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
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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