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  • AndrewUzilov
    Junior Member
    • Mar 2011
    • 3
    #1

    tophat (or tophat-fusion) v2.0.6 error on: Joining segment hits (long_spanning_reads)

    Hi all,

    (First, I emailed the tophat.cufflinks mailing list about this, but posting here in parallel in case anyone has some insight. Thanks!! I will post updates if anything is learned.)

    This may not necessarily be a tophat-fusion error --- maybe it is just tophat regardless of the fusion flag. But I don't know, I haven't run tophat on this data in a non-fusion pipeline.

    The situation: 6 RNA-Seq samples sequenced on Illumina HiSeq 2000, in the same run. 2 samples run just fine (including the "post" step), but 4 samples crash for no clear reason, all in the same way (below). I've run previous samples from other runs, and they worked great, too.

    I am running on Amazon Linux 64-bit in EC2, using pre-compiled binaries I downloaded just a few days ago (v 2.0.6 release). Using against bowtie1 version 0.12.8 which I compiled myself.

    It looks like a seg fault bug related to long_spanning_reads was fixed in this release, and I'm wondering if this is related.

    The output trace with error:

    [2012-12-06 14:42:04] Beginning TopHat run (v2.0.6)
    -----------------------------------------------
    [2012-12-06 14:42:04] Checking for Bowtie
    Bowtie version: 0.12.8.0
    [2012-12-06 14:42:04] Checking for Samtools
    Samtools version: 0.1.18.0
    [2012-12-06 14:42:04] Checking for Bowtie index files
    [2012-12-06 14:42:04] Checking for reference FASTA file
    [2012-12-06 14:42:04] Generating SAM header for /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome
    format: fastq
    quality scale: phred33 (default)
    [2012-12-06 14:42:33] Preparing reads
    left reads: min. length=30, max. length=101, 32578716 kept reads (328730 discarded)
    right reads: min. length=30, max. length=101, 32513191 kept reads (394255 discarded)
    [2012-12-06 15:11:17] Mapping left_kept_reads to genome genome with Bowtie
    [2012-12-06 15:59:10] Mapping left_kept_reads_seg1 to genome genome with Bowtie (1/4)
    [2012-12-06 16:12:50] Mapping left_kept_reads_seg2 to genome genome with Bowtie (2/4)
    [2012-12-06 16:23:17] Mapping left_kept_reads_seg3 to genome genome with Bowtie (3/4)
    [2012-12-06 16:36:51] Mapping left_kept_reads_seg4 to genome genome with Bowtie (4/4)
    [2012-12-06 16:47:37] Mapping right_kept_reads to genome genome with Bowtie
    [2012-12-06 17:31:10] Mapping right_kept_reads_seg1 to genome genome with Bowtie (1/4)
    [2012-12-06 17:44:15] Mapping right_kept_reads_seg2 to genome genome with Bowtie (2/4)
    [2012-12-06 17:57:19] Mapping right_kept_reads_seg3 to genome genome with Bowtie (3/4)
    [2012-12-06 18:08:51] Mapping right_kept_reads_seg4 to genome genome with Bowtie (4/4)
    [2012-12-06 18:21:06] Searching for junctions via segment mapping
    [2012-12-06 19:11:05] Retrieving sequences for splices
    [2012-12-06 19:14:54] Indexing splices
    [2012-12-06 20:01:23] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie (1/4)
    [2012-12-06 20:25:49] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie (2/4)
    [2012-12-06 20:48:54] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie (3/4)
    [2012-12-06 21:13:03] Mapping left_kept_reads_seg4 to genome segment_juncs with Bowtie (4/4)
    [2012-12-06 21:36:26] Joining segment hits
    [FAILED]
    Error running 'long_spanning_reads':Loading fusions...done


    Other log files that may be useful:

    $ cat tophat_out/logs/long_spanning_reads.segs.log
    long_spanning_reads v2.0.6 (3670M)
    --------------------------------------------
    Opening ./tophat_out/tmp/segment.fusions for reading
    [samopen] SAM header is present: 84 sequences.
    Loading reference sequences...
    reference sequences loaded.
    Loading junctions...done
    Loading deletions...done
    Loading insertions...done
    Loading fusions...done


