# Gffcompare issue: 0 reference transcripts loaded

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 all_38.gtf.

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.


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
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:<outprefix>.Q_discarded.lst, <outprefix>.missed_introns.gff, <outprefix>.R_missed.lst.

However, in my output folder, I only have the missed_introns.gff and 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.

• hi! could you try to run it again in debug mode? (adding --debug to your command)? – aechchiki Jun 12 '20 at 7:33
• @aechchiki thanks very much for your help. I ran with --debug option as suggested. The error file includes more information but only about duplicated transfrags and not about reference transcripts. I've edited the main question above with a few lines of the error file and other information. Thanks for your time. – nattzy Jun 14 '20 at 3:04
• perfect ;) maybe you can add an answer to your own question (and accept it), in case someone else runs into the same issue in the future – aechchiki Jun 15 '20 at 4:04