Overview
The purpose of this protocol is to take the sequencing data that was retrieved from eurofins and aligning the sequences to get a consensus sequence and then aligning that consensus sequence with the wildtype to confirm the knockout and seeing if you could predict a frameshift.
Protocol
- On benchling.com, create a new alignment (“+” → “DNA Sequence” → “New Alignment”)
- Upload the forward and reverse sequence files that you received from eurofins. The files that I recommend uploading are the .ab1 format files. These files have sufficient data needed in order to make an accurate alignment.
- Export the consensus sequence as a FASTA file and save it. This is done to back up your consensus sequence.
- Ensure that your file is saved as “[Insert CRISPR combination and replicate] MM### Consensus sequence.” Note that automatically, this is all saved as “Untitled sequence” and it is difficult to rename the file on the same page.
- Once you have all your sample consensus sequences, it is time to compare your consensus sequences to your wildtype sequences. Before doing so, note the reading frame of translation that happens in your wildtype. Double and triple-check that it matches the online resources!
- Now create a new alignment with the WT WTF4 from the internet, your WT consensus sequence, and all the sequences that you sequences. Translate them. When done, export the pdf and save it. As of this point in time, I do not know how to save them so you can access this page again, so screenshot and save to show your framshifts and deletions!