Low mapping rate for Tophat2

Question

[fadhil.abubaker]

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?

[WouterDeCoster]

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.

Answer 1

[Carlo Yague]

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.

[fadhil.abubaker]

The files in /home/fadhil/Bowtie2Index/genome are all .bt2 files and not fasta. I'm not too sure if this makes any difference.

[WouterDeCoster]

The bowtie index was build using fasta files, or where did you get the index?

[Carlo Yague]

I would suggest that you download the reference in fasta and rebuild the index with the above command. Your reference and indexes seem corrupted.

[fadhil.abubaker]

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!