Question: RNAseq problems with tophat
0
gravatar for clizama
3.5 years ago by
clizama0
clizama0 wrote:

HI

I'm working with a RNAseq pair end dataset. I'm running tophat in galaxy using the amazon cloud. My problem is when I ran tophat I was able to analyze just the half of the data (6 files). For some reason with the other 6 files tophat showed me this msn:

error An error occurred with this dataset: Fatal error: Tool execution failed [2016-03-23 04:49:57] Beginning TopHat run (v2.0.13) ----------------------------------------------- [2016-03-23 04:49:57] Checking for Bowtie Bowtie version: 2.2.3.0 [2016-03-23 04:49:58] Checking for Bowtie inde

I have 600 gb of free space, I tried several times ran tophat but all the time fail and show the same error.

Thanks

rna-seq • 1.3k views
ADD COMMENTlink modified 2.1 years ago by akhilesh0 • written 3.5 years ago by clizama0

Hi clizama, Are you sure the error message you posted here is complete ? It seems truncated to me. Could we have the complete recording of tophat's logging, so that we'll be able to help you ?

ADD REPLYlink written 3.5 years ago by Thibault D.650

2016-03-24 07:39:10] Checking for Bowtie Bowtie version: 2.2.3.0 [2016-03-24 07:39:10] Checking for Bowtie index files (genome).. Found both Bowtie1 and Bowtie2 indexes. [2016-03-24 07:39:10] Checking for reference FASTA file [2016-03-24 07:39:10] Generating SAM header for /data/refs/mm10/genome.fa [2016-03-24 07:39:34] Reading known junctions from GTF file [2016-03-24 07:39:36] Preparing reads left reads: min. length=20, max. length=100, 41391601 kept reads (96792 discarded) right reads: min. length=20, max. length=100, 41365359 kept reads (123034 discarded) [2016-03-24 08:06:07] Building transcriptome data files ./tophat_out/tmp/genes [2016-03-24 08:06:31] Building Bowtie index from genes.fa [2016-03-24 08:09:56] Mapping left_kept_reads to transcriptome genes with Bowtie2 [2016-03-24 08:44:53] Mapping right_kept_reads to transcriptome genes with Bowtie2 [2016-03-24 09:19:32] Resuming TopHat pipeline with unmapped reads [2016-03-24 09:19:32] Mapping left_kept_reads.m2g_um to genome genome.fa with Bowtie2 [2016-03-24 10:18:51] Mapping left_kept_reads.m2g_um_seg1 to genome genome.fa with Bowtie2 (1/4) [2016-03-24 10:25:00] Mapping left_kept_reads.m2g_um_seg2 to genome genome.fa with Bowtie2 (2/4) [2016-03-24 10:31:38] Mapping left_kept_reads.m2g_um_seg3 to genome genome.fa with Bowtie2 (3/4) [2016-03-24 10:37:52] Mapping left_kept_reads.m2g_um_seg4 to genome genome.fa with Bowtie2 (4/4) [2016-03-24 10:43:53] Mapping right_kept_reads.m2g_um to genome genome.fa with Bowtie2 [2016-03-24 11:45:39] Mapping right_kept_reads.m2g_um_seg1 to genome genome.fa with Bowtie2 (1/4) [2016-03-24 11:52:27] Mapping right_kept_reads.m2g_um_seg2 to genome genome.fa with Bowtie2 (2/4) [2016-03-24 12:00:02] Mapping right_kept_reads.m2g_um_seg3 to genome genome.fa with Bowtie2 (3/4) [2016-03-24 12:07:14] Mapping right_kept_reads.m2g_um_seg4 to genome genome.fa with Bowtie2 (4/4) [2016-03-24 12:13:35] Searching for junctions via segment mapping [2016-03-24 12:26:03] Retrieving sequences for splices [2016-03-24 12:27:17] Indexing splices Building a SMALL index [2016-03-24 12:27:35] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/4) [2016-03-24 12:29:17] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/4) [2016-03-24 12:31:55] Mapping left_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/4) [2016-03-24 12:34:29] Mapping left_kept_reads.m2g_um_seg4 to genome segment_juncs with Bowtie2 (4/4) [2016-03-24 12:36:54] Joining segment hits [2016-03-24 12:42:45] Mapping right_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/4) [2016-03-24 12:44:46] Mapping right_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/4) [2016-03-24 12:47:38] Mapping right_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/4) [2016-03-24 12:50:32] Mapping right_kept_reads.m2g_um_seg4 to genome segment_juncs with Bowtie2 (4/4) [2016-03-24 12:53:30] Joining segment hits [2016-03-24 12:59:36] Reporting output tracks [FAILED] Error running /software/tophat/2.0.13/x86_64-linux-ubuntu14.04/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 70 --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 1000 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p4 --inner-dist-mean 200 --inner-dist-std-dev 20 --gtf-annotations /data/refs/mm10/genes.gtf --gtf-juncs ./tophat_out/tmp/genes.juncs --no-closure-search --no-coverage-search --no-microexon-search --library-type fr-unstranded --sam-header ./tophat_out/tmp/genome.fa_genome.bwt.samheader.sam --report-mixed-alignments --samtools=/software/tophat/2.0.13/x86_64-linux-ubuntu14.04/bin/samtools_0.1.18 --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 /data/refs/mm10/genome.fa.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.m2g.bam,./tophat_out/tmp/left_kept_reads.m2g_um.mapped.bam,./tophat_out/tmp/left_kept_reads.m2g_um.candidates ./tophat_out/tmp/left_kept_reads.bam ./tophat_out/tmp/right_kept_reads.m2g.bam,./tophat_out/tmp/right_kept_reads.m2g_um.mapped.bam,./tophat_out/tmp/right_kept_reads.m2g_um.candidates ./tophat_out/tmp/right_kept_reads.bam Loaded 209433 junctions

