Tophat2 output error

Question

[K.Nijbroek]

I am trying to run a basic Tophat2 command but it goes wrong somewhere. Perhaps anyone has experience with this error message? I'm running Tophat2 within a Virtual Box with BioLinux, writing output to shared directories with the windows computer. When specifying output directory to a non-shared directory the same error occurs.

Command:

tophat2 \
  -o /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq/ \
  --keep-tmp \
  -p 8 \
  /home/koen/Host/Stage_Enschede/methods_Bowtie2/indexes_chromFA/genome /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq/3_S3_L001_R1_001_accepted.fq

Error:

[FAILED] Error running /usr/bin/tophat_reports

Log:

[2014-06-02 13:37:09] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2014-06-02 13:37:09] Checking for Bowtie
          Bowtie version:     2.1.0.0
[2014-06-02 13:37:09] Checking for Samtools
        Samtools version:     0.1.19.0
[2014-06-02 13:37:09] Checking for Bowtie index files (genome)..
[2014-06-02 13:37:09] Checking for reference FASTA file
[2014-06-02 13:37:09] Generating SAM header for /home/koen/Host/Stage_Enschede/methods_Bowtie2/indexes_chromFA/genome
    format:         fastq
    quality scale:     phred33 (default)
[2014-06-02 13:38:08] Preparing reads
     left reads: min. length=242, max. length=300, 541 kept reads (0 discarded)
[2014-06-02 13:38:08] Mapping left_kept_reads to genome genome with Bowtie2 
[2014-06-02 13:38:39] Mapping left_kept_reads_seg1 to genome genome with Bowtie2 (1/12)
[2014-06-02 13:39:09] Mapping left_kept_reads_seg2 to genome genome with Bowtie2 (2/12)
[2014-06-02 13:39:40] Mapping left_kept_reads_seg3 to genome genome with Bowtie2 (3/12)
[2014-06-02 13:40:11] Mapping left_kept_reads_seg4 to genome genome with Bowtie2 (4/12)
[2014-06-02 13:40:42] Mapping left_kept_reads_seg5 to genome genome with Bowtie2 (5/12)
[2014-06-02 13:41:13] Mapping left_kept_reads_seg6 to genome genome with Bowtie2 (6/12)
[2014-06-02 13:41:43] Mapping left_kept_reads_seg7 to genome genome with Bowtie2 (7/12)
[2014-06-02 13:42:15] Mapping left_kept_reads_seg8 to genome genome with Bowtie2 (8/12)
[2014-06-02 13:42:46] Mapping left_kept_reads_seg9 to genome genome with Bowtie2 (9/12)
[2014-06-02 13:43:16] Mapping left_kept_reads_seg10 to genome genome with Bowtie2 (10/12)
[2014-06-02 13:43:46] Mapping left_kept_reads_seg11 to genome genome with Bowtie2 (11/12)
[2014-06-02 13:44:16] Mapping left_kept_reads_seg12 to genome genome with Bowtie2 (12/12)
[2014-06-02 13:44:46] Searching for junctions via segment mapping
[2014-06-02 13:47:10] Retrieving sequences for splices
[2014-06-02 13:49:33] Indexing splices
[2014-06-02 13:49:34] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie2 (1/12)
[2014-06-02 13:49:40] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie2 (2/12)
[2014-06-02 13:49:44] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie2 (3/12)
[2014-06-02 13:49:49] Mapping left_kept_reads_seg4 to genome segment_juncs with Bowtie2 (4/12)
[2014-06-02 13:49:53] Mapping left_kept_reads_seg5 to genome segment_juncs with Bowtie2 (5/12)
[2014-06-02 13:49:57] Mapping left_kept_reads_seg6 to genome segment_juncs with Bowtie2 (6/12)
[2014-06-02 13:50:02] Mapping left_kept_reads_seg7 to genome segment_juncs with Bowtie2 (7/12)
[2014-06-02 13:50:06] Mapping left_kept_reads_seg8 to genome segment_juncs with Bowtie2 (8/12)
[2014-06-02 13:50:10] Mapping left_kept_reads_seg9 to genome segment_juncs with Bowtie2 (9/12)
[2014-06-02 13:50:14] Mapping left_kept_reads_seg10 to genome segment_juncs with Bowtie2 (10/12)
[2014-06-02 13:50:18] Mapping left_kept_reads_seg11 to genome segment_juncs with Bowtie2 (11/12)
[2014-06-02 13:50:22] Mapping left_kept_reads_seg12 to genome segment_juncs with Bowtie2 (12/12)
[2014-06-02 13:50:26] Joining segment hits
[2014-06-02 13:52:51] Reporting output tracks
    [FAILED]
Error running /usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq// --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-coverage-search --no-microexon-search --sam-header /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//tmp/genome_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/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 /home/koen/Host/Stage_Enschede/methods_Bowtie2/indexes_chromFA/genome.fa /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//junctions.bed /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//insertions.bed /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//deletions.bed /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//fusions.out /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//tmp/accepted_hits  /home/koen/Host/Stage_Enschede/data_RNA-SampleA3/3_S3_L001_R1_001.fastq//tmp/left_kept_reads.bam
    Loading ...done

[Devon Ryan]

This usually happens when you don't have enough RAM. Look at the run log to find the last command run (it'll start with "/usr/bin/tophat_reports") and then execute that manually to see what the actual error message is.

[K.Nijbroek]

There should be around +- 16GB of RAM available.. The last command in run.log:

#>tophat_reports:
/usr/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq// --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-coverage-search --no-microexon-search --sam-header /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//tmp/genome_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/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 /home/koen/Host/Stage_Enschede/methods_Bowtie2/indexes_chromFA/genome.fa /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//junctions.bed /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//insertions.bed /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//deletions.bed /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//fusions.out /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//tmp/accepted_hits  /home/koen/Desktop/Output/3_S3_L001_R1_001.fastq//tmp/left_kept_reads.bam

[K.Nijbroek]

Problem solved. It wasn't due to the RAM but due to the -p 8. Apparently it only works with -p 1.

Answer 1

[K.Nijbroek]

Problem solved. It wasn't due to the RAM but due to the -p 8. Apparently it only works with -p 1.

[Devon Ryan]

That's quite odd, sounds like a bug. If you get a chance, you might post this to the tophat bug tracker or the google group.