HT-Seq Count (Banging Head Against Wall)

[bstamps]

So I've seen this error crop up in the forums several times, but attempting to solve with the suggestions (find/replace within the gff file, use the -i option) doesn't seem to help...

When I run

htseq-count /media/lab/Glacier/Completed_RNAseq/12.sam pf5.gff

I get the error

Error occured in line 5200 of file pf5.gff.
Error: Failure parsing GFF attribute line
[Exception type: ValueError, raised in __init__.py:177]


Checking the gff line 5200 looks like this (and a few after)

NC_004129 img_core_v400 CDS 6045540 6045707 . + 0 gene_id=637322614;locus_tag=PFL_5279;product=aspartate 1-decarboxylase precursor; PanD
NC_004129 img_core_v400 CDS 6045954 6047618 . + 0 gene_id=637322615;locus_tag=PFL_5280;product=glucose-6-phosphate isomerase
NC_004129 img_core_v400 CDS 6047747 6049684 . + 0 gene_id=637322616;locus_tag=PFL_5281;product=acetyl-coenzyme A synthetase
NC_004129 img_core_v400 CDS 6049738 6050604 . + 0 gene_id=637322617;locus_tag=PFL_5282;product=hypothetical protein
NC_004129 img_core_v400 CDS 6050665 6051942 . + 0 gene_id=637322618;locus_tag=PFL_5283;product=acetyl-CoA acetyltransferase

I'm lost as to how to proceed- There doesn't seem to be any error with the file

[dpryan]

I expect the semi-colon between "precursor" and "PanD" is causing an issue. PanD isn't its own field, but coming after a semicolon, I suspect htseq-count is expecting it to be. You might just remove the semi-colon and see if that fixes things.

[bstamps]

That did the trick.

New problem...

Warning: No features of type 'exon' found.
Warning: Malformed SAM line: MRNM != '*' although flag bit &0x0008 set
Warning: Malformed SAM line: RNAME != '*' although flag bit &0x0004 set
Segmentation fault (core dumped)




Sample is bacterial, so is there a way to suppress this? Pass "-t CDS" or something similar?

[dpryan]

If you only have "CDS" as opposed to both that and "exon", then yeah. That will at least remove the first warning. The second and third warnings can likely be ignored, they're warning you about something that's not actually incorrect. It's odd that htseq-count segfaults, hopefully "-t CDS" will clear that up.

[bstamps]

Trying again, converted my sorted.bam to a sorted.sam and had this error crop up.

Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1111:18163:68474 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1113:13764:65962 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1115:17060:20187 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1205:13812:3203 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1205:1284:98106 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1214:20402:7870 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1306:3086:83183 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1308:8208:59054 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1316:10565:55061 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2202:12622:48507 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2203:11480:33264 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2204:4697:77559 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2212:16668:56587 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2212:20141:98589 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2216:19574:46777 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:2307:18351:66925 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1102:11861:9301 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Malformed SAM line: MRNM != '*' although flag bit &0x0008 set
Warning: Read D3NH4HQ1:160:C1UJ8ACXX:7:1102:10738:17694 claims to have an aligned mate which could not be found. (Is the SAM file properly sorted?)
Warning: Malformed SAM line: RNAME != '*' although flag bit &0x0004 set

[dpryan]

Oh, you really want to use a name sorted, rather than coordinate sorted BAM file. One could write a version of htseq-count to work with coordinate sorted BAM files, but I expect it'd be slower.

Give "samtools sort -n" a try.