RNAseq Tophat crashes

Question

[wisewillwin]

Hello everyone, I have encountered the following errors and it has puzzled me for more than a week, I am wondering how can I fix that?

Thanks very much for your help.

[2016-06-04 07:24:25] Beginning TopHat run (v2.0.14)


[2016-06-04 07:24:25] Checking for Bowtie

          Bowtie version:     0.12.9.0

[2016-06-04 07:24:25] Checking for Bowtie index files (genome)..

[2016-06-04 07:24:25] Checking for reference FASTA file

[2016-06-04 07:24:25] Generating SAM header for /home/Research/Util/Genome/genome

[2016-06-04 07:24:25] Reading known junctions from GTF file

[2016-06-04 07:24:25] Preparing reads

     left reads: min. length=90, max. length=90, 24320620 kept reads (21406 discarded)

[2016-06-04 07:32:31] Building transcriptome data files ../BAM/mapped/patientr2/tmp/genome

[2016-06-04 07:32:31] Building Bowtie index from genome.fa

[2016-06-04 07:32:32] Mapping left_kept_reads to transcriptome genome with Bowtie

    [FAILED]

Error running:

/usr/local/Cellar/tophat-binary/2.0.14/bin/bam2fastx --all ../BAM/mapped/patientr2/tmp/left_kept_reads.bam|/home/Programs/bin/bowtie -v 2 -k 60 -m 60 -S -p 32 --sam-nohead --max /dev/null ../BAM/mapped/patientr2/tmp/genome -|/usr/local/Cellar/tophat-binary/2.0.14/bin/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --bowtie1 --sam-header ../BAM/mapped/patientr2/tmp/genome.bwt.samheader.sam - - ../BAM/mapped/patientr2/tmp/left_kept_reads.m2g_um.bam | /usr/local/Cellar/tophat-binary/2.0.14/bin/map2gtf --sam-header ../BAM/mapped/patientr2/tmp/genome_genome.bwt.samheader.sam ../BAM/mapped/patientr2/tmp/genome.fa.tlst - ../BAM/mapped/patientr2/tmp/left_kept_reads.m2g.bam > ../BAM/mapped/patientr2/logs/m2g_left_kept_reads.out

[GenoMax]

You appear to be running a year-old version of Tophat (v.2.0.14). In general first thing to try would be to update to the latest (v. 2.1.1) and see if the error goes away.

As for this error can you check to see if bam2fastx is present and has execute permissions ls -l /usr/local/Cellar/tophat-binary/2.0.14/bin/bam2fastx ?

[wisewillwin]

Thanks. Yeah, it is present and executable, codes$ ls -l /usr/local/Cellar/tophat-binary/2.0.14/bin/bam2fastx -r-xr-xr-x 1 2612 5032 976644 Mar 24 2015 /usr/local/Cellar/tophat-binary/2.0.14/bin/bam2fastx

I have updated to the latest version of tophat v 2.1.1. But just get exactly the same error.

If I switch the reference genome to hg19, there is no problem at all. I am wondering if the genome.gtf file has some problem.

[GenoMax]

I also noticed that your original run of TopHat was using bowtie (v. 0.12.9) which is again very old at this point. Are you using bowtie1 for some reason? In any case update to the latest bowtie v.1/2.

It would also help if you can provide the command line you are using to run TopHat. Remove real names/directory paths if you wish to.

[wisewillwin]

Thanks very much for your help. I will update bowtie now to see if that helps.

Here is my commandline, tophat --bowtie1 -p 32 -G ~/Research/Util/Genome/hgenome.gtf -o ../BAM/mapped/Patientr1 --library-type=fr-firststrand ~/Research/Util/Genome/genome ../Source/Patient/SeqR1.fq.gz

If I use hg19 it runs very well, if use this genome, program crashes as above.

Thanks very much.

[GenoMax]

See this post for an explanation of the G option and how to use it: C: tophat2 cannot find transcript file

Unless you have a specific need why not use bowtie v.2?