ADD REPLYlink written 3.5 years ago by clizama0

I got the exact same error while using Tophat on Galaxy [2017-07-24 11:08:21] Joining segment hits [2017-07-24 11:18:01] Reporting output tracks [FAILED]

Antonio were you able to resolve this issue?

ADD REPLYlink written 2.1 years ago by akhilesh0

If this error is from PSU Galaxy then consider posting this over at Galaxy Biostars.

ADD REPLYlink written 2.1 years ago by genomax71k
0
gravatar for Antonio R. Franco
3.5 years ago by
Spain. Universidad de Córdoba
Antonio R. Franco4.1k wrote:

The error message is incomplete. It start indicating that it checks for the Bowtie index, but after the "x" the message is gone, so no clues about what is happening

One caution note: You need to create the bowtie index using the same prefix that the fasta reference sequence. For example, if your fasta sequence is named my_fasta.fa, you need to create the index with bowtie-build using my_fasta. It is also important to have in the same directory of the index file a copy of my_fasta.fa

Then, you need to invoque the bowtie index in Tophat by indicating the path of your indexes using the prefix only. For example path/to_your_indexed_reference/my_fasta

Bowtie will notice the prefix name my_fasta, and then, it will look into the directory for the indexed files and the reference fasta name having the same prefixes

ADD COMMENTlink written 3.5 years ago by Antonio R. Franco4.1k

I added the full error

ADD REPLYlink written 3.5 years ago by clizama0
0
gravatar for clizama
3.5 years ago by
clizama0
clizama0 wrote:

[2016-03-24 07:39:10] Beginning TopHat run (v2.0.13)

