Question: rsem-calculate-expression fails to open iso_res file
gravatar for emyers
3.1 years ago by
emyers0 wrote:

I am new to any kind of sequence analysis, and am trying to use RSEM for mapping (with STAR) and expression estimates. The step of preparing a reference completed successfully. The call to rsem-calculate-expression gets through the mapping, but hits an error before producing the expression estimates.

This is the call to rsem-calculate-expression:

sudo /opt/RSEM-1.3.0/rsem-calculate-expression $trimmedFile $refName BF_RORbHTp200_1 -p 8 --star --star-path $starPath --star-output-genome-bam

And the output:

/opt/STAR/bin/MacOSX_x86_64//STAR --genomeDir /Volumes/CodingClub1/STAR_stuff/rsem_reference  --outSAMunmapped Within  --outFilterType BySJout  --outSAMattributes NH HI AS NM MD  --outFilterMultimapNmax 20  --outFilterMismatchNmax 999  --outFilterMismatchNoverLmax 0.04  --alignIntronMin 20  --alignIntronMax 1000000  --alignMatesGapMax 1000000  --alignSJoverhangMin 8  --alignSJDBoverhangMin 1  --sjdbScore 1  --runThreadN 8  --genomeLoad NoSharedMemory  --outSAMtype BAM Unsorted  --quantMode TranscriptomeSAM  --outSAMheaderHD \@HD VN:1.4 SO:unsorted  --outFileNamePrefix BF_RORbHTp200_1.temp/BF_RORbHTp200_1  --readFilesIn /Volumes/CodingClub1/RNAseq/RORb/trimmed/trimmed_m20_q20/BF_RORbHTp200_1_trimmed_m20_q20.fastq 
Dec 18 09:36:35 ..... started STAR run
Dec 18 09:36:35 ..... loading genome
Dec 18 09:39:16 ..... started mapping
Dec 18 09:43:09 ..... finished successfully

rsem-parse-alignments /Volumes/CodingClub1/STAR_stuff/rsem_reference/mm10_ucsc_ BF_RORbHTp200_1.temp/BF_RORbHTp200_1 BF_RORbHTp200_1.stat/BF_RORbHTp200_1 BF_RORbHTp200_1.temp/BF_RORbHTp200_1.bam 1 -tag XM

rsem-build-read-index 32 1 0 BF_RORbHTp200_1.temp/BF_RORbHTp200_1_alignable.fq

rsem-run-em /Volumes/CodingClub1/STAR_stuff/rsem_reference/mm10_ucsc_ 1 BF_RORbHTp200_1 BF_RORbHTp200_1.temp/BF_RORbHTp200_1 BF_RORbHTp200_1.stat/BF_RORbHTp200_1 -p 8 -b BF_RORbHTp200_1.temp/BF_RORbHTp200_1.bam 0

Fail to open file BF_RORbHTp200_1.temp/BF_RORbHTp200_1.iso_res!

RSEM appears to be writing other files to the directory BF_RORbHTp200_1.temp just fine. The other possibility that occured to me is the number of files that can be opened at the same time, in case RSEM was opening a lot of temporary files. I set ulimit -n to 2048 and got the same problem. Any help would be greatly appreciated.

rna-seq • 1.5k views
ADD COMMENTlink written 3.1 years ago by emyers0

sounds like a permissions issue. why are you running as root?

ADD REPLYlink modified 3.1 years ago • written 3.1 years ago by mforde841.3k

Whoops - I shouldn't have been. But I tried again under a regular user and got the same error.

ADD REPLYlink written 3.1 years ago by emyers0

look at the permissions of the folder in question. check that it's readable, and writable to the user running the process 'ls -l'. check that you have available space on the mount point 'df -h'.

if all else fails, delete the temporary folders and files associated with the RSEM analysis, and rerun the analysis from scratch from a normal user.

ADD REPLYlink modified 3.1 years ago • written 3.1 years ago by mforde841.3k

Thanks very much for your suggestions. The directory is read/writeable to the user, and I tried again on a drive with terabytes of available space, starting from scratch, from a regular user. I'm getting the same strange error.

I have been able to do the mapping part with STAR entirely separately from RSEM. . .maybe for now I should use another tool for getting expression estimates, like Cufflinks.

I hate to admit defeat, so if you do think of any other possibilities I'd love to hear it! Thanks again for your help.

ADD REPLYlink written 3.1 years ago by emyers0
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