TopHat2 Help-Error: segment-based junction search failed with err =-11

[adisaxena]

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

[pepperoni]

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