Hi,

I think seem to be doing something very basically wrong in running TopHat2 so it's probably an easy answer.

Here's my command:

tophat \
  --read-mismatches 1 \
  --output-dir /n/user/rubi/data/tophat.alns/s1/s1 \
  --num-threads 8 \
  --transcriptome-index /n/user/rubi/refdata/tophat.2.gencode.vM2.annotation.2wayconspseudos.mm10/mm10 \
  /n/user/rubi/refdata/bowtie.2.gencode.vM2.annotation.2wayconspseudos.mm10/mm10 \
  /n/user/rubi/data/fq/s1.fq

Here's the log file:

[2015-03-20 00:07:34] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2015-03-20 00:07:34] Checking for Bowtie
Bowtie version:  2.0.0.6
[2015-03-20 00:07:34] Checking for Samtools
Samtools version:  0.1.12.a
[2015-03-20 00:07:34] Checking for Bowtie index files
[2015-03-20 00:07:34] Checking for Bowtie index files
[2015-03-20 00:07:34] Checking for reference FASTA file
[2015-03-20 00:07:34] Generating SAM header for /n/user/rubi/refdata/tophat.2.gencode.vM2.annotation.2wayconspseudos.mm10/mm10
format:  fastq
quality scale:  phred33 (default)
[2015-03-20 00:07:35] Reading known junctions from GTF file
[2015-03-20 00:08:03] Preparing reads
 left reads: min. length=20, max. length=59, 128844351 kept reads (192369 discarded)
[2015-03-20 00:49:52] Using pre-built transcriptome index..
[2015-03-20 00:50:20] Mapping left_kept_reads to transcriptome mm10 with Bowtie2 
[2015-03-20 03:33:13] Resuming TopHat pipeline with unmapped reads
[2015-03-20 03:33:15] Mapping left_kept_reads.m2g_um to genome mm10 with Bowtie2 
[FAILED]
Error running bowtie:
Error reading _plen[] array: 1776466139, 4294967292
Error: Encountered internal Bowtie 2 exception (#1)
Command: /n/sw/bowtie2-2.0.0-beta6/bowtie2-align -q -k 20 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min L,-0.6,-0.6 -p 8 --sam-no-hd -x /n/user/rubi/refdata/bowtie.2.gencode.vM2.annotation.2wayconspseudos.mm10/mm10 -

As far as I can tell BOWTIE_INDEXES is defined as an environment variable:

rubi@rclogin09 10:25 ~ >echo $BOWTIE_INDEXES
/n/user/rubi/refdata/bowtie.2.gencode.vM2.annotation.2wayconspseudos.mm10

And the transcriptome and genome index folders seem to be found - according to the log. So what's wrong?