RSEM Error - rsem-calculate-expression

Question

[anoo]

Hello,

I tried to calculate the expression using RSEM rsem-calculate-expression and came across few errors. The below given are the errors:

Cannot open /.../.../../RSEM/.grp! It may not exist.

/....sorted.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

I used the below given command:

rsem-calculate-expression -p 8 \
    --paired-end \
    /data/mmliver_1.fq \
    /data/mmliver_2.fq \
    /ref/mouse_125 \
    mmliver_paired_end_quals

Ref: http://deweylab.biostat.wisc.edu/rsem/rsem-calculate-expression.html

Any help regarding this is appreciated.

Thanks

[adam.faranda]

There are a couple of things I can think of. Check your reference directory ../ref/mouse_125; was this file generated when you built the reference?

Also -- which aligner are you using, Bowtie/TopHat or STAR ?

[anoo]

Hello Adam, thanks for your response.

The organism I am working on is Rat. When i tried building the index using the following command, it worked.

rsem-prepare-reference --gtf /home/user/data/genome/Rattus_6/Rattus_norvegicus.Rnor_6.0.98.gtf \ --bowtie2 --bowtie2-path /home/user/data/bowtie2-2.3.4.1-linux-x86_64/bowtie2 \ /home/user/data/genome/Rattus_6/Rattus_norvegicus.Rnor_6.0.dna_sm.toplevel.fa \ /home/user/data/RSEM_UPDATED/Rat

The below given files were generated, 1. Rat.grp 2. Rat.ti 3. Rat.chrlist 4. Rat.transcripts.fa 5. Rat.seq 6. Rat.idx.fa 7. Rat.n2g.idx.fa

Any help is appreciated!

Answer 1

[svp]

There is an error in the command used. You should use the index name correctly. In the following command mouse_ref is reference index name which should used in rsem-calculate-expression also.

For preparing the reference

rsem-prepare-reference --gtf ref/Mus_musculus.GRCm38.99.gtf --bowtie2 --bowtie2-path /home/ngs/bin/bowtie2 ref/Mus_musculus.GRCm38.dna.primary_assembly.fa ref/mouse_ref

For calculating the expression

rsem-calculate-expression --paired-end -p 8 --bowtie2 --estimate-rspd --append-names -output-genome-bam fastq/sample_R1.fastq fastq/sample_R2.fastq ref/mouse_ref sample_name