    $ cat tophat_out/logs/run.log
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/tophat -p 15 --fusion-search --no-coverage-search --bowtie1 --mate-inner-dist 0 --mate-std-dev 80 --max-intron-length 100000 --fusion-min-dist 100000 --fusion-anchor-length 13 --fusion-ignore-chromosomes chrM /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome ../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_001.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_002.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_003.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_004.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_005.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_006.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_007.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_008.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_009.fastq.gz ../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_001.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_002.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_003.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_004.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_005.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_006.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_007.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_008.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_009.fastq.gz
    #>prep_reads:
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/prep_reads --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 100000 --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 100000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 --bowtie1 --fusion-search --fusion-anchor-length 13 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p15 --inner-dist-mean 0 --inner-dist-std-dev 80 --no-closure-search --no-coverage-search --no-microexon-search --fastq --aux-outfile=./tophat_out/prep_reads.info --index-outfile=./tophat_out/tmp/%side%_kept_reads.bam.index --sam-header=./tophat_out/tmp/genome_genome.bwt.samheader.sam --outfile=./tophat_out/tmp/%side%_kept_reads.bam ../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_001.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_002.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_003.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_004.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_005.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_006.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_007.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_008.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R1_009.fastq.gz ../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_001.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_002.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_003.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_004.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_005.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_006.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_007.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_008.fastq.gz,../noPolyA.trimmed.US-1430655-RD_truseq_ACAGTG_L006_R3_009.fastq.gz
    #>map_start:
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/bam2fastx --all --fastq ./tophat_out/tmp/left_kept_reads.bam|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 20 -m 20 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads.mapped.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_unmapped.bam
    #>map_segments:
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg1.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg1.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg1.bam ./tophat_out/tmp/left_kept_reads_seg1_unmapped.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg2.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg2.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg2.bam ./tophat_out/tmp/left_kept_reads_seg2_unmapped.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg3.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg3.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg3.bam ./tophat_out/tmp/left_kept_reads_seg3_unmapped.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg4.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg4.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg4.bam ./tophat_out/tmp/left_kept_reads_seg4_unmapped.bam
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/bam2fastx --all --fastq ./tophat_out/tmp/right_kept_reads.bam|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 20 -m 20 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/right_kept_reads.mapped.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/right_kept_reads.mapped.bam ./tophat_out/tmp/right_kept_reads_unmapped.bam
    #>map_segments:
    gzip -cd< ./tophat_out/tmp/right_kept_reads_seg1.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/right_kept_reads_seg1.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/right_kept_reads_seg1.bam ./tophat_out/tmp/right_kept_reads_seg1_unmapped.bam
    gzip -cd< ./tophat_out/tmp/right_kept_reads_seg2.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/right_kept_reads_seg2.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/right_kept_reads_seg2.bam ./tophat_out/tmp/right_kept_reads_seg2_unmapped.bam
    gzip -cd< ./tophat_out/tmp/right_kept_reads_seg3.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/right_kept_reads_seg3.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/right_kept_reads_seg3.bam ./tophat_out/tmp/right_kept_reads_seg3_unmapped.bam
    gzip -cd< ./tophat_out/tmp/right_kept_reads_seg4.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/right_kept_reads_seg4.bam.index --bowtie1 --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam - ./tophat_out/tmp/right_kept_reads_seg4.bam ./tophat_out/tmp/right_kept_reads_seg4_unmapped.bam
    #>find_juncs:
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/segment_juncs --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 100000 --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 100000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 --bowtie1 --fusion-search --fusion-anchor-length 13 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p15 --inner-dist-mean 0 --inner-dist-std-dev 80 --no-closure-search --no-coverage-search --no-microexon-search --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam --ium-reads ./tophat_out/tmp/left_kept_reads_seg1_unmapped.bam,./tophat_out/tmp/left_kept_reads_seg2_unmapped.bam,./tophat_out/tmp/left_kept_reads_seg3_unmapped.bam,./tophat_out/tmp/left_kept_reads_seg4_unmapped.bam,./tophat_out/tmp/right_kept_reads_seg1_unmapped.bam,./tophat_out/tmp/right_kept_reads_seg2_unmapped.bam,./tophat_out/tmp/right_kept_reads_seg3_unmapped.bam,./tophat_out/tmp/right_kept_reads_seg4_unmapped.bam /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome.fa ./tophat_out/tmp/segment.juncs ./tophat_out/tmp/segment.insertions ./tophat_out/tmp/segment.deletions ./tophat_out/tmp/segment.fusions ./tophat_out/tmp/left_kept_reads.bam ./tophat_out/tmp/left_kept_reads.mapped.bam ./tophat_out/tmp/left_kept_reads_seg1.bam,./tophat_out/tmp/left_kept_reads_seg2.bam,./tophat_out/tmp/left_kept_reads_seg3.bam,./tophat_out/tmp/left_kept_reads_seg4.bam ./tophat_out/tmp/right_kept_reads.bam ./tophat_out/tmp/right_kept_reads.mapped.bam ./tophat_out/tmp/right_kept_reads_seg1.bam,./tophat_out/tmp/right_kept_reads_seg2.bam,./tophat_out/tmp/right_kept_reads_seg3.bam,./tophat_out/tmp/right_kept_reads_seg4.bam
    #>juncs_db:
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/juncs_db 3 26 ./tophat_out/tmp/segment.juncs ./tophat_out/tmp/segment.insertions ./tophat_out/tmp/segment.deletions ./tophat_out/tmp/segment.fusions /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome.fa > ./tophat_out/tmp/segment_juncs.fa
    /usr/local/BinfTools/bowtie-0.12.8/bin/bowtie-build ./tophat_out/tmp/segment_juncs.fa ./tophat_out/tmp/segment_juncs
    #>map2juncs:
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg1.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null ./tophat_out/tmp/segment_juncs -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg1.to_spliced.bam.index --bowtie1 --sam-header ./tophat_out/tmp/segment_juncs.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg1.to_spliced.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg2.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null ./tophat_out/tmp/segment_juncs -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg2.to_spliced.bam.index --bowtie1 --sam-header ./tophat_out/tmp/segment_juncs.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg2.to_spliced.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg3.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null ./tophat_out/tmp/segment_juncs -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg3.to_spliced.bam.index --bowtie1 --sam-header ./tophat_out/tmp/segment_juncs.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg3.to_spliced.bam
    gzip -cd< ./tophat_out/tmp/left_kept_reads_seg4.fq.z|/usr/local/BinfTools/bowtie-0.12.8/bin/bowtie -q -v 2 -k 40 -m 40 -S -p 15 --sam-nohead --max /dev/null ./tophat_out/tmp/segment_juncs -|/usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile ./tophat_out/tmp/left_kept_reads_seg4.to_spliced.bam.index --bowtie1 --sam-header ./tophat_out/tmp/segment_juncs.bwt.samheader.sam - ./tophat_out/tmp/left_kept_reads_seg4.to_spliced.bam
    /usr/local/BinfTools/tophat-2.0.6.Linux_x86_64/long_spanning_reads --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 100000 --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 100000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 --bowtie1 --fusion-search --fusion-anchor-length 13 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p15 --inner-dist-mean 0 --inner-dist-std-dev 80 --no-closure-search --no-coverage-search --no-microexon-search --sam-header ./tophat_out/tmp/genome_genome.bwt.samheader.sam --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 /mnt/ref/Homo_sapiens/UCSC/hg19/Sequence/BowtieIndex/genome.fa ./tophat_out/tmp/left_kept_reads.bam ./tophat_out/tmp/segment.juncs ./tophat_out/tmp/segment.insertions ./tophat_out/tmp/segment.deletions ./tophat_out/tmp/segment.fusions ./tophat_out/tmp/left_kept_reads.candidates.bam ./tophat_out/tmp/left_kept_reads_seg1.bam,./tophat_out/tmp/left_kept_reads_seg2.bam,./tophat_out/tmp/left_kept_reads_seg3.bam,./tophat_out/tmp/left_kept_reads_seg4.bam ./tophat_out/tmp/left_kept_reads_seg1.to_spliced.bam,./tophat_out/tmp/left_kept_reads_seg2.to_spliced.bam,./tophat_out/tmp/left_kept_reads_seg3.to_spliced.bam,./tophat_out/tmp/left_kept_reads_seg4.to_spliced.bam


