RNAseq

[Shuksi]

Hi,

I ran following command:

/path/to/rsem/rsem-calculate-expression -p 2 --output-genome-bam -star --star-path /path/to/STAR_index_files --paired-end path/to/SRR925687_1.fastq path/to/SRR925687_2.fastq human.refseq.gff.gtf path/to/RNA.test

I got following error msg:
dFilesIn SRR925687_1.fastq SRR925687_2.fastq" failed! Plase check if you provide correct parameters/options for the pipeline!

STAR index folder consists of following files:

drwxrwxr-x 2 stuser stuser        4096 Apr 17 18:16 ./
drwxrwxr-x 7 stuser stuser        4096 Apr 18 11:12 ../
-rw-r--r-- 1 root   root          1194 Apr  9 18:37 chrLength.txt
-rw-r--r-- 1 root   root          3532 Apr  9 18:37 chrNameLength.txt
-rw-r--r-- 1 root   root          2338 Apr  9 18:37 chrName.txt
-rw-r--r-- 1 root   root          2108 Apr  9 18:37 chrStart.txt
-rw-r--r-- 1 root   root      51816787 Apr  9 19:41 exonGeTrInfo.tab
-rw-r--r-- 1 root   root      23213627 Apr  9 19:41 exonInfo.tab
-rw-r--r-- 1 root   root        380243 Apr  9 19:41 geneInfo.tab
-rw-r--r-- 1 root   root    3202741093 Apr  9 21:34 Genome
-rw-r--r-- 1 root   root           858 Apr  9 21:25 genomeParameters.txt
-rw-r--r-- 1 root   root   12693443564 Apr  9 22:47 SA
-rw-r--r-- 1 root   root    1565873619 Apr  9 22:54 SAindex
-rw-r--r-- 1 root   root       9414412 Apr  9 19:41 sjdbInfo.txt
-rw-r--r-- 1 root   root      10724637 Apr  9 19:41 sjdbList.fromGTF.out.tab
-rw-r--r-- 1 root   root      10723973 Apr  9 19:41 sjdbList.out.tab
-rw-r--r-- 1 root   root       7215534 Apr  9 19:41 transcriptInfo.tab

I have provided correct path to the files generated by STAR indexing. Dont understand the problem.

[Alexander Dobin]

Hi Shuksi,

if you are using the -star option from RSEM, RSEM calls STAR on its own, and I cannot tell what goes wrong - this is a question better asked to RSEM authors. However, you can run STAR mapping yourself with --quantMode TranscriptomeSAM option, and then input the Aligned.toTranscriptome.bam file into RSEM (first you need to prepare reference for RSEM):

$ ./rsem-prepare-reference --gtf annot.gtf  genome.fa RSEMprepRef

$ ./rsem-calculate-expression --bam -p 2 --output-genome-bam --paired-end Aligned.toTranscriptome.out.bam /path/to/RSEMprepRef

Cheers

Alex