Error running bowtie:

[Nextgenanalysis]

Hi I am new user for TOPHAT. I am trying to align my RNASeq single end reads from illumina with human genome refernce GRCH37. My system specifications are 32 GB RAM and 1TB allotment to ubuntu in a total of 2 TB harddisk. But still I hget the following error. Please suggest me the solution.

[2014-01-18 13:30:47] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2
[FAILED]
Error running bowtie:
Out of memory allocating the ebwt[] array for the Bowtie index. Please try
again on a computer with more memory.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /usr/local/bin/bowtie2-align -k 1 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 4 --sam-no-hd -x /home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome -

Thanks

[GenoMax]

Have you checked to see if there are any "limits" on your user account by running the following command?

$ limit or ulimit

Does the program quit right away or does it run for some time before it does?

[Nextgenanalysis]

Thank you so much for the reply.,
The program is running for pretty long time before it shows the error. I am currently running this again removing -p option as suggested by one person in internet. i will check the limit immediately after the run is over

[Nextgenanalysis]

Have you checked to see if there are any "limits" on your user account by running the following command?

$ limit or ulimit

Does the program quit right away or does it run for some time before it does?

Thank you so much for the reply.,
The program is running for pretty long time before it shows the error. I am currently running this again removing -p option as suggested by one person in internet. i will check the limit immediately after the run is over

[Nextgenanalysis]

Thank you so much for the reply.,
The program is running for pretty long time before it shows the error. I am currently running this again removing -p option as suggested by one person in internet. i will check the limit immediately after the run is over

I have run the command and it has shown unlimited

[Nextgenanalysis]

how much memory is required to align reads from human RNA sequencing data to reference genome using tophat2(Bowtie2).

I am worried that I keep getting the error as i have posted earlier what ever I do. 32GB memory is not enough for this?!!

root@linux-Precision-T3600:~# tophat -g 1 -G '/home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf' -o '/home/linux/Desktop/NGSsampledata/tophat_output_1' '/home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome' '/home/linux/Desktop/NGSsampledata/Normal/normal.fastq'

[2014-01-18 18:25:14] Beginning TopHat run (v2.0.10)
-----------------------------------------------
[2014-01-18 18:25:14] Checking for Bowtie
Bowtie version: 2.1.0.0
[2014-01-18 18:25:14] Checking for Samtools
Samtools version: 0.1.19.0
[2014-01-18 18:25:14] Checking for Bowtie index files (genome)..
[2014-01-18 18:25:14] Checking for reference FASTA file
[2014-01-18 18:25:14] Generating SAM header for /home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome
[2014-01-18 18:25:16] Reading known junctions from GTF file
[2014-01-18 18:25:32] Preparing reads
left reads: min. length=35, max. length=35, 15669232 kept reads (5155 discarded)
[2014-01-18 18:28:12] Building transcriptome data files /home/linux/Desktop/NGSsampledata/tophat_output_1/tmp/genes
[2014-01-18 18:28:45] Building Bowtie index from genes.fa
[2014-01-18 18:56:21] Mapping left_kept_reads to transcriptome genes with Bowtie2
[2014-01-18 19:29:21] Resuming TopHat pipeline with unmapped reads
Warning: you have only one segment per read.
If the read length is greater than or equal to 45bp,
we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments
[2014-01-18 19:29:21] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2
[FAILED]
Error running bowtie:
Out of memory allocating the ebwt[] array for the Bowtie index. Please try
again on a computer with more memory.
Error: Encountered internal Bowtie 2 exception (#1)
Command: /usr/local/bin/bowtie2-align -k 1 -D 15 -R 2 -N 0 -L 20 -i S,1,1.25 --gbar 4 --mp 6,2 --np 1 --rdg 5,3 --rfg 5,3 --score-min C,-14,0 -p 1 --sam-no-hd -x /home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome -

for getting the error message it is taking a very long time too!!!

Please suggest me a solution

[GenoMax]

Based on the time stamps in your log the job appears to be failing after an hour which is not a "long" time for NGS data analysis. Expect the tophat job to run for a few hours (depending on the hardware you have) when things do get working.

Getting back to your error. A few things to check.

1. What flavor of 64-bit linux distro are you using?
2. How big is your temp (/tmp) partition? Is is filling up during the run (something to monitor). If you are the admin see if there are any messages in /var/syslog (or an appropriate location).
3. While running the alignment job try to capture the standard error/out to a file. This is generally different for different shells but if you are using bash then here is how you would do it:

root@linux-Precision-T3600:~# tophat -g 1 -G '/home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Annotation/Genes/genes.gtf' -o '/home/linux/Desktop/NGSsampledata/tophat_output_1' '/home/linux/Desktop/NGSsampledata/Homo_sapiens_Ensembl_GRCh37/Homo_sapiens/Ensembl/GRCh37/Sequence/Bowtie2Index/genome' '/home/linux/Desktop/NGSsampledata/Normal/normal.fastq' [COLOR="Red"]>file.log 2>&1[/COLOR]

4. Let us know what the file.log contains if the job fails again.

It appears that you running regular jobs as the "root" user. This is NOT a good practice. You should use a regular user account for normal jobs.