Hello, new to RNA-seq here. I have mapped my fastq reads to a reference genome (transcript.fasta) with HISAT2. The BAM files look very good with >85% reads mapped. However, when I try to use featurecounts, both local and on Galaxy, the results always yield 0 mapped reads. From lurking around the web, it seems there may be a disconnect between my BAM file Headers and the .GFF file that I am using in feature counts.
Example line of .GFF Seqname Source Feature Start End Score Strand Frame Group Niben101Ctg00054 maker exon 167 487 . + . ID=Niben101Ctg00054g00001.1:exon:001;Parent=Niben101Ctg00054g00001.1;Alias=snap_masked-Niben101Ctg00054-processed-gene-0.0-mRNA-1:exon:4812 Example line of .BAM A00247:109:GW190602181st_novaxp:1:1402:19651:8030 419 Niben101Ctg00109Ctg001 108 3 141M = 120 151 CTTCAGACATCTTGGCATGGCATTTGACATGTATCAATCGTTGACACCATAAACAATACCAAGTTGGAGATGCATCAAGGAAAGGAACACCACAAGGTTCATCACAGTAGAAACAAAAGGCAGACATCTCAGGATTCTCAT FFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFF AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:141 YT:Z:UU NH:i:2 CC:Z:Niben101Scf04375Ctg112 CP:i:5129 HI:i:0
Here is a sample output summary:
Status accepted_hits_Mock-1.bam Assigned 0 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_Chimera 7331650 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 10893853 Unassigned_Secondary 0 Unassigned_Nonjunction 0 Unassigned_NoFeatures 26615011 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 0
Is there any obvious discrepancy that could cause this? In featurecounts, I have used 'ID' as my gene identifier, not gene_id as it is not present in my .gff file. I'm hoping there is a simple error that is causing this complete failure.