What I’m trying to do: DEG with wheat sequences by using HISAT2 -> StringTie -> Deseq2.
The problem: StringTie failing to use my provided GTF file, giving the warning:
WARNING: no reference transcripts were found for the genomic sequences where reads were mapped!
Please make sure the -G annotation file uses the same naming convention for the genome sequences.
I’ve noticed several other threads about this, often involving wheat, but with no clear (to me) follow-up solutions.
What I did before this point:
Upload full fasta sequence of cDNA for 1 chromosome from Ensembl and ran NormalizeFasta as per these instructions. Using the full genome fasta will result in memory failures, and mapping to a single chromosome’s fasta results in failure to set metadata, every time.
Upload GFF3 file, also from Ensembl, that was converted to GTF format.
Run StringTie using the HiSat2 BAM file and GTF file.
My thought is the problem is because I have to align to cDNA otherwise the metadata will not bet set. I was hoping StringTie would take the cDNA fasta file gene names from the Hisat2 BAM file, which are the exact same as the GTF gene_id attribute, and make the connection. But that seems wrong now:
Is it possible to change my GTF file to get StringTie to work with it and a BAM file made from a cDNA fasta file? I’ve tried everything to get the alignment with a full chromosome sequence FASTA, but I can’t get it to work without the cDNA file, so I’m at a loss.