How to use RSEM to quantify genes and transcript expression from STAR outpute file (Aligned.toTranscriptome.out.bam)
Have already run normal Star alignment and obtained Aligned.toTranscriptome.out.bam file which should be compatible with RSEM tool. However, when I use RSEM to quantify genes and transcript expression I do not any result. I think I do not get what one part rsem-calculate-expression.
If this is the command:
rsem-calculate-expression --bam --no-bam-output -p 10 \
--paired-end --forward-prob 0 \
RNASEQ_data/star_GM12878_rep1/Aligned.toTranscriptome.out.bam # (star output file)
GENOME_data/rsem/GRCh38 \ #(rsem reference genome)
RNASEQ_data/rsem_GM12878_rep1/rsem \ # What is this suppose to be? I think this is what I am missing)
>& RNASEQ_data/rsem_GM12878_rep1/rsem.log
Any help please.
Erick
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Ram
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otieno43 •
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otieno43 •
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The "what is this supposed to be" part is the output prefix. Are you using
>&when you mean to use&>? In any case, use>rsem.out 2>rsem.errand show us the contents of both files.Hello Ram,
I have tried what you suggested, I still got an error. The error reads, "-tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!"
Another error is found in rsem.err file reads, "The SAM/BAM file declares more reference sequences (25014) than RSEM knows (20519)!"
This seems strange because I used the same reference genome (fasta) and annotation (gft) file to generate rsem reference genome and star index. I used stand alone star to build index same to rsem ref genome.
Can you share your STAR command as well? See the STAR command line used by RSEM here: https://github.com/deweylab/RSEM/blob/master/rsem-calculate-expression#L457
You will need to dig deeper to understand why your STAR generated BAM was not compatible with RSEM. I also think that your STAR and RSEM are not being run against the same prepared index or at least not with compatible parameters.