RNA-Seq : merged.gtf file from cuffmerge where exons are duplicated (two exons with the same coordinates and the same gene_id but without the same oId)

Question

[JoannaF]

Hi, I want to analyse RNA-seq data files. The pipeline used is HISAT2 > Stringtie (.gtf files) > cuffmerge (merged.gtf file). But I have an issue with the merged.gtf file produced by cuffmerge. I have two lines with the same coordinates (chromosome, start, end), width, strand, etc, except the transcript_id and the oId which are not the same. In the "contained_in" column, there is <na> for one exon and TCONS_00000003 for the other exon. Here there is an exemple of this issue but there are a lot of lines like that for others exons in this file :

seqnames   start     end width strand    source type score phase     gene_id  transcript_id exon_number     gene_name                  oId          nearest_ref class_code tss_id   contained_in p_id
chr1 3252757 3253236   480      + Cufflinks exon    NA    NA XLOC_000003 TCONS_00000004           1       Gm18956 ENSMUST00000192857.1 ENSMUST00000192857.1          =   TSS3 TCONS_00000003 <NA>
4     chr1 3252757 3253236   480      + Cufflinks exon    NA    NA XLOC_000003 TCONS_00000003           1       Gm18956             CUFF.3.2 ENSMUST00000192857.1          =   TSS3           <NA> <NA>

It seems that cuffmerge thinks that these two exons are not the same exon because the first comes from a transcript de novo (transcript_id : "CUFF.3.2") and the other comes from a known transcript (transcript_id : "ENSMUST00000192857.1").

The only message I have when I run cuffmerge is : [bam_header_read] EOF marker is absent. The input is probably truncated. [bam_header_read] invalid BAM binary header (this is not a BAM file).

Do you have a solution for this issue ?

Thank you for your answers !

Answer 1

[Satyajeet Khare]

I think it is quite expected. Many exons will be repeated in a merged GTF file or even in the reference GTF file.

It just means that there are more than one transcripts which harbour this exon. These transcripts are distinct because they differ in rest of the exons.

[JoannaF]

Thanks a lot for your answer Satya ! The issue is that these exons are part of mono-exonic transcripts so I don't understand why they are duplicated and why they have not the same transcript_id...