How to quantify mRNA transcripts from RNA-Seq data?

Question

[Neu]

Hi, I am analyzing total transcriptome RNA-Seq data. I want to quantify expression of mRNA transcripts. What should be the correct approach. I have aligned the reads with reference genome (sequence file -Mus_musculus.GRCm38.dna.primary_assembly.fa and GTF file - Mus_musculus.GRCm38.95.chr.gtf) using STAR. Please guide me to do the quantification.

Thank you!

Answer 1

[Buffo]

There are some biotools to quantify gene expression or transcripts, such as ht-seq, or strintie ( from the new protocolo tuxedo). However I recommend you to read about RNAseq-analysis protocols before to continue, in order to know "what" and "why" are you doing.

Answer 2

[Friederike]

Gene expression can be quantified either by pseudo-aligners such as kallisto and salmon, which Kristoffer mentioned, or, following read alignment as you just did, using tools such as featureCounts or HTSeq-Counts.

The main difference between the pseudo-aligners and the counting tools is that the counting tools absolutely need the BAM files and there is some evidence that suggests that the results may be more accurate for complex and well-annotated transcriptomes with the pseudo-aligners (see this paper for more details).

The main difference between featureCounts and HTSeq-Counts is speed (featureCounts is way faster).

For more details on how to proceed after the quantification, see Michael Love's tutorial.

Answer 3

[Kristoffer Vitting-Seerup]

I wrote a long section about the different things to think about when quantifying RNASeq data (including tools to use) here.