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HTSeq-count in RNA-Seq

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.

rna-seq htseq-count
• 4,522 views • link
updated by Balatheskulter • Balatheskulter • written by mlemusfuentes • mlemusfuentes

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.

• 1 views • link
written by Eric Lim Eric Lim

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.

• 1 views • link
written by mlemusfuentes • mlemusfuentes

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 .

• 1 views • link
written by Eric Lim Eric Lim

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

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written by GenoMax GenoMax

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.

• 1 views • link
written by WouterDeCoster WouterDeCoster

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

• 1 views • link
written by ATpoint ATpoint

That is @WouterTopHatBot :-)

• 1 views • link
written by GenoMax GenoMax

1 answer

kindly help me out bro, C: htseq count error

• 0 views • link
written by Balatheskulter • Balatheskulter

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