A method for confirming CRISPR knockouts of genes from sanger sequencing data. I have not used this method for many typical use cases, so I make no guarantees about its accuracy.
Inputs are:
- a gene name (either ensembl or hgnc), only tested for human
- an .ab1 sanger trace file
Two folders are used:
- ABIfiles
- contains all .ab1 files of interest
- gene names are associated with .ab1 files as the second field, seperated by underscore (e.g. xxxx_YFG_yyy.ab1) will analyze a gene YFG, while output will be tagged with xxx_YFK_yyy
- Output
- for storing results
General approach is:
- call homozygous and heterozygous region of sanger sequence
- identify indels of the first allele
- subtract first allele from degenerate call
- identify indels of the second allele
Output will be:
- a chromatogram indicating called homozygous and heterozygous regions
- a QC file indicating regions of the sanger sequence that are relatively high quality
- an alignment - calling predicted indels for both alleles