Hello everyone,
I am trying to use tophat to align my fastq reads to the reference genome.
I first used bowtie to create index files. I have trimmed my read1 and read2 fastq files. All output files of bowtie index and read files are in the same directory. Here you can see the screen shot of my directory.
Then I am running the following command to run tophat.
tophat -p 8 -G genome.gtf -o sample_thout genome SAMPLE_R1.fq SAMPLE_R2.fq
I do not know what I am doing wrong since my command runs but after almost 45 minutes it stops.
The sample_thout directory only contains the following files.
Inside the tmp folder I can see the following:
inside the logs folder I can see the following files:
So I can not run tophat properly to get the desired output. I am pretty new to RNA seq and using tophat. Does anybody have any idea what is my mistake?
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Please run STAR or HISAT2 or so. Tophat is not recommended these days.
Thanks for your reply. Actually I have to use tophat because it is part of my research pipeline. Do you have any idea what is the issue?
OK. What is the genome given in the command line?
The genome is my index which is called ToxoDB35. I mean that is the name for out out of bowtie index.
The exact command that I am running is as follows:
DON'T USE TOPHAT - LIOR PACHTER TWEET
Hello yasaman.rezvani66,
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.