HTSeq-count in RNA-Seq

Question

[mlemusfuentes]

strong texthello everybody,

I'm doing for the first time an analysis of differential gene expression. already perform the alignment with tophat, then use samtools sort for the reads and now I want to count the readings with the HTSeq-count.

htseq-count -f bam -r name filesorted.bam hg19_genes.gtf > directoryoutput/file.HTSeq-count.txt

I get a message that says the following:

**0 GFF lines processed**

**Warning: no features of type 'exon' found**

and the output file that generates have not name of the genes.

I would appreciate any help or guidance.

[WouterDeCoster]

already perform the alignment with tophat

You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon followed by DESEq2 or edgeR.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

[ATpoint]

I like you are not getting tired of posting this. Maybe a bot posting this to everything new post containing 'tophat' would be helpful :-D

[GenoMax]

That is @WouterTopHatBot :-)

[WouterDeCoster]

Yeah I've thought of that

[Eric Lim]

htseq-count -h

-t FEATURETYPE, --type=FEATURETYPE
                    feature type (3rd column in GFF file) to be used, all
                    features of other type are ignored (default, suitable
                    for Ensembl GTF files: exon)

The warning message suggests your hg19_genes.gtf doesn't contain exon records.

[mlemusfuentes]

hi Eric Lim, thanks for you answer, but I'm confused, how to use -t?

htseq-count -f bam -r name -t?...

Watch for your comments

thanks.

[Eric Lim]

Hi, you should add a reply or comment instead of an answer.

I don't know where you're getting the gtf from and the contents of the file. I'd recommend using the same gtf/gff you used for tophat. You can also download gtf for hg19 at https://www.gencodegenes.org/releases/28lift37.html .

[GenoMax]

mlemusfuentes : Show us 10 lines from your GTF file by given us output of head -10 your.gtf.