FeatureCounts Not Reading GTF File Correctly

Question

[dec986]

Hello,

I am writing my own GTF file, and featureCounts will only process some of the reads and not others, I have no idea why, as far as I can tell, the lines are identical.

why does featureCounts recognize lines like

chrM    ENSEMBL gene    15356   15422   .   -   .   gene_id "ENSMUSG00000064372.1"; gene_type "Mt_tRNA"; gene_status "KNOWN"; gene_name "mt-Tp"; level 3;
chrM    ENSEMBL transcript  15356   15422   .   -   .   gene_id "ENSMUSG00000064372.1"; transcript_id "ENSMUST00000082423.1"; gene_type "Mt_tRNA"; gene_status "KNOWN"; gene_name "mt-Tp"; transcript_type "Mt_tRNA"; transcript_status "KNOWN"; transcript_name "mt-Tp-201"; level 3; tag "basic"; transcript_support_level "NA";
chrM    ENSEMBL exon    15356   15422   .   -   .   gene_id "ENSMUSG00000064372.1"; transcript_id "ENSMUST00000082423.1"; gene_type "Mt_tRNA"; gene_status "KNOWN"; gene_name "mt-Tp"; transcript_type "Mt_tRNA"; transcript_status "KNOWN"; transcript_name "mt-Tp-201"; exon_number 1; exon_id "ENSMUSE00000521550.1"; level 3; tag "basic"; transcript_support_level "NA";

but not read my added lines, written like this?

chr1    ENSEMBL transcript  13139159    13142763    .   -   .   gene_id "Unknown7"; transcript_id "Unknown7"; gene_type "TEC"; gene_status "PUTATIVE"; gene_name "Unknown7"; transcript_type "TEC"; transcript_status "PUTATIVE"; transcript_name "Unknown7"; exon_number 1; exon_id "Unknown7"; level 3;
chr1    ENSEMBL transcript  13139159    13142763    .   -   .   gene_id "Unknown8"; transcript_id "Unknown8"; gene_type "TEC"; gene_status "PUTATIVE"; gene_name "Unknown8"; transcript_type "TEC"; transcript_status "PUTATIVE"; transcript_name "Unknown8"; exon_number 1; exon_id "Unknown8"; level 3;
chr1    ENSEMBL transcript  13139159    13142763    .   -   .   gene_id "Unknown9"; transcript_id "Unknown9"; gene_type "TEC"; gene_status "PUTATIVE"; gene_name "Unknown9"; transcript_type "TEC"; transcript_status "PUTATIVE"; transcript_name "Unknown9"; exon_number 1; exon_id "Unknown9"; level 3;

I realize this is a tedious question... but I've spent hours on this and I can't see the problem :(

[WouterDeCoster]

Could you add the command you use for featurecounts with these gtf files?

[dec986]

the command I use is:

featureCounts -g transcript_id -a ~/GENE_DATA/mm10/embryo_novel_transcripts_only.gtf -o transcript_id_featureCount.tsv sorted.bam

[WouterDeCoster]

Default behavior is to count only for feature 'exon', you have to specify the -t flag, in your case

-t transcript

[dec986]

yes! this solves my problem, instead of using -g.

Answer 1

[dec986]

The key here is that

-t

means the 3rd column in Gencode GTF.

Also,

-g

means 9th column. I think -t option works better.