Hi all, I'm trying to figure out how to assess the editing/point mutation efficiency of a Cas9 targeting experiment. I have amplified a (human) locus from cells that were previously targeted with a method that is predicted to produce point mutations and indels in this locus. This was then sequenced via NGS and the results are now in fastq form. If I align these files to the human genome then I assume I won't be able to assess the fraction of reads with mutations because they'll be aligned to the 'standard' genome build. Can you recommend a programme/method to extract reads from fasta files and quantify point mutations and indels? (Essentially something that will allow me to align these "non-canonical" reads to my locus of interest.) I have very briefly looked at a tool suite called Biopieces but I'm not 100% sure whether it's the optimal/most straightforward method to go about this. Any help will be appreciated, thanks!
