CRISPRessoPooled with --alternate_alleles

[GreenSeaBug]

Hello,

When using CRISPRessoPooled with the --alternate_alleles parameter, what goes in the AMPLICON_NAME and AMPLICON_SEQUENCE columns of the description file? Should all alleles go in those columns or just one? Does the region_name column of the alternate alleles file correspond to the AMPLICON_NAME of the description file and, if so, must they be exactly the same?

And what happens if the gRNA is mismatched on non-target alleles? Will this affect the analysis in a bad way?

Thank you

[kclem]

The --alternate_alleles function is used when two (or more) alleles are amplified by a primer pair. This could be because a SNP exists in your amplicon, and you want to quantify editing on each allele separately.

The --alternate_alleles parameter accepts a separate file as a parameter. The names in 'region_name' must exactly match with the -f name. Your primary amplicon should be present in the -f file, and other amplicons can be provided in the --alternate_alleles file.

I find it simplest to create a file with potential alternate alleles by running CRISPRessoPooled once with --compile_postrun_references. You can then change the names and delete amplicon sequences in this file as appropriate, and pass this file in as --alternate_alleles.

If the gRNA is mismatched on non-target alleles, CRISPResso will align the reference and alternate alleles, and use the corresponding positions of the reference allele in the alternate alleles.

[GreenSeaBug]

Thank you for clarifying that. All makes sense.

Just one thing: I found in my testing is that all alleles must be provided in the alternate_alleles file. If you only provide the other alleles (i.e. all alleles except the primary target allele), then it doesn't appear to make any output files for the primary target allele, even though the primary target allele is in the -f file.