tophat2/2.1.0 error running from bam2fastx

[natalie_1]

I am running tophat2.1.0 (bowtie 2.2.6.0) on SE RNAseq data and encountering the following error:


[2016-02-29 09:38:34] Beginning TopHat run (v2.1.0)
-----------------------------------------------
[2016-02-29 09:38:34] Checking for Bowtie
Bowtie version: 2.2.6.0
[2016-02-29 09:38:34] Checking for Bowtie index files (transcriptome)..
[2016-02-29 09:38:34] Checking for Bowtie index files (genome)..
[2016-02-29 09:38:34] Checking for reference FASTA file
[2016-02-29 09:38:34] Generating SAM header for PGSC_DM_v4.03_index
[2016-02-29 09:38:40] Reading known junctions from GTF file
[2016-02-29 09:38:44] Preparing reads
left reads: min. length=30, max. length=40, 47957373 kept reads (864 discarded)
[2016-02-29 09:48:09] Using pre-built transcriptome data..
[2016-02-29 09:48:11] Mapping left_kept_reads to transcriptome known with Bowtie2
[FAILED]
Error running:
/opt/software/TopHat2/2.1.0--GCC-4.4.5/bin/bam2fastx --all tophat_Kalkaska/tmp/left_kept_reads.bam|/opt/software/bowtie2/2.2.6--GCC-4.4.5/bin/bowtie2 -k 60 -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 C,-62,0 -p 1 --sam-no-hd -x transcriptome_data/known -|/opt/software/TopHat2/2.1.0--GCC-4.4.5/bin/fix_map_ordering --bowtie2-min-score 55 --read-mismatches 3 --read-gap-length 10 --read-edit-dist 10 --read-realign-edit-dist 11 --sam-header tophat_Kalkaska/tmp/known.bwt.samheader.sam - - tophat_Kalkaska/tmp/left_kept_reads.m2g_um.bam | /opt/software/TopHat2/2.1.0--GCC-4.4.5/bin/map2gtf --sam-header tophat_Kalkaska/tmp/PGSC_DM_v4.03_index_genome.bwt.samheader.sam transcriptome_data/known.fa.tlst - tophat_Kalkaska/tmp/left_kept_reads.m2g.bam > tophat_Kalkaska/logs/m2g_left_kept_reads.out


When I run the error output, I get:


Error at parsing .tlst line (invalid strand):
53551 PGSC0003DMT400030180 ST4.03ch12. 56298573-56298656
(ERR): bowtie2-align died with signal 13 (PIPE)


Looking at the GTF file (PGSC_DM_V403_genes.gff from SpudDB), there are a fair number of entries where the strand is recorded as “.”. Online forums report the same issue but no solutions, save for deleting these entries, where are very numerous. Are there any other solutions I could try?

Thank you,
Natalie

[GenoMax]

For reference cross-posted: https://www.biostars.org/p/179178/