High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing.

ABSTRACT: Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any a

SUBMITTER: Gleeson D 

PROVIDER: S-EPMC8205115 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature