I'm trying to find the best approach to fill my goal. I would like to measure the expression of specific set of kmers - 10 aa long peptide sequences across different samples. I have RNA-Seq data for each sample. I'm planing to use alignment-independent methods such as
Salmon. My first question is:
1) How can I measure the expression of specific Kmer when this Kmer is not unique in the genome? Let's say that it can be coded by different transcripts in the same gene or in different genes sharing the sequence... How can I measure the global abundance of that Kmer in the sample?
For other hand...:
2) Which method should I choose for measuring the expression? I have read that
TPM has overcome
FPKM. But both
FPKM measure the relative abundance, maybe I should consider measuring absolute abundance with
TMM. Is there any specific case when relative is preferred over absolute measurement?
Thank you in advance,