Hi,
I'm somewhat new to bioinformatics, so please bear with me.
I'm running tophat2 on some fastq files using the HG38 as reference. This is the command that I ran:
tophat2 --b2-sensitive -G /home/fadhil/hg38_ref/lib/hg38.refGene.gtf -p 16 -o /home/data/mcf10_tophat_output /home/fadhil/Bowtie2Index/genome ./SRR925720_mcf10a.fastq
It takes about 8 hours, but in the end the mapping rate is almost 0%, it maps 3997 out of 31898079 reads. I'm not sure I understand why this is happening, although tophat emitted the following error consecutively as it was running:
Warning: Encountered reference sequence with only gaps
Ignoring any potential errors with the fastq files themselves, what could possibly be the problem here?
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fadhil.abubaker •
Warning: Encountered reference sequence with only gaps
Have you checked if your reference genome is correct ? Locate the fasta file in
ls /home/fadhil/Bowtie2Index/genome
Lets say it's called "genome.fa". Then check if it looks good :
head /home/fadhil/Bowtie2Index/genome.fa
If it is ok, you can try to rebuild the index
bowtie2-build /home/fadhil/Bowtie2Index/genome.fa /home/fadhil/Bowtie2Index/genome
hope this helps.
The files in /home/fadhil/Bowtie2Index/genome are all .bt2 files and not fasta. I'm not too sure if this makes any difference.
The bowtie index was build using fasta files, or where did you get the index?
I would suggest that you download the reference in fasta and rebuild the index with the above command. Your reference and indexes seem corrupted.
In the end rebuilding indices helped a bit but I was still getting low mapping rate. Turns out the fastq files were corrupted and re-downloading them helped. Thanks for everyone's help!
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You should know that the old 'Tuxedo' pipeline of Tophat 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. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.