Hi All,

I'm new to RNAseq, so I'm confused with a couple of terms.

As I understand, raw counts are generated by sequence aligning. Is it right?

And then, expected counts are generated by running RSEM with raw counts, right? 

This is to correct sequence reads mapped to more than one spot or alternatively spliced variants. Is this right?

Then, expected counts are not normalized by length of genes. Did I understand correct?

I guess, this is pretty simple things, but I'm still a little confused when someone asks me..-.-v

Please correct, if I have misunderstanding.

Thanks in advance.
MH

Raw counts are what you get when you align to the genome and count how many reads (generally discounting multimappers) align to each gene.

What you refer to as "expected counts" are better called "estimated counts". This involves using expectation-maximization (or similar) to try to apportion multimapping reads in the most likely manner. This can also be used to get isoform-level metrics. Aside from RSEM, salmon and kallisto can also do this.

Estimated counts are typically presented in TPMs and/or estimated counts. TPMs are length normalized, while estimated counts are not.