featureCounts isn't working how I expect

Question

[jrbustosm]

I'm runnig next command

featureCounts -s 1 -t exon -g Parent -a test.gff -o test.counts test.sam

in test.sam

HWI-ST568:115:D04H3ACXX:7:1101:5518:2079    0   gi|448814763|ref|NC_000962.3|   2125761 255 61M *   0   0   TGGACAAGCTAGGAGAGGAGCTTTACTCGGCGCAGTGCGTCTCTCGCCCACCGACCTGATG   CCCFFFFFHDBHHIGFHIEGHIIJCCGHHJHHJBHGHGIGGIDGGGGHGAEFFDDDDDDDD   XA:i:0  MD:Z:61 NM:i:0

in test.gff

TEST    GenBank exon    2125761 2504608 .   -   .   Parent "XXX" 

I get in test.counts,

# Program:featureCounts v1.5.0; Command:"featureCounts" "-s" "1" "-t" "exon" "-g" "Parent" "-a" "test.gff" "-o" "test.counts" "test.sam" 
Geneid  Chr Start   End Strand  Length  test.sam
XXX TEST    2125761 2504608 -   378848  0

I expected,

# Program:featureCounts v1.5.0; Command:"featureCounts" "-s" "1" "-t" "exon" "-g" "Parent" "-a" "test.gff" "-o" "test.counts" "test.sam" 
Geneid  Chr Start   End Strand  Length  test.sam
XXX TEST    2125761 2504608 -   378848  1

What's the mistake that I'm making?

[Devon Ryan]

Aside from the chromosome name issue, I imagine that the strandedness setting would cause problems as well.

[jrbustosm]

You are right ..... this is the problem thanks

Answer 1

[Martombo]

The chromosome should be "TEST" in the sam file

[jrbustosm]

"gi|448814763|ref|TEST|"

instead of

"gi|448814763|ref|NC_000962.3|"

?

[Devon Ryan]

Just "TEST", unless you edited things to remove gi|448814763|ref|NC_000962.3| from the GFF file snippet you posted.

[jrbustosm]

thanks @Martombo, @Devon Ryan this solved my problem