High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing.
CRISPR
Genotyping
Mouse
Mutant
NGS
QC
Journal
Methods (San Diego, Calif.)
ISSN: 1095-9130
Titre abrégé: Methods
Pays: United States
ID NLM: 9426302
Informations de publication
Date de publication:
07 2021
07 2021
Historique:
received:
25
07
2020
revised:
25
09
2020
accepted:
18
10
2020
pubmed:
24
10
2020
medline:
5
1
2022
entrez:
23
10
2020
Statut:
ppublish
Résumé
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 assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.
Identifiants
pubmed: 33096238
pii: S1046-2023(20)30226-7
doi: 10.1016/j.ymeth.2020.10.011
pmc: PMC8205115
pii:
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
78-86Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Wellcome Trust
ID : WT206194
Pays : United Kingdom
Informations de copyright
Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.
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