So I am trying to process some RNA-Seq data. I have built an annotated reference genome and mapped my reads against it using STAR, outputting the results into sorted BAM files. The issue I am facing right now is quantifying the read numbers, with subsequent calculation of differential expression(DE). After reviewing a lot of literature and posts on here, I decided to go with RSEM for calculating expression values and Limma for determining DE.
The issue I am having is that I can't figure out how to get RSEM to quantify my expression values from the sorted BAM files. Full disclaimer, as it stands I am just giving it the annotated reference genome I built using STAR. Does it really want me to rebuild a reference genome just for quantifying counts? Because while that is not the end of the world, that does not seem like a good use of resources for such a relatively simplistic task.
rsem-calculate-expression -p 8 --time --bam --paired-end /home/peter/Documents/Files/Output/s_A/Aligned.sortedByCoord.out.bam /home/peter/Documents/Files/STAR_Reference s_A
Thank you for the all the help! If you have a different recommendation on a pipeline feel free to share it. I have two(2) replicates per sample, in case that influences anything. I am so close to finishing this project, so it is frustrating to be held up near the finish line.