Error RNA-seq data, TOPHAT

Question

[David_emir]

Hi All,

I tried aligning the RNA-seq reads to the genome using tophat, used the following command

[root@BIO-DT-415 RNA_SEQ]# tophat -p 8 -G genes.gtf -o C1_R1_thout genome GSM794483_C1_R1_1.fq GSM794483_C1_R1_2.fq

It's throwing error:

/usr/local/bin/tophat -p 8 -G genes.gtf -o C1_R1_thout genome GSM794483_C1_R1_1.fq GSM794483_C1_R1_2.fq
/usr/local/bin/gtf_juncs genes.gtf  > C1_R1_thout/tmp/genes.juncs
2014-11-26 16:25:31] Reporting output tracks
    [FAILED]

Error running /usr/local/bin/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir C1_R1_thout/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 2 --read-gap-length 2 --read-edit-dist 2 --read-realign-edit-dist 3 --max-insertion-length 3 --max-deletion-length 3 -z gzip -p8 --inner-dist-mean 50 --inner-dist-std-dev 20 --gtf-annotations genes.gtf --gtf-juncs C1_R1_thout/tmp/genes.juncs --no-closure-search --no-coverage-search --no-microexon-search --sam-header C1_R1_thout/tmp/genome_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/usr/local/bin/samtools_0.1.18 --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 genome.fa C1_R1_thout/junctions.bed C1_R1_thout/insertions.bed C1_R1_thout/deletions.bed C1_R1_thout/fusions.out C1_R1_thout/tmp/accepted_hits C1_R1_thout/tmp/left_kept_reads.m2g.bam C1_R1_thout/tmp/left_kept_reads.bam C1_R1_thout/tmp/right_kept_reads.m2g.bam C1_R1_thout/tmp/right_kept_reads.bam
Loaded 51950 junctions

I am not able to decode this, Please let me know if you have any solution for this.

Thanks a lot

-Ateeq Khaliq

[Devon Ryan]

Try running that command manually (you'll need to be in the same directory) and then see what the actual error message is. Often this means you've run out of memory, though tophat isn't without its own bugs.

[David_emir]

Thanks Devon , Thanks for coming to my rescue again !!! I have increased the RAM size to 16 gigs, now its running fine . Hoping to finish this protocol by this week!!! I am sure I will be back again to this forum.. Thank you so much ...its great help for me !!!

Answer 1

[Manvendra Singh]

how much memory are you providing on 8 threads?

[David_emir]

Thanks Manu, you pointed it right, I have increased the ram size, now its working fine. Thanks a lot !!!

[Manvendra Singh]

Welcome !

Good!