    Finally, here are the tmp files produced, if they are useful at all:

    $ lat tophat_out/tmp
    total 22G
    -rw-r--r-- 1 auzilov ec2-user 580K Dec 6 21:42 left_kept_reads.candidates14.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates0.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates0.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates10.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates10.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates11.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates11.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates12.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates12.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates13.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates13.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates14.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates1.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates1.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates2.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates2.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates3.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates3.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates4.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates4.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates5.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates5.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates6.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates6.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates7.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates7.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates8.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates8.bam.index
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates9.bam
    -rw-r--r-- 1 auzilov ec2-user 0 Dec 6 21:38 left_kept_reads.candidates9.bam.index
    -rw-r--r-- 1 auzilov ec2-user 465K Dec 6 21:36 left_kept_reads_seg4.to_spliced.bam.index
    -rw-r--r-- 1 auzilov ec2-user 397M Dec 6 21:36 left_kept_reads_seg4.to_spliced.bam
    -rw-r--r-- 1 auzilov ec2-user 423K Dec 6 21:13 left_kept_reads_seg3.to_spliced.bam.index
    -rw-r--r-- 1 auzilov ec2-user 383M Dec 6 21:12 left_kept_reads_seg3.to_spliced.bam
    -rw-r--r-- 1 auzilov ec2-user 450K Dec 6 20:48 left_kept_reads_seg2.to_spliced.bam.index
    -rw-r--r-- 1 auzilov ec2-user 384M Dec 6 20:48 left_kept_reads_seg2.to_spliced.bam
    -rw-r--r-- 1 auzilov ec2-user 452K Dec 6 20:25 left_kept_reads_seg1.to_spliced.bam.index
    -rw-r--r-- 1 auzilov ec2-user 388M Dec 6 20:25 left_kept_reads_seg1.to_spliced.bam
    -rw-r--r-- 1 auzilov ec2-user 457M Dec 6 20:01 segment_juncs.bwt.samheader.sam
    -rw-r--r-- 1 auzilov ec2-user 457M Dec 6 19:59 temp.samheader.sam
    -rw-r--r-- 1 auzilov ec2-user 577M Dec 6 19:42 segment_juncs.rev.1.ebwt
    -rw-r--r-- 1 auzilov ec2-user 42M Dec 6 19:41 segment_juncs.rev.2.ebwt
    -rw-r--r-- 1 auzilov ec2-user 577M Dec 6 19:28 segment_juncs.1.ebwt
    -rw-r--r-- 1 auzilov ec2-user 42M Dec 6 19:28 segment_juncs.2.ebwt
    -rw-r--r-- 1 auzilov ec2-user 63M Dec 6 19:15 segment_juncs.3.ebwt
    -rw-r--r-- 1 auzilov ec2-user 84M Dec 6 19:15 segment_juncs.4.ebwt
    -rw-r--r-- 1 auzilov ec2-user 714M Dec 6 19:14 segment_juncs.fa
    -rw-r--r-- 1 auzilov ec2-user 65M Dec 6 19:11 segment.juncs
