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I am very new to RNAseq analysis, please correct me if i am doing something wrong. i have the TPM values of 48 samples (6 conditions each with 8 repeats) from salmon. i want to calculate the absolute value of transcripts, which is the abundance of specific RNA in the cell. Should i calculate it for each sample, or for each condition (this makes more sense to me)? But if i do the calculation for each group then i should take the average TPM values from 8 samples. how do i include the multiple testing correction while performing the calculation for average TPM? My collaborator obtained the TPM values from salmon. we had added ercc spike in RNAs during lib prep, I have the TPM for ERCC also.
Multiple testing correction has nothing to do with calculating abundances. By the way, RNA-seq is a relative measurement, it does not allow to calculate absolute values of RNA molecules in terms of concentrations. This does not really have anything to do with Bioconductor. In any case, I recommend to work with a local person experienced in this because you are currently mixing up several concepts and things could go quite wrong. Please be sure to talk to someone with experience-
Yes i understand that I have spike in RNA, the ERCC RNAs. the confusion for me is regarding taking the average of TPM values. thank you for the suggestion, I try to reach back to my collaborator.