High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing
| العنوان: | High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing |
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| المؤلفون: | Shaheen Akhtar, Joanna Bottomley, Edