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Old 08-06-2013, 05:52 PM   #1
julio.fernandez.banet
Junior Member
 
Location: San Diego

Join Date: Jun 2011
Posts: 2
Default tophat2 fail while running long_spanning_reads with out_of_range error.

Hi all,

I am running tophat v2.0.9 on a set of samples using the --fusion-search option.
While I managed to successfully run tophat and the -post step in most samples there is one that keep failing with the same error while trying to run the long_spanning_reads step.

Here is the output I get from the log files:

tophat.log:
[2013-08-05 19:47:56] Beginning TopHat run (v2.0.9)
-----------------------------------------------
[2013-08-05 19:47:56] Checking for Bowtie
Bowtie version: 1.0.0.0
[2013-08-05 19:47:57] Checking for Samtools
Samtools version: 0.1.16.0
[2013-08-05 19:47:57] Checking for Bowtie index files (genome)..
[2013-08-05 19:47:58] Checking for reference FASTA file
[2013-08-05 19:47:58] Generating SAM header for /LJ/CompBio/data/common/references/tophat/hg19
format: fastq
quality scale: phred33 (default)
[2013-08-05 19:48:07] Preparing reads
left reads: min. length=101, max. length=101, 73046615 kept reads (24832 discarded)
right reads: min. length=101, max. length=101, 72925819 kept reads (145628 discarded)
[2013-08-05 20:57:05] Mapping left_kept_reads to genome hg19 with Bowtie
[2013-08-05 23:03:01] Mapping left_kept_reads_seg1 to genome hg19 with Bowtie (1/4)
[2013-08-06 00:08:27] Mapping left_kept_reads_seg2 to genome hg19 with Bowtie (2/4)
[2013-08-06 01:13:12] Mapping left_kept_reads_seg3 to genome hg19 with Bowtie (3/4)
[2013-08-06 02:17:24] Mapping left_kept_reads_seg4 to genome hg19 with Bowtie (4/4)
[2013-08-06 03:12:17] Mapping right_kept_reads to genome hg19 with Bowtie
[2013-08-06 04:57:01] Mapping right_kept_reads_seg1 to genome hg19 with Bowtie (1/4)
[2013-08-06 06:02:53] Mapping right_kept_reads_seg2 to genome hg19 with Bowtie (2/4)
[2013-08-06 07:09:10] Mapping right_kept_reads_seg3 to genome hg19 with Bowtie (3/4)
[2013-08-06 08:14:16] Mapping right_kept_reads_seg4 to genome hg19 with Bowtie (4/4)
[2013-08-06 09:08:03] Searching for junctions via segment mapping
[2013-08-06 10:41:46] Retrieving sequences for splices
[2013-08-06 10:44:13] Indexing splices
[2013-08-06 10:55:30] Mapping left_kept_reads_seg1 to genome segment_juncs with Bowtie (1/4)
[2013-08-06 11:19:57] Mapping left_kept_reads_seg2 to genome segment_juncs with Bowtie (2/4)
[2013-08-06 11:44:35] Mapping left_kept_reads_seg3 to genome segment_juncs with Bowtie (3/4)
[2013-08-06 12:09:25] Mapping left_kept_reads_seg4 to genome segment_juncs with Bowtie (4/4)
[2013-08-06 12:30:49] Joining segment hits
[FAILED]
Error running 'long_spanning_reads': what(): basic_string::substr

long_spanning_reads.log
long_spanning_reads v2.0.9 (4112M)
--------------------------------------------
Opening /LJ/CompBio/data/julio/CCLE/RNASEQ/LUNG_secondary/LXF-289/tophat_LXF-289/tmp/segment.fusions for reading
[samopen] SAM header is present: 25 sequences.
Loading reference sequences...
reference sequences loaded.
Loading junctions...done
Loading deletions...done
Loading insertions...done
Loading fusions...done
terminate called after throwing an instance of 'std:ut_of_range'
what(): basic_string::substr

