Question: tophat2 0% mapping rate for strand-specific RNA-seq
gravatar for ag1194
3.8 years ago by
ag11940 wrote:


I am new tophat user, I run my first RNA-seq, by using an already published data to test. However I have got 0.0% mapping rate. Obviously I am making a mistake.

The data that I am using is strand specific RNA-seq. I used the GEOdatabase to access the data. I run my tophat based on options they provide in the paper with following command:

tophat2 -r 25 --coverage-search -G UCSC/refGene_mm9.gtf --library-type fr-firststrand /bowtie2/mm9  subset697.fastq &> tophat.log

I was looking at the Nature Protocols paper for tophat and I realize that in the paper, for strand specific data, they provide 2 different fastq file.I think that is the problem in my case. I only have left_reads in my tmp folder. Does anybody have experience on strand-specific RNA-seq? And if I need two fastq file, any ideas what can I do about it since the data on GEOdatabase contains 1 big fastq file?

Thank you!! -A

rna-seq • 1.4k views
ADD COMMENTlink modified 3.8 years ago by Devon Ryan94k • written 3.8 years ago by ag11940

Hi, I am using tophat2 to align fastq downloaded from dbGaP, and have the same issue. Have you figured out the solution yet? Thanks.

ADD REPLYlink written 3.4 years ago by wwu2220
gravatar for Devon Ryan
3.8 years ago by
Devon Ryan94k
Freiburg, Germany
Devon Ryan94k wrote:

Depending on when this was sequenced, fr-firststrand may not be appropriate. You might try fr-secondstrand in case this is an old dataset. Also, don't use -r 25, since you don't have paired-end data (to answer your question regarding that, no, you don't need two files). Your full command should be:

tophat2 --coverage-search -G UCSC/refGene_mm9.gtf --library-type fr-secondstrand /bowtie2/mm9  subset697.fastq &> tophat.log

You could alternatively try fr-unstranded, which should always work.

As an aside, I'd recommend ditching tophat2 and switching to hisat2, which has replaced it.

ADD COMMENTlink modified 3.8 years ago • written 3.8 years ago by Devon Ryan94k
Please log in to add an answer.


Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 774 users visited in the last hour