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tophat2 stopped at bowtie step

Here is the problem

I have two paired read files, each with only 250 reads (so memory is not an issue)

the quality score is in illumina 1.8 format, has concerted to illumina 1.3 with command

sed -ie '4~4y/!"#$%&'\''()*+,-.\/0123456789:;<=>?@ABCDEFGHIJ/@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\\]^_`abcdefghi/'

Then I run

tophat2 --solexa1.3-quals -r -70 --mate-std-dev 35 --library-type fr-firststrand -p 10 -o tophat_output allchr read1.fastq read2.fastq

which reported the following error

[2014-06-06 13:29:08] Beginning TopHat run (v2.0.11)
-----------------------------------------------
[2014-06-06 13:29:08] Checking for Bowtie
          Bowtie version:     2.2.3.0
[2014-06-06 13:29:08] Checking for Samtools
        Samtools version:     0.1.18.0
[2014-06-06 13:29:08] Checking for Bowtie index files (genome)..
    Found both Bowtie1 and Bowtie2 indexes.
[2014-06-06 13:29:08] Checking for reference FASTA file
[2014-06-06 13:29:08] Generating SAM header for allchr
[2014-06-06 13:29:08] Preparing reads
     left reads: min. length=101, max. length=101, 250 kept reads (0 discarded)
    right reads: min. length=101, max. length=101, 250 kept reads (0 discarded)
[2014-06-06 13:29:08] Mapping left_kept_reads to genome allchr with Bowtie2 
    [FAILED]
Error running bowtie:
Error while flushing and closing output
terminate called after throwing an instance of 'int'
(ERR): bowtie2-align died with signal 6 (ABRT)

Removing --solexa1.3-quals does not help

Can anyone help me?

tophat
• 5,570 views • link
updated by Ram Ram • written by ruansun1983 • ruansun1983

Why not just keep the quality scores as is? You don't need to do anything special to get your fastq files to be handled properly.

• 0 views • link
updated by Ram Ram • written by Devon Ryan Devon Ryan

original quality score report the same error. That is why I convert it

• 0 views • link
written by ruansun1983 • ruansun1983

Try using a non-negative -r value. If that doesn't work, run the last command from the run log yourself and see if you get a different error.

• 0 views • link
written by Devon Ryan Devon Ryan

just tried, does not work. the same error

• 0 views • link
written by ruansun1983 • ruansun1983

1 answer

Are you sure about the library type you used? Illumina's library type is fr-unstranded.

It might be that tophat is trying to align the reads in the opposite direction and running out of memory. And hence, throwing that error.

• 0 views • link
updated by Ram Ram • written by Jordan Jordan

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