rsem prepare-reference error

Question

[amithasandur]

Hi there,

I am trying to run "rsem-prepare-reference" using the options below:

rsem-prepare-reference commands:

  rsem-prepare-reference --gtf path/to/gtf_file \
    --star \
    --star-path path/to/STAR \
    --star-sjdboverhang 99 \
     -p 8 \
      path/to/primary_assembly_fasta_file \
      /ref/mouse_ensembl \

But I get error below:

In the ".err" file:

Cannot open mouse_ensembl.transcripts.fa! It may not exist.

In the ".out" file:

rsem-preref /ref/mouse_ensembl.transcripts.fa 1 /ref/mouse_ensembl
"rsem-preref /ref/mouse_ensembl.transcripts.fa 1 /ref/mouse_ensembl" failed! Plase check if you provide correct parameters/options for the pipeline!

Has anyone faced the same error or know how to fix this?

Thank you,

Amitha

Answer 1

[h.mon]

The error message is pretty clear. The path to the transcripts fasta is incorrect. What is that "1" between the path to the file and the reference name?

The command line you have shown has nothing to do with the error message, as you edited files and paths.

[amithasandur]

What is the transcripts fasta here?

For my case in command line, I have just provided the mouse gtf file (on 1st line) and primary assembly fa file (on 6th line).. Not sure what "/ref/mouse_ensembl.transcripts.fa" is supposed to mean??

Is it trying to generate these files or locate them? If so which transcript files is it trying to locate?

In the ".out" file, I can see the following:

"path to primary_assembly.fa is processed!

136630 transcripts are extracted.

Extracting sequences is done!

Group File is generated!

Transcript Information File is generated!

Chromosome List File is generated!

Extracted Sequences File is generated!"

rsem-preref ref/mouse_ensembl.transcripts.fa 1 ref/mouse_ensembl

"rsem-preref ref/mouse_ensembl.transcripts.fa 1 ref/mouse_ensembl" failed! Plase check if you provide correct parameters/options for the pipeline!

So are these "group file, transcript information file, chromosome list file and extracted sequences file" be generated in the directory, if so, I do not see them.

Any clarification is much appreciated!

Thanks,

Amitha

[gokce.ouz]

Hi Amithasandur,

Were you able to solve this problem ? If so, could you share with us ?

Many thanks,

Gokce

[wanle159]

Hi Gokce

Have you solved this problem?

Thanks, Ledong

[Kai_Qi]

Hi Amithasandur, Gokce and Wanle:

I have met the same problem. In my situation, I just did mkdir ref/mouse_ensembl and ran the command. It finished successfully.

Hope it helps,

Answer 2

[Abhishek]

Hi! I had the same error. Upon calling rsem-prepare-reference --help and looking at the examples, I found the following example very helpful. Please see below:

Suppose we have mouse RNA-Seq data and want to use the UCSC mm9 version of the mouse genome. We have downloaded the UCSC Genes transcript annotations in GTF format (as mm9.gtf) using the Table Browser and the knownIsoforms.txt file for mm9 from the UCSC Downloads. We also have all chromosome files for mm9 in the directory '/data/mm9'. We want to put the generated reference files under '/ref' with name 'mouse_0'. We do not add any poly(A) tails. Please note that GTF files generated from UCSC's Table Browser do not contain isoform-gene relationship information. For the UCSC Genes annotation, this information can be obtained from the knownIsoforms.txt file. Suppose we want to build Bowtie indices and Bowtie executables are found in '/sw/bowtie'.

 rsem-prepare-reference --gtf mm9.gtf \
                        --transcript-to-gene-map knownIsoforms.txt \
                        --bowtie \
                        --bowtie-path /sw/bowtie \                  
                        /data/mm9/chr1.fa,/data/mm9/chr2.fa,...,/data/mm9/chrM.fa \
                        /ref/mouse_0

I hope this helps.