We have the RNAseq data of oral cancer(OSCC) tumor samples without matched normal. The filtered reads were of high base quality and more than 95% of base matching was considered for mutation calling. We have found several mutations in these reads (with reference to hg38). For a particular position, we have 2% of reads having mutations. For others, we have less than 2% of the reads showing mutations. On what basis do we validate the functionality of the mutation? Is there a specific range (number of reads having the mutation) which implies that the mutation has a consequence on the function.
It is true that we cannot ascertain the protein function by mutations in RNAseq. However, we should know which mutation to consider for experimental validation. This selection of the mutation must be based on some criteria, one of them that we want to consider, is its frequency. So is there a range which can tell us valid mutations and help in ruling out transcription/sequencing errors?