    -rw-r--r-- 1 auzilov ec2-user 142M Dec 6 19:10 segment.fusions
    -rw-r--r-- 1 auzilov ec2-user 2.2M Dec 6 19:09 segment.deletions
    -rw-r--r-- 1 auzilov ec2-user 1.1M Dec 6 19:09 segment.insertions
    -rw-r--r-- 1 auzilov ec2-user 497M Dec 6 18:21 right_kept_reads_seg4.bam
    -rw-r--r-- 1 auzilov ec2-user 513K Dec 6 18:21 right_kept_reads_seg4.bam.index
    -rw-r--r-- 1 auzilov ec2-user 543M Dec 6 18:08 right_kept_reads_seg3.bam
    -rw-r--r-- 1 auzilov ec2-user 551K Dec 6 18:08 right_kept_reads_seg3.bam.index
    -rw-r--r-- 1 auzilov ec2-user 601M Dec 6 17:57 right_kept_reads_seg2.bam
    -rw-r--r-- 1 auzilov ec2-user 602K Dec 6 17:57 right_kept_reads_seg2.bam.index
    -rw-r--r-- 1 auzilov ec2-user 597M Dec 6 17:44 right_kept_reads_seg1.bam
    -rw-r--r-- 1 auzilov ec2-user 596K Dec 6 17:44 right_kept_reads_seg1.bam.index
    -rw-r--r-- 1 auzilov ec2-user 283M Dec 6 17:31 right_kept_reads_seg1.fq.z
    -rw-r--r-- 1 auzilov ec2-user 292M Dec 6 17:31 right_kept_reads_seg2.fq.z
    -rw-r--r-- 1 auzilov ec2-user 277M Dec 6 17:31 right_kept_reads_seg3.fq.z
    -rw-r--r-- 1 auzilov ec2-user 239M Dec 6 17:31 right_kept_reads_seg4.fq.z
    -rw-r--r-- 1 auzilov ec2-user 1.9G Dec 6 17:10 right_kept_reads.mapped.bam
    -rw-r--r-- 1 auzilov ec2-user 549K Dec 6 17:10 right_kept_reads.mapped.bam.index
    -rw-r--r-- 1 auzilov ec2-user 922M Dec 6 17:10 right_kept_reads_unmapped.bam
    -rw-r--r-- 1 auzilov ec2-user 297K Dec 6 17:10 right_kept_reads_unmapped.bam.index
    -rw-r--r-- 1 auzilov ec2-user 564M Dec 6 16:47 left_kept_reads_seg4.bam
    -rw-r--r-- 1 auzilov ec2-user 562K Dec 6 16:47 left_kept_reads_seg4.bam.index
    -rw-r--r-- 1 auzilov ec2-user 612M Dec 6 16:36 left_kept_reads_seg3.bam
    -rw-r--r-- 1 auzilov ec2-user 616K Dec 6 16:36 left_kept_reads_seg3.bam.index
    -rw-r--r-- 1 auzilov ec2-user 585M Dec 6 16:23 left_kept_reads_seg2.bam
    -rw-r--r-- 1 auzilov ec2-user 584K Dec 6 16:23 left_kept_reads_seg2.bam.index
    -rw-r--r-- 1 auzilov ec2-user 604M Dec 6 16:12 left_kept_reads_seg1.bam
    -rw-r--r-- 1 auzilov ec2-user 626K Dec 6 16:12 left_kept_reads_seg1.bam.index
    -rw-r--r-- 1 auzilov ec2-user 258M Dec 6 15:59 left_kept_reads_seg1.fq.z
    -rw-r--r-- 1 auzilov ec2-user 290M Dec 6 15:59 left_kept_reads_seg2.fq.z
    -rw-r--r-- 1 auzilov ec2-user 280M Dec 6 15:59 left_kept_reads_seg3.fq.z
    -rw-r--r-- 1 auzilov ec2-user 261M Dec 6 15:59 left_kept_reads_seg4.fq.z
    -rw-r--r-- 1 auzilov ec2-user 1.8G Dec 6 15:38 left_kept_reads.mapped.bam
    -rw-r--r-- 1 auzilov ec2-user 552K Dec 6 15:38 left_kept_reads.mapped.bam.index
    -rw-r--r-- 1 auzilov ec2-user 932M Dec 6 15:38 left_kept_reads_unmapped.bam
    -rw-r--r-- 1 auzilov ec2-user 297K Dec 6 15:38 left_kept_reads_unmapped.bam.index
    -rw-r--r-- 1 auzilov ec2-user 2.5G Dec 6 15:11 left_kept_reads.bam
    -rw-r--r-- 1 auzilov ec2-user 771K Dec 6 15:11 left_kept_reads.bam.index
    -rw-r--r-- 1 auzilov ec2-user 2.6G Dec 6 15:11 right_kept_reads.bam
    -rw-r--r-- 1 auzilov ec2-user 773K Dec 6 15:11 right_kept_reads.bam.index
    -rw-r--r-- 1 auzilov ec2-user 4.2K Dec 6 14:42 genome_genome.bwt.samheader.sam
    Tags: fusion, rna seq, tophat 2, tophat error
  • AndrewUzilov
    Junior Member
    • Mar 2011
    • 3
    #2
    Oh, if it matters, I'm using the hg19 bowtie index that comes with Illumina's iGenomes.

