Hello, I want to calculate the Tumor mutational burden of my responders and non responders to treatment. I have many question regarding this matter:

1. Should I calculate a TMB for responders and another TMB for non responders or only a global TMB for all samples?

2. samtools mpileup , bcftools norm and bcftools view have to be applied to all bams together or to each bam of each sample? Right now I am doing it for all bams at the same time to get one vcf. Then, I would annotate the vcf file and count the number of snps that are non synonymous, and divide by region size to get the TMB.

3. I am doing this with RNA-Seq data for orientative purpose and practice, I know it is not the ideal situation. But, what is the size of the coding region of the targeted territory by which I have to divide my mutation number in RNA-Seq? Is it the length of the reads ( 100b) or something like 3099750718 bp (got it from a coverage reference file when mapping), or 30 (length used when TMB is calculated for whole exon sequencing)?

Thank you so much for your help.