIJEHIJ=@AEA?AHFBE@7;@CCCCEEEDCB@@>?CBB@BDDCDDDBBBCDCDC<@ AS:i:-9 XN:i:0 XM:i:1 XO:i:1 XG:i:1 NM:i:2 MD:Z:31A66 YT:Z:UU NH:i:1 XS:A:-
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:10782 0 2L 2191942 50 100M * 0 0 CGCTCATTCACATAGGTCACCTTGGTGGCCGNTTGACCCTGGCCCTGGCTCTTGTCCTTCTCCTTTTCGGATATCGTCCTCTCGATGGAGCAGGAGGCCG CC@FFFFFGDFHHJJJEGGJI@HIJHJJJJJ#1?DFHIGIJIIIGHIIGIJJJJIJIJJJJJIJEEHDE6BFCCE=CBDDDDDDCDDDCBDDDDD?BBB5 AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:31C68 YT:Z:UU NH:i:1 XS:A:-
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:30995 16 3R 7900087 50 100M * 0 0 AAGGCAAGGATCCACCTTTCCTGCTAGAAGTCAGCGCACAAATCACTTTTGTTCTGTTCCTGCTGTTCNTGACCATCATTCTGATGAACCTGCTCGTGGG @:?CA>ACA><B@:9DDDBADDCA@CDDCEADFFHCHCIHG@@@7FHDHJIJIJIIHIGFIHEGB?11#IHGIIIIHFCHJJHGFEBHHHFFDDFFFCC@ AS:i:-1 XN:i:0 XM:i:1 XO:i:0 XG:i:0 NM:i:1 MD:Z:68G31 YT:Z:UU NH:i:1 XS:A:+
HWI-ST1146:240:C5HD2ACXX:1:1101:1041:48103 16 2L 14617161 50 100M * 0 0 AACCAGAACGGAGCCATCTGGAAACTGGACTTGGGCGCCCTGGAGGCCATCCAGTGGACCAAGCACTGNGACTCCGGCATCTAAGAAGTGATACTCCCAA
>
BBBABBBBBBA4>BAA>;A@;5>@:BB?B?695(ECECEE@FAC@CCFEFFFDEFIEF??;F?:0#IIFGFCBAIHHEBEEIIIFFFA:=DDDD@@@ AS:i:-3 XN:i:0 XM:i:2 XO:i:0 XG:i:0 NM:i:2 MD:Z:36A31G31 YT:Z:UU NH:i:1 XS:A:+If I use another experiment with paired-end reads it works without a problem. As far as I can get it is an error related to "paired-end handling", although my reads are single-end.
It would appear that htseq-count recognises my reads as paired-end rather than single-end. It also does not matter if I have selected stranded= yes,no or reverse, same error.
Would really appreciate any help.
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