    Comment

    • jonas76
      Junior Member
      • Dec 2012
      • 6
      #3
      Similar problem here with 2.0.6:

      [2012-12-18 18:53:06] Joining segment hits
      [FAILED]
      Error running 'long_spanning_reads':Loading insertions...done

      Comment

      • RocheKermit
        Member
        • Nov 2011
        • 15
        #4
        Exactly the same issue there!
        The TopHat-Fusion "getting started" works like a charm (after a piece of pain..).
        Now with my own data, it crashes exactly the same as upper.
        It seems to be a memory issue.
        Now I'm trying to reduce from -p 12 to -p 6 and -p 2 (2GB RAM per core).
        Next I'll try to be more stringent in my parameters.
        Any suggestions from the community or developpers?

        Comment

        • adamyao
          Member
          • Feb 2011
          • 18
          #5
          My lab members have recorded memory consumption by TopHat-fusion. The following is the outcome

          4 million pair-end reads (100bp read length):
          Max memory used: 10G

          88 million:
          Max memory used: 60G

          220 million:
          Max memory used: 107G

          Comment

          • RocheKermit
            Member
            • Nov 2011
            • 15
            #6
            Update: I've relaunched a crashed tophat --fusion-search with --resume and it crashed again:
            [2013-01-16 17:34:30] Joining segment hits
            [FAILED]
            Error running 'long_spanning_reads':Loading fusions...done
            What is new is that the processed has died, rather than the server itself, as before...

            In parallel, I've also launched the same command line with option -p 2 and another one with -p 6 (instead of 12 cores available).
            The -p 2 is still runing but the -p 6 has finished without any problem.
            [2013-01-17 04:35:05] Run complete: 11:55:13 elapsed
            I'll try to catch a system admin to ask him for tools related to memory usage/consumation...

            Comment

            • RocheKermit
              Member
              • Nov 2011
              • 15
              #7
              Update: the run with -p 2 finished without any problem, after 1 day...
              Please note our versions:
              TopHat v2.0.6
              bowtie version 0.12.8

              htop reveals a heavy load on the memory.

              Comment

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