How to run HISAT2 / Stringtie / Ballgown analysis starting with gff file

Question

[al-ash]

Hi! I'm trying to extract exon and splice site information from the gff file (which I downloaded from NCBI: ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF_000214255.1_Bter_1.0/GCF_000214255.1_Bter_1.0_genomic.gff.gz) using the python scripts provided in HISAT2 package extract_splice_sites.py and extract_exons.py) for downstream analysis starting with HISAT2.

The scripts work fine for the example gtf file which I downloaded as supplementary file from the Nature Protocols article ( http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html ) but when I use these on the gff file the scripts run without returning any error but the output files are empty.

I guess it is because of the gtf fromat (although I see that the information which should be extracted from the gtf file is present in the same columns also in my gff file so I thought the scripts might work as well on the gff file). I simply tried to rename the *.gff to *. gtf, but the python scripts outputed again empty files.

I'll be thankfull for any suggestions on how to extract the exon and splice site information from the gff file!

Answer 1

[Jeffin Rockey]

Please do not simply rename a gff file to gtf or vice versa. There are quite serious differences especially in 9th column.

If you find that a tool works with gtf and not gff3 , an approach to resolution is to convert the gff3 to gtf and then try the tool again.

Please see biostars dicussions like this to get a suitable way to convert the gff to gtf.

Another method to convert is from ucsc

Step1: gff3ToGenePred yourfile.gff yourgenemodel.genePred

Step2: genePredToGtf file yourgenemodel.genePred yourgenemodel.gtf

Also , there is a program in genometools

I hope if the conversion happens successfully for your file, the extraction program should also work as expected with the converted gtf.

[al-ash]

Thanks! I used gffread to convert my gff to gtf and the new gtf was processed by the scripts without any apparent problem and the output files containing extracted exon and splice site information look reasonable.

[Jeffin Rockey]

Good to know that it worked for you.

Jf