I am trying to use gffcompare to compare my assembled transcriptome to a reference gtf that contains information about small open reading frames (sORFs). The reference gtf was obtained by processing downloaded data from several sORF databases and is called
I used the command:
gffcompare -r path/to/all_38.gtf -o /output_folder my_transcriptome.gtf.
mytranscriptome.gtf is generated by assembling my Bam files with reference to ensembl hg38 v99 reference.
Upon completion, the output of the error message log file reads:
0 reference transcripts loaded. 237788 query transfrags loaded. 2714 duplicate query transfrags discarded.
The expected output files are generated except for the .refmap file which I need for downstream analysis. I assume this is because 0 reference transcripts were loaded into the program. Has anyone encountered similar issues? And is possibly something wrong with my custom reference gtf file?
My reference gtf can be found here. The first few lines of my input gtf look like this:
# StringTie version 2.1.2 1 StringTie transcript 11869 14409 1000 + . gene_id "MSTRG.1"; transcript_id "ENST00000456328"; gene_name "DDX11L1"; ref _gene_id "ENSG00000223972"; 1 StringTie exon 11869 12227 1000 + . gene_id "MSTRG.1"; transcript_id "ENST00000456328"; exon_number "1"; gene_name "DDX1 1L1"; ref_gene_id "ENSG00000223972"; 1 StringTie exon 12613 12721 1000 + . gene_id "MSTRG.1"; transcript_id "ENST00000456328"; exon_number "2"; gene_name "DDX1 1L1"; ref_gene_id "ENSG00000223972"; 1 StringTie exon 13221 14409 1000 + . gene_id "MSTRG.1"; transcript_id "ENST00000456328"; exon_number "3"; gene_name "DDX1 1L1"; ref_gene_id "ENSG00000223972";
EDIT: Output when running with --debug option
Running with debug option set results in an error file with more information about duplicated transfrags but not about reference transcripts. I've included a few lines of the error file:
Prefix for output files: norfs Loading reference transcripts.. 0 reference transcripts loaded. 237788 query transfrags loaded. Query transcript ENST00000655384 discarded (duplicate of ENST00000667728) Query transcript ENST00000415295 discarded (duplicate of ENST00000670824) Query transcript ENST00000263741 discarded (duplicate of ENST00000660930) Query transcript ENST00000473215 discarded (duplicate of ENST00000347370)
The --debug option also generates 3 additional files:
However, in my output folder, I only have the
R_missed.gff files. The
Q_discard.lst is missing.
One thing that I'm not sure matters is that
my_transcriptome.gtf was generated by aligning and assembling with reference to an
Ensembl gtf file. The gtf that I am using as reference input in gffcompare is downloaded from norfs.org. It contains information about small Open Reading Frames (sORFs). Could the different gtf files used be causing this issue?
If so, the solution would be to assemble my transcripts using the sORFs gtf as reference. However, I'm not sure if this is a good idea since sORFs have relatively short lengths (< 300bp) and this might screw up the assembly.