[2016-03-24 07:39:10] Checking for Bowtie Bowtie version: 2.2.3.0 [2016-03-24 07:39:10] Checking for Bowtie index files (genome).. Found both Bowtie1 and Bowtie2 indexes. [2016-03-24 07:39:10] Checking for reference FASTA file [2016-03-24 07:39:10] Generating SAM header for /data/refs/mm10/genome.fa [2016-03-24 07:39:34] Reading known junctions from GTF file [2016-03-24 07:39:36] Preparing reads left reads: min. length=20, max. length=100, 41391601 kept reads (96792 discarded) right reads: min. length=20, max. length=100, 41365359 kept reads (123034 discarded) [2016-03-24 08:06:07] Building transcriptome data files ./tophat_out/tmp/genes [2016-03-24 08:06:31] Building Bowtie index from genes.fa [2016-03-24 08:09:56] Mapping left_kept_reads to transcriptome genes with Bowtie2 [2016-03-24 08:44:53] Mapping right_kept_reads to transcriptome genes with Bowtie2 [2016-03-24 09:19:32] Resuming TopHat pipeline with unmapped reads [2016-03-24 09:19:32] Mapping left_kept_reads.m2g_um to genome genome.fa with Bowtie2 [2016-03-24 10:18:51] Mapping left_kept_reads.m2g_um_seg1 to genome genome.fa with Bowtie2 (1/4) [2016-03-24 10:25:00] Mapping left_kept_reads.m2g_um_seg2 to genome genome.fa with Bowtie2 (2/4) [2016-03-24 10:31:38] Mapping left_kept_reads.m2g_um_seg3 to genome genome.fa with Bowtie2 (3/4) [2016-03-24 10:37:52] Mapping left_kept_reads.m2g_um_seg4 to genome genome.fa with Bowtie2 (4/4) [2016-03-24 10:43:53] Mapping right_kept_reads.m2g_um to genome genome.fa with Bowtie2 [2016-03-24 11:45:39] Mapping right_kept_reads.m2g_um_seg1 to genome genome.fa with Bowtie2 (1/4) [2016-03-24 11:52:27] Mapping right_kept_reads.m2g_um_seg2 to genome genome.fa with Bowtie2 (2/4) [2016-03-24 12:00:02] Mapping right_kept_reads.m2g_um_seg3 to genome genome.fa with Bowtie2 (3/4) [2016-03-24 12:07:14] Mapping right_kept_reads.m2g_um_seg4 to genome genome.fa with Bowtie2 (4/4) [2016-03-24 12:13:35] Searching for junctions via segment mapping [2016-03-24 12:26:03] Retrieving sequences for splices [2016-03-24 12:27:17] Indexing splices Building a SMALL index [2016-03-24 12:27:35] Mapping left_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/4) [2016-03-24 12:29:17] Mapping left_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/4) [2016-03-24 12:31:55] Mapping left_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/4) [2016-03-24 12:34:29] Mapping left_kept_reads.m2g_um_seg4 to genome segment_juncs with Bowtie2 (4/4) [2016-03-24 12:36:54] Joining segment hits [2016-03-24 12:42:45] Mapping right_kept_reads.m2g_um_seg1 to genome segment_juncs with Bowtie2 (1/4) [2016-03-24 12:44:46] Mapping right_kept_reads.m2g_um_seg2 to genome segment_juncs with Bowtie2 (2/4) [2016-03-24 12:47:38] Mapping right_kept_reads.m2g_um_seg3 to genome segment_juncs with Bowtie2 (3/4) [2016-03-24 12:50:32] Mapping right_kept_reads.m2g_um_seg4 to genome segment_juncs with Bowtie2 (4/4) [2016-03-24 12:53:30] Joining segment hits [2016-03-24 12:59:36] Reporting output tracks [FAILED] Error running /software/tophat/2.0.13/x86_64-linux-ubuntu14.04/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 70 --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 1000 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p4 --inner-dist-mean 200 --inner-dist-std-dev 20 --gtf-annotations /data/refs/mm10/genes.gtf --gtf-juncs ./tophat_out/tmp/genes.juncs --no-closure-search --no-coverage-search --no-microexon-search --library-type fr-unstranded --sam-header ./tophat_out/tmp/genome.fa_genome.bwt.samheader.sam --report-mixed-alignments --samtools=/software/tophat/2.0.13/x86_64-linux-ubuntu14.04/bin/samtools_0.1.18 --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 /data/refs/mm10/genome.fa.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.m2g.bam,./tophat_out/tmp/left_kept_reads.m2g_um.mapped.bam,./tophat_out/tmp/left_kept_reads.m2g_um.candidates ./tophat_out/tmp/left_kept_reads.bam ./tophat_out/tmp/right_kept_reads.m2g.bam,./tophat_out/tmp/right_kept_reads.m2g_um.mapped.bam,./tophat_out/tmp/right_kept_reads.m2g_um.candidates ./tophat_out/tmp/right_kept_reads.bam Loaded 209433 junctions

ADD COMMENTlink written 3.5 years ago by clizama0
0
gravatar for fanli.gcb
3.5 years ago by
fanli.gcb690
Los Angeles, CA
fanli.gcb690 wrote:

This has been reported on and off for various Tophat versions. See:

https://groups.google.com/forum/#!topic/tuxedo-tools-users/0pcEVAOCBaw

What causes tophat_reports error?

In my experience, the simplest way to workaround is to change the number of threads used.

ADD COMMENTlink written 3.5 years ago by fanli.gcb690

I'm running the tophat from galaxy I'm not sure how to change the number of threads?

Can you explain me a little more please

Thanks carlos

ADD REPLYlink written 3.5 years ago by clizama0

I don't know how to do this in Galaxy, but the tophat option -p 8 would specify 8 processors. Changing this to -p 4 or -p 1 for example, generally fixes this issue in my experience.

ADD REPLYlink written 3.5 years ago by fanli.gcb690

I'm trying to find how to do it. still working on it.

ADD REPLYlink written 3.5 years ago by clizama0
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