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Old 05-29-2016, 02:31 PM   #1
adisaxena
Junior Member
 
Location: USA

Join Date: Aug 2015
Posts: 6
Default TopHat2 Help-Error: segment-based junction search failed with err =-11

Hi all,

I am new to Tuxedo tools and want to use TopHat + Cufflinks for differential gene expression analysis with RNASeq data.

I have ~77.7 million mouse SR-50 illumina reads (libraries prepared with strand-specific TrueSeq mRNA, illumina kit) that I want to map back to the Mouse genome. I use prebuild iGenome mouse index (NCBI_Build37.2) as a reference and have tried to run the Tophat pipeline on a cluster (two attempt with memory=120Gb;CPU=24 and memory=700Gb;CPU-30) and got this error:

[2016-05-29 13:08:56] Beginning TopHat run (v2.1.0)
-----------------------------------------------

[2016-05-29 13:08:56] Checking for Bowtie Bowtie version: 2.2.4.0

[2016-05-29 13:08:56] Checking for Bowtie index files (genome)..

[2016-05-29 13:08:56] Checking for reference FASTA file

[2016-05-29 13:08:56] Generating SAM header for /oasis/projects/nsf/csd473/adi/Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome

[2016-05-29 13:09:05] Reading known junctions from GTF file

[2016-05-29 13:09:07] Preparing reads

left reads: min. length=40, max. length=50, 77774302 kept reads (3761 discarded)

[2016-05-29 13:18:23] Building transcriptome data files M1M_TopHat2_MusGenBuild37.2/tmp/genes

[2016-05-29 13:18:47] Building Bowtie index from genes.fa

[2016-05-29 13:21:03] Mapping left_kept_reads to transcriptome genes with Bowtie2

[2016-05-29 13:39:09] Resuming TopHat pipeline with unmapped reads

[2016-05-29 13:39:09] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2

[2016-05-29 13:41:31] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)

[2016-05-29 13:42:03] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)

[2016-05-29 13:42:34] Searching for junctions via segment mapping

[FAILED]

Error: segment-based junction search failed with err =-11

My run script is:-

tophat2 -p 30 -G Build37.2_iGenome_Mus/Annotation/Archives/archive-2015-07-17-14-32-40/Genes/genes.gtf --library-type fr-secondstrand --no-coverage-search -o TopHat2_MusGenBuild37.2 Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome M1M_Trimmed.fastq.gz

Could someone please help me understand the error 'segment-based junction search failed with err =-11'? The 'segment_juncs.log' file is blank.

Shall be very grateful for your help with troubleshoot advice!

thanks in advance,
Adi
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Old 10-14-2017, 07:41 AM   #2
pepperoni
Member
 
Location: US

Join Date: Oct 2011
Posts: 57
Default

Quote:
Originally Posted by adisaxena View Post
Hi all,

I am new to Tuxedo tools and want to use TopHat + Cufflinks for differential gene expression analysis with RNASeq data.

I have ~77.7 million mouse SR-50 illumina reads (libraries prepared with strand-specific TrueSeq mRNA, illumina kit) that I want to map back to the Mouse genome. I use prebuild iGenome mouse index (NCBI_Build37.2) as a reference and have tried to run the Tophat pipeline on a cluster (two attempt with memory=120Gb;CPU=24 and memory=700Gb;CPU-30) and got this error:

[2016-05-29 13:08:56] Beginning TopHat run (v2.1.0)
-----------------------------------------------

[2016-05-29 13:08:56] Checking for Bowtie Bowtie version: 2.2.4.0

[2016-05-29 13:08:56] Checking for Bowtie index files (genome)..

[2016-05-29 13:08:56] Checking for reference FASTA file

[2016-05-29 13:08:56] Generating SAM header for /oasis/projects/nsf/csd473/adi/Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome

[2016-05-29 13:09:05] Reading known junctions from GTF file

[2016-05-29 13:09:07] Preparing reads

left reads: min. length=40, max. length=50, 77774302 kept reads (3761 discarded)

[2016-05-29 13:18:23] Building transcriptome data files M1M_TopHat2_MusGenBuild37.2/tmp/genes

[2016-05-29 13:18:47] Building Bowtie index from genes.fa

[2016-05-29 13:21:03] Mapping left_kept_reads to transcriptome genes with Bowtie2

[2016-05-29 13:39:09] Resuming TopHat pipeline with unmapped reads

[2016-05-29 13:39:09] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2

[2016-05-29 13:41:31] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)

[2016-05-29 13:42:03] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)

[2016-05-29 13:42:34] Searching for junctions via segment mapping

[FAILED]

Error: segment-based junction search failed with err =-11

My run script is:-

tophat2 -p 30 -G Build37.2_iGenome_Mus/Annotation/Archives/archive-2015-07-17-14-32-40/Genes/genes.gtf --library-type fr-secondstrand --no-coverage-search -o TopHat2_MusGenBuild37.2 Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome M1M_Trimmed.fastq.gz

Could someone please help me understand the error 'segment-based junction search failed with err =-11'? The 'segment_juncs.log' file is blank.

Shall be very grateful for your help with troubleshoot advice!

thanks in advance,
Adi
Hi, did you solved this?
Any advice will be appreciated
pepperoni is offline   Reply With Quote
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