run.log
/tools//BowTieSuite/tophat/tophat -p 4 --fusion-search --keep-fasta-order --bowtie1 --fusion-min-dist 100000 -o /user/sample289/tophat_sample289 --no-coverage-search --fusion-ignore-chromosomes chrM /references/tophat/hg19 /user/sample289/sample289__1.fastq /user/sample289/sample289__2.fastq
#>prep_reads:
/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/prep_reads --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /user/sample289/ --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 --bowtie1 --fusion-search --fusion-anchor-length 20 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p4 --inner-dist-mean 50 --inner-dist-std-dev 20 --no-closure-search --no-coverage-search --no-microexon-search --fastq --aux-outfile=/user/sample289/prep_reads.info --index-outfile=/user/sample289/tmp/%side%_kept_reads.bam.index --sam-header=/user/sample289/tmp/hg19_genome.bwt.samheader.sam --outfile=/user/sample289/tmp/%side%_kept_reads.bam /user/CCLE/RNASEQ/LUNG_secondary/LXF-289/LXF-289__1.fastq /user/CCLE/RNASEQ/LUNG_secondary/LXF-289/LXF-289__2.fastq
#>map_start:
/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/bam2fastx --all --fastq /user/sample289/tmp/left_kept_reads.bam|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 20 -m 20 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads.mapped.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads.mapped.bam /user/sample289/tmp/left_kept_reads_unmapped.bam
#>map_segments:
gzip -cd< /user/sample289/tmp/left_kept_reads_seg1.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools/BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg1.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg1.bam /user/sample289/tmp/left_kept_reads_seg1_unmapped.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg2.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools/BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg2.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg2.bam /user/sample289/tmp/left_kept_reads_seg2_unmapped.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg3.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools/BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg3.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg3.bam /user/sample289/tmp/left_kept_reads_seg3_unmapped.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg4.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools/BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg4.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg4.bam /user/sample289/tmp/left_kept_reads_seg4_unmapped.bam
/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/bam2fastx --all --fastq /user/sample289/tmp/right_kept_reads.bam|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 20 -m 20 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/right_kept_reads.mapped.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/right_kept_reads.mapped.bam /user/sample289/tmp/right_kept_reads_unmapped.bam
#>map_segments:
gzip -cd< /user/sample289/tmp/right_kept_reads_seg1.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/right_kept_reads_seg1.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/right_kept_reads_seg1.bam /user/sample289/tmp/right_kept_reads_seg1_unmapped.bam
gzip -cd< /user/sample289/tmp/right_kept_reads_seg2.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/right_kept_reads_seg2.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/right_kept_reads_seg2.bam /user/sample289/tmp/right_kept_reads_seg2_unmapped.bam
gzip -cd< /user/sample289/tmp/right_kept_reads_seg3.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/right_kept_reads_seg3.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/right_kept_reads_seg3.bam /user/sample289/tmp/right_kept_reads_seg3_unmapped.bam
gzip -cd< /user/sample289/tmp/right_kept_reads_seg4.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /LJ/CompBio/data/common/references/tophat/hg19 -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/right_kept_reads_seg4.bam.index --bowtie1 --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam - /user/sample289/tmp/right_kept_reads_seg4.bam /user/sample289/tmp/right_kept_reads_seg4_unmapped.bam
#>find_juncs:
/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/segment_juncs --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /user/sample289/ --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 --bowtie1 --fusion-search --fusion-anchor-length 20 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p4 --inner-dist-mean 50 --inner-dist-std-dev 20 --no-closure-search --no-coverage-search --no-microexon-search --sam-header /user/sample289/tmp/hg19_genome.bwt.samheader.sam --ium-reads /user/sample289/tmp/left_kept_reads_seg1_unmapped.bam,/user/sample289/tmp/left_kept_reads_seg2_unmapped.bam,/user/sample289/tmp/left_kept_reads_seg3_unmapped.bam,/user/sample289/tmp/left_kept_reads_seg4_unmapped.bam,/user/sample289/tmp/right_kept_reads_seg1_unmapped.bam,/user/sample289/tmp/right_kept_reads_seg2_unmapped.bam,/user/sample289/tmp/right_kept_reads_seg3_unmapped.bam,/user/sample289/tmp/right_kept_reads_seg4_unmapped.bam /LJ/CompBio/data/common/references/tophat/hg19.fa /user/sample289/tmp/segment.juncs /user/sample289/tmp/segment.insertions /user/sample289/tmp/segment.deletions /user/sample289/tmp/segment.fusions /user/sample289/tmp/left_kept_reads.bam /user/sample289/tmp/left_kept_reads.mapped.bam /user/sample289/tmp/left_kept_reads_seg1.bam,/user/sample289/tmp/left_kept_reads_seg2.bam,/user/sample289/tmp/left_kept_reads_seg3.bam,/user/sample289/tmp/left_kept_reads_seg4.bam /user/sample289/tmp/right_kept_reads.bam /user/sample289/tmp/right_kept_reads.mapped.bam /user/sample289/tmp/right_kept_reads_seg1.bam,/user/sample289/tmp/right_kept_reads_seg2.bam,/user/sample289/tmp/right_kept_reads_seg3.bam,/user/sample289/tmp/right_kept_reads_seg4.bam
#>juncs_db:
/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/juncs_db 3 26 /user/sample289/tmp/segment.juncs /user/sample289/tophat_sample289/tmp/segment.insertions /user/sample289/tmp/segment.deletions /user/sample289/tmp/segment.fusions /references/tophat/hg19.fa > /user/sample289/tmp/segment_juncs.fa
/tools/bowtie-1.0.0/bowtie-build /user/sample289/tmp/segment_juncs.fa /user/sample289/tmp/segment_juncs
#>map2juncs:
gzip -cd< /user/sample289/tmp/left_kept_reads_seg1.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /user/sample289/tmp/segment_juncs -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg1.to_spliced.bam.index --bowtie1 --sam-header /user/sample289/tmp/segment_juncs.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg1.to_spliced.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg2.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /user/sample289/tmp/segment_juncs -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg2.to_spliced.bam.index --bowtie1 --sam-header /user/sample289/tmp/segment_juncs.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg2.to_spliced.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg3.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /user/sample289/tmp/segment_juncs -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg3.to_spliced.bam.index --bowtie1 --sam-header /user/sample289/tmp/segment_juncs.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg3.to_spliced.bam
gzip -cd< /user/sample289/tmp/left_kept_reads_seg4.fq.z|/tools/bowtie-1.0.0/bowtie -q -v 2 -k 40 -m 40 -S -p 4 --sam-nohead --max /dev/null /user/sample289/tmp/segment_juncs -|/tools//BowTieSuite/tophat-2.0.9.Linux_x86_64/fix_map_ordering --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --index-outfile /user/sample289/tmp/left_kept_reads_seg4.to_spliced.bam.index --bowtie1 --sam-header /user/sample289/tmp/segment_juncs.bwt.samheader.sam - /user/sample289/tmp/left_kept_reads_seg4.to_spliced.bam
/tools/BowTieSuite/tophat-2.0.9.Linux_x86_64/long_spanning_reads --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /user/sample289/ --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 --bowtie1 --fusion-search --fusion-anchor-length 20 --fusion-min-dist 100000 --fusion-read-mismatches 2 --fusion-multireads 2 --fusion-multipairs 2 --fusion-ignore-chromosomes chrM -z gzip -p4 --inner-dist-mean 50 --inner-dist-std-dev 20 --no-closure-search --no-coverage-search --no-microexon-search --sam-header /user/sample289/tmp/hg19_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 /LJ/CompBio/data/common/references/tophat/hg19.fa /user/sample289/tmp/left_kept_reads.bam /user/sample289/tmp/segment.juncs /user/sample289/tmp/segment.insertions /user/sample289/tmp/segment.deletions /user/sample289/tmp/segment.fusions /user/sample289/tmp/left_kept_reads.candidates.bam /user/sample289/tmp/left_kept_reads_seg1.bam,/user/sample289/tmp/left_kept_reads_seg2.bam,/user/sample289/tmp/left_kept_reads_seg3.bam,/user/sample289/tmp/left_kept_reads_seg4.bam /user/sample289/tmp/left_kept_reads_seg1.to_spliced.bam,/user/sample289/tmp/left_kept_reads_seg2.to_spliced.bam,/user/sample289/tmp/left_kept_reads_seg3.to_spliced.bam,/user/sample289/tmp/left_kept_reads_seg4.to_spliced.bam


For reference I also tried running the just the long_spanning_reads part removing the flag --fusion-search and the run finished successfully.

Has anyone experience a similar issue and have any advice on how to solve it?.

Thanks a lot,

Julio
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Old 08-09-2013, 08:29 AM   #2
julio.fernandez.banet
Junior Member
 
Location: San Diego

Join Date: Jun 2011
Posts: 2
Default Problem solved

Hi all,

As I managed to fix the issue I would like to give a quick update.
After trying multiple things I decided to redownload all the precalculated indexes and annotation files.
I also switch from using bowtie1 in tophat to using bowtie2

So either was an error with the indexes I was using (downloaded from a previous version of tophat) or i has something to do with bowtie

Not sure why this fixed the problem but my samples that keep on failing run without an issue after I did these two changes.

Thanks,

Julio
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