htseq-count for sam and gff3

[sofia17]

Hi everyone,

I need some help with running htseq-count.

I am running htseq-count for a sam file obtained as a result of Illumina reads (not paired) aligned to the genome. I am getting the following Warning for all my reads:

"Warning: Skipping read 'S5_057841196', because chromosome 'SL2.40ch00', to which it has been aligned, did not appear in the GFF file."

and of course, all of my reads go in the "alignment_not_unique" pile, so there is no gene count at the end.

I read a similar problem posted by hibachings2013 in 2010, but unlike his problem, my sam and gff3 files BOTH have the correct chromosome names.

What am I doing wrong?

Below are examples of entries in my sam and gff3 files:

@HD VN:1.0 SO:coordinate
@SQ SN:SL2.40ch00 LN:21805821
@SQ SN:SL2.40ch01 LN:90304244
@SQ SN:SL2.40ch02 LN:49918294
@SQ SN:SL2.40ch03 LN:64840714
@SQ SN:SL2.40ch04 LN:64064312
@SQ SN:SL2.40ch05 LN:65021438
@SQ SN:SL2.40ch06 LN:46041636
@SQ SN:SL2.40ch07 LN:65268621
@SQ SN:SL2.40ch08 LN:63032657
@SQ SN:SL2.40ch09 LN:67662091
@SQ SN:SL2.40ch10 LN:64834305
@SQ SN:SL2.40ch11 LN:53386025
@SQ SN:SL2.40ch12 LN:65486253
@PG ID:TopHat VN:1.3.1 CL:/usr/local/bin/tophat -I 5000 --segment-mismatches 1 -o ./Output/tophatSA1_S6/ -G ./Annotation/ITAG2.3_gene_models.gff3 ./Reference/S_lycopersicm_genome ./Input/SA1_S6
S6_021828409 272 SL2.40ch00 96314 0 49M * 0 0 GACTCTTGAATACAATCTTACAATTTTTCCTCACAAATTGCTACACCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 NH:i:7 CC:Z:SL2.40ch02 CP:i:13220492 HI:i:0
S6_028644958 256 SL2.40ch00 225372 0 49M * 0 0 CCGACACACTAAATAAAAGAACAATATCACATTGCATATCAAACTAATA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:1 NH:i:11 CC:Z:= CP:i:3434611 HI:i:0
S6_008579902 272 SL2.40ch00 251575 0 49M * 0 0 CTTATGATTTTAATATGAACACATTTATCACTTTCATCATTCTTCGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 NH:i:10 CC:Z:SL2.40ch01 CP:i:5753251 HI:i:0
S6_033284153 272 SL2.40ch00 251575 0 49M * 0 0 CTTATGATTTTAATATGAACACATTTATCACTTTCATCATTCTTCGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 NH:i:10 CC:Z:SL2.40ch01 CP:i:5753251 HI:i:0
S6_084652203 272 SL2.40ch00 251582 0 49M * 0 0 TTTTAATATGAACACATTTATCACTTTCATCATTCTTCGATCCATTTGC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 NH:i:8 CC:Z:SL2.40ch01 CP:i:5753258 HI:i:0
S6_015020076 272 SL2.40ch00 251583 0 49M * 0 0 TTTAATATGAACACATTTATCACTTTCATCATTCTTCGATCCATTTGCC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII NM:i:2 NH:i:9 CC:Z:SL2.40ch01 CP:i:5753259 HI:i:0

##gff-version 3
##feature-ontology http://song.cvs.sourceforge.net/*che...?revision=1.93
##sequence-region SL2.40ch00 1 21805821
SL2.40ch00 ITAG_eugene gene 16437 18189 . + . Alias=Solyc00g005000;ID=gene:Solyc00g005000.2;Name=Solyc00g005000.2;from_BOGAS=1;length=1753
SL2.40ch00 ITAG_eugene mRNA 16437 18189 . + . ID=mRNA:Solyc00g005000.2.1;Name=Solyc00g005000.2.1;Note=Aspartic proteinase nepenthesin I (AHRD V1 **-- A9ZMF9_NEPAL)%3B contains Interpro domain(s) IPR001461 Peptidase A1 ;Ontology_term=GO:0006508;Parent=gene:Solyc00g005000.2;from_BOGAS=1;interpro2go_term=GO:0006508;length=1753;nb_exon=2
SL2.40ch00 ITAG_eugene exon 16437 17275 . + . ID=exon:Solyc00g005000.2.1.1;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene five_prime_UTR 16437 16479 . + . ID=five_prime_UTR:Solyc00g005000.2.1.0;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene CDS 16480 17275 . + 0 ID=CDS:Solyc00g005000.2.1.1;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene intron 17276 17335 . + . ID=intron:Solyc00g005000.2.1.1;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene exon 17336 18189 . + 0 ID=exon:Solyc00g005000.2.1.2;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene CDS 17336 17940 . + 2 ID=CDS:Solyc00g005000.2.1.2;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene three_prime_UTR 17941 18189 . + . ID=three_prime_UTR:Solyc00g005000.2.1.0;Parent=mRNA:Solyc00g005000.2.1;from_BOGAS=1
###
SL2.40ch00 ITAG_eugene gene 68062 68764 . + . Alias=Solyc00g005020;ID=gene:Solyc00g005020.1;Name=Solyc00g005020.1;from_BOGAS=1;length=703
SL2.40ch00 ITAG_eugene mRNA 68062 68764 . + . ID=mRNA:Solyc00g005020.1.1;Name=Solyc00g005020.1.1;Note=Unknown Protein (AHRD V1);Parent=gene:Solyc00g005020.1;from_BOGAS=1;length=703;nb_exon=3;eugene_evidence_code=10F0H0E0IEG
SL2.40ch00 ITAG_eugene exon 68062 68211 . + 0 ID=exon:Solyc00g005020.1.1.1;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene CDS 68062 68211 . + 0 ID=CDS:Solyc00g005020.1.1.1;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene intron 68212 68343 . + . ID=intron:Solyc00g005020.1.1.1;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene exon 68344 68568 . + 0 ID=exon:Solyc00g005020.1.1.2;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene CDS 68344 68568 . + 0 ID=CDS:Solyc00g005020.1.1.2;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene intron 68569 68653 . + . ID=intron:Solyc00g005020.1.1.2;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene exon 68654 68764 . + 0 ID=exon:Solyc00g005020.1.1.3;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1
SL2.40ch00 ITAG_eugene CDS 68654 68764 . + 0 ID=CDS:Solyc00g005020.1.1.3;Parent=mRNA:Solyc00g005020.1.1;from_BOGAS=1