I have installed RNA-SeQC on our cluster and have been trying to run a number of jobs for RNA-SeQC (version 1.18) on 5-6 Gb BAM files of human RNA-seq data.
Info about the BAM files: human paired-end sequences, aligned to hg38 build genome using STAR, read groups added, sorted and indexed.
The command file for RNA-SeQC is generally:
/share/apps/jdk1.7.0_71/bin/java -Xmx60g -jar /user/tools/RNA-SeQC/RNA-SeQC_v1.1.8.jar \ -bwa /user/bwa-0.7.10/bwa -BWArRNA \ /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/rRNA.gtf -t \ /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Annotation/Archives/archive-2015-08-14-08-18-15/Genes/genes.gtf -r \ /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa -s \ /user/bam/bam_samples.txt -o /user/bam/RNA-SeQC
The bam_samples.txt only includes one BAM now as I was having the same error with many so I am trying to get it sorted just to work with one for now.
From the output it looks like the job is working fine to a certain stage and then I am getting memory errors. I have given the cluster job memory ranging from 16Gb to 128Gb with no luck at all.
The error I am getting is:
RNA-SeQC v184.108.40.206 07/11/14 Retriving contig names from reference contig names in reference: 195 Loading GTF for Read Counting Converting to refGene Transcript objects to RefGen format: 1 s Running IntronicExpressionReadBlock Walker .... Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, /user/bam/RNA-SeQC/NC101/NC101.metrics.tmp.txt, -R, /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa, -I, /user/bam/NC101/NC101_unique.RG.bam, -refseq, /user/bam/RNA-SeQC/refGene.txt, -l, ERROR] Finished writing /user/bam/RNA-SeQC/NC101/NC101.metrics.tmp.txt.intronReport.txt Finished writing /user/bam/RNA-SeQC/NC101/NC101.metrics.tmp.txt.intronReport.txt_intronOnly.txt, now creating RPKM values for introns .. GATK command result code: 0 ... GATK CoutReadMetrics Analysis DONE CountReadMetricsWalker Runtime: 12 min Counting rRNA reads with BWA and /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/rRNA.gtf Downsampling before aligning at rate: 0.009106594123052198 INFO 2016-09-15 21:23:02 DownsampleSam Read 10000000 reads, kept 91285 INFO 2016-09-15 21:23:34 DownsampleSam Read 20000000 reads, kept 182434 INFO 2016-09-15 21:23:56 DownsampleSam Read 30000000 reads, kept 272798 INFO 2016-09-15 21:24:18 DownsampleSam Read 40000000 reads, kept 364148 INFO 2016-09-15 21:24:39 DownsampleSam Read 50000000 reads, kept 455860 INFO 2016-09-15 21:25:01 DownsampleSam Read 60000000 reads, kept 547155 INFO 2016-09-15 21:25:24 DownsampleSam Read 70000000 reads, kept 639031 INFO 2016-09-15 21:25:50 DownsampleSam Read 80000000 reads, kept 730499 INFO 2016-09-15 21:26:19 DownsampleSam Read 90000000 reads, kept 822225 INFO 2016-09-15 21:26:41 DownsampleSam Read 100000000 reads, kept 912608 INFO 2016-09-15 21:27:10 DownsampleSam Finished! Kept 1001492 out of 109810538 reads. Downsampling exited with code: 0 BWA on end 1 Running BWA on /user/bam/RNA-SeQC/NC101/dSample.bam Command: [/user/bwa-0.7.10/bwa, aln, /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/rRNA.gtf, -b1, /user/bam/RNA-SeQC/NC101/dSample.bam] # # There is insufficient memory for the Java Runtime Environment to continue. # pthread_getattr_np /opt/gridengine/default/spool/lum-7-13/job_scripts/246455: line 28: 57179 Aborted /share/apps/jdk1.7.0_71/bin/java -Xmx61440M -jar /user/tools/RNA-SeQC/RNA-SeQC_v1.1.8.jar -bwa /user/bwa-0.7.10/bwa -BWArRNA /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/rRNA.gtf -t /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Annotation/Archives/archive-2015-08-14-08-18-15/Genes/genes.gtf -r /user/ref_genome/hg38_ucsc/Homo_sapiens/UCSC/hg38/Sequence/WholeGenomeFasta/genome.fa -s /user/bam/bam_samples.txt -o /user/bam/RNA-SeQC
Apologies for the length of the post but I wanted to get as much useful information in place.
If anyone has any suggestions or advice it would be greatly appreciated!