RSEM - sorting and error with calculate expression

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Rashmi Kallath

Oct 30, 2022, 8:02:48 AM10/30/22
to RSEM Users
Dear RSEM group,
   I am new to rnaseq. I am going through the tutorial
I am following star-rsem pipeline, where I aligned the using star with the following options-
    --outSAMunmapped Within \
     --outFilterType BySJout \
     --outSAMattributes NH HI AS NM MD \
     --outSAMtype BAM Unsorted \
     --quantMode TranscriptomeSAM GeneCounts \
     --quantTranscriptomeBan IndelSoftclipSingleend

Then I ran RSEM, suing the Aligned.toTranscriptome.out.bam file, a sample script used for RSEM quatification is given below.


rsem-calculate-expression -p 8 --paired-end --alignments \
--estimate-rspd \
--calc-ci \
--ci-memory 32000 \
--seed 123456 \
--no-bam-output \
--strandedness reverse \
$bam db/refanno/ensembl_rsem $out > $log

So, my question is -Do I need to sort the bam files before running RSEM quantification?
I also learned from Harvard edx tutorial that RSEM needs and exon only file for successive running.

So next question is do I need to make exon only gtf file in/for the above steps I see that RSEM alignment requires exon only gtf.

Thank you for your time and consideration.

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