The annotation I am trying to use is the Zm-B73-REFERENCE-NAM-5.0_Zm00001eb.1.gff3.gz file linked here: Index of /Zm-B73-REFERENCE-NAM-5.0. I suspect this gff3 file is the source of the problems and needs to be altered somehow to work with featurecounts
I have paired-end RNA-seq reads from maize that I am trying to convert to counts using featurecounts. I basically followed the steps outlined in this tutorial, but with a few differences (e.g. my RNA library is forward-stranded and has longer reads): Reference-based RNA-Seq data analysis
The creation of a collection of 2 paired ends, quality control, and initial mapping with RNA-star seemed to work, but then when I tried to do featurecounts and got an error: “failed to find the gene identifier attribute in the 9th column of the provided GTF file. The specified gene identifier attribute is ‘gene_id’. An example of attributes included in your GTF annotation is ‘Parent=Zm00001eb000010_T001;Name=Zm00001eb000010_T001.exon.1;ensembl_end_phase=0;ensembl_phase=0;exon_id=Zm00001eb000010_T001.exon.1;rank=1’.”
Then I looked into my file and saw that it was using “gene_id” as an identifier, so I tried GFF gene identifier: gene_id, which is used in the 9th column of my file; however, I still got “failed to find the gene identifier attribute in the 9th column of the provided GTF file” error.
Then I tried leaving the “GFF feature type filter” and “GFF gene identifier” fields empty, but I got an error saying “no features were loaded in format GTF. The annotation format can be specified by the ‘-F’ option, and the required feature type can be specified by the ‘-t’ option”
What I tried next: I learned that featurecounts requires a genome to be specified, so I created a custom genome by uploading the fasta file Zm-B73-REFERENCE-NAM-5.0.fa.gzfrom the page linked above. I ran NormalizeFasta as suggested to create a uniform line length of 80 and created a custom genome, maize_v5. I went back in my history and tagged the gff3 file, the reads, and the RNA Star outputs as being associated with maize_v5 and ran it again, but the same errors occurred.
What should I do next to troubleshoot this problem?