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Old 07-19-2012, 09:32 AM   #1
caddymob
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Location: USA

Join Date: Apr 2009
Posts: 36
Unhappy tophat2 splice sequence has more than 2^32-1 characters

Hello,

I have 60 samples that I've tried on tophat2 and about half have all failed at Indexing splices saying "Error: Reference sequence has more than 2^32-1 characters".

Anyone else get this and have a solution? Any ideas?

Comand used:

Code:
 
${TOPHAT} \
--mate-inner-dist ${INNERDIST} \
--mate-std-dev ${STDEV} \
--output-dir $PBS_O_WORKDIR/tophat2 \
--num-threads 12 \
--transcriptome-index ${HUMAN_TRANS_FA} \
--no-coverage-search \
--fusion-search \
--fusion-ignore-chromosomes MT \
--b2-sensitive \
--keep-fasta-order \
--rg-id ${SAMPLE_ID}_${DATE} \
--rg-sample ${SAMPLE_ID} \
--rg-library ${SAMPLE_ID}_Lib1 \
--rg-platform ILLUMINA \
${HUMAN_REF_FA} \
${read1} ${read2}
And error output:

Code:
[2012-07-19 00:40:43] Beginning TopHat run (v2.0.4)
-----------------------------------------------
[2012-07-19 00:40:43] Checking for Bowtie
		  Bowtie version:	 2.0.0.7
[2012-07-19 00:40:43] Checking for Samtools
		Samtools version:	 0.1.18.0
[2012-07-19 00:40:43] Checking for Bowtie index files
[2012-07-19 00:40:43] Checking for Bowtie index files
[2012-07-19 00:40:43] Checking for reference FASTA file
[2012-07-19 00:40:43] Generating SAM header for ~/resources/GRCh37/bowtie2_b37/Homo_sapiens.GRCh37.62
	format:		 fastq
	quality scale:	 phred33 (default)
[2012-07-19 00:41:19] Reading known junctions from GTF file
[2012-07-19 00:41:33] Preparing reads
	 left reads: min. length=51, max. length=51, 82100145 kept reads (7919 discarded)
	right reads: min. length=51, max. length=51, 81990155 kept reads (117909 discarded)
[2012-07-19 01:11:51] Using pre-built transcriptome index..
[2012-07-19 01:11:57] Mapping left_kept_reads to transcriptome Homo_sapiens.GRCh37.66 with Bowtie2 
[2012-07-19 02:28:44] Mapping right_kept_reads to transcriptome Homo_sapiens.GRCh37.66 with Bowtie2 
[2012-07-19 03:44:34] Resuming TopHat pipeline with unmapped reads
[2012-07-19 03:44:34] Mapping left_kept_reads.m2g_um to genome Homo_sapiens.GRCh37.62 with Bowtie2 
[2012-07-19 03:59:51] Mapping left_kept_reads.m2g_um_seg1 to genome Homo_sapiens.GRCh37.62 with Bowtie2 (1/2)
[2012-07-19 04:02:30] Mapping left_kept_reads.m2g_um_seg2 to genome Homo_sapiens.GRCh37.62 with Bowtie2 (2/2)
[2012-07-19 04:05:18] Mapping right_kept_reads.m2g_um to genome Homo_sapiens.GRCh37.62 with Bowtie2 
[2012-07-19 04:20:59] Mapping right_kept_reads.m2g_um_seg1 to genome Homo_sapiens.GRCh37.62 with Bowtie2 (1/2)
[2012-07-19 04:24:50] Mapping right_kept_reads.m2g_um_seg2 to genome Homo_sapiens.GRCh37.62 with Bowtie2 (2/2)
[2012-07-19 04:28:48] Searching for junctions via segment mapping
[2012-07-19 05:04:34] Retrieving sequences for splices
[2012-07-19 05:18:32] Indexing splices
Error: Reference sequence has more than 2^32-1 characters!  Please divide the
reference into batches or chunks of about 3.6 billion characters or less each
and index each independently.
Error: Encountered internal Bowtie 2 exception (#1)
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Old 07-23-2012, 11:40 AM   #2
caddymob
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Location: USA

Join Date: Apr 2009
Posts: 36
Default

Turns out this was due to an upgrade to our isilon NFS disk... Rebooting machines fixed the error. Very strange, but moving on...
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