Problem with TopHat

[mblanche]

Dear all,

For some reason TopHat (v2.0.6) is dying on me with a FAILED error at the report stage (see output bellow). It use to run fine on my Mac OS X server (v 10.7.5). I have been installing a few patches and updates using HomeBrew and I must have broken something but I can't figure what and not sure what's the best strategy to clean up my mess. I try reinstalling topHat from scratch using home brew but it failed.

Any suggestions will be greatly appreciated.

thanks
[2013-06-07 13:51:03] Beginning TopHat run (v2.0.6)
-----------------------------------------------
[2013-06-07 13:51:03] Checking for Bowtie
Bowtie version: 2.1.0.0
[2013-06-07 13:51:03] Checking for Samtools
Samtools version: 0.1.19.0
[2013-06-07 13:51:03] Checking for Bowtie index files
[2013-06-07 13:51:03] Checking for reference FASTA file
Warning: Could not find FASTA file /Users/lab_project/Genomes/bowtie_indexes/bwt2/Users/lab_project/Genomes/current_genome/ensembl/release-69/drosophila_melanogaster/bowtie2_indexes/Drosophila_melanogaster.BDGP5.69.fa
[2013-06-07 13:51:03] Reconstituting reference FASTA file from Bowtie index
Executing: /usr/local/bin/bowtie2-inspect /Users/lab_project/Genomes/current_genome/ensembl/release-69/drosophila_melanogaster/bowtie2_indexes/Drosophila_melanogaster.BDGP5.69 > ./tophat_out/tmp/Drosophila_melanogaster.BDGP5.69.fa
[2013-06-07 13:51:11] Generating SAM header for /Users/lab_project/Genomes/current_genome/ensembl/release-69/drosophila_melanogaster/bowtie2_indexes/Drosophila_melanogaster.BDGP5.69
format: fastq
quality scale: phred33 (default)
[2013-06-07 13:51:13] Preparing reads
left reads: min. length=50, max. length=50, 10519226 kept reads (496 discarded)
[2013-06-07 13:53:14] Mapping left_kept_reads to genome Drosophila_melanogaster.BDGP5.69 with Bowtie2
[2013-06-07 13:57:59] Mapping left_kept_reads_seg1 to genome Drosophila_melanogaster.BDGP5.69 with Bowtie2 (1/2)
[2013-06-07 13:58:28] Mapping left_kept_reads_seg2 to genome Drosophila_melanogaster.BDGP5.69 with Bowtie2 (2/2)
[2013-06-07 13:58:57] Searching for junctions via segment mapping
Coverage-search algorithm is turned on, making this step very slow
Please try running TopHat again with the option (--no-coverage-search) if this step takes too much time or memory.
[2013-06-07 14:02:52] Retrieving sequences for splices
[2013-06-07 14:03:03] Indexing splices
[2013-06-07 14:03:39] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/2)
[2013-06-07 14:04:08] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/2)
[2013-06-07 14:04:37] Joining segment hits
[2013-06-07 14:05:06] Reporting output tracks
[FAILED]
Error running /usr/local/Cellar/tophat/2.0.6/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir ./tophat_out/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p8 --no-closure-search --no-microexon-search --sam-header ./tophat_out/tmp/Drosophila_melanogaster.BDGP5.69_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/local/bin/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 ./tophat_out/tmp/Drosophila_melanogaster.BDGP5.69.fa ./tophat_out/junctions.bed ./tophat_out/insertions.bed ./tophat_out/deletions.bed ./tophat_out/fusions.out ./tophat_out/tmp/accepted_hits ./tophat_out/tmp/left_kept_reads.mapped.bam,./tophat_out/tmp/left_kept_reads.candidates ./tophat_out/tmp/left_kept_reads.bam
Loaded 42711 junctions

[catbus]

I am encountering the same problem in Tophat 2.0.9.

This problem:
Reporting output tracks
[FAILED]
Error running [... ] tophat_report

Other posters suggest re-downloading the genome fasta file(s) and re-calculating the bowtie2 index, so I am going to try that. Unclear if that actually is going to solve things.

[catbus]

Downloading a totally fresh "iGenome" hg19 fasta + gtf annotation + pre-computed bowtie 2 index did not fix the problem.

I have tried it with many different input files (but all from this one project), so either there is something systematically wrong with all the input fasta files for this project, or there is a bug in Tophat that only occurs in rare conditions.

This is the error I get:

[2013-08-08 03:38:30] Reporting output tracks
        [FAILED]
Error running /work/Apps/Bio/tophat/tophat-2.0.9/tophat_reports  [...]

I hav also cross-posted this in a couple other threads here on seq-answers.

[GenoMax]

Pardon an obvious question but have you checked to make sure that the executable (/work/Apps/Bio/tophat/tophat-2.0.9/tophat_reports) is ok?

[catbus]

Unfortunately, there doesn't appear to be anything wrong with the executable, which is happy to run if I invoke it directly.

That was a pretty good idea, though, and I hadn't thought to check it before.

[catbus]

I solved this problem by removing "--no-discordant" from my tophat parameters.

I tried a TON of combinations of parameters, and the "tophat_reports" only gave an error when "--no-discordant" was present.

So I think it's a tophat bug that only occurs under specific conditions and with --no-discordant activated.

This is true for at least Tophat 2.0.8b and Tophat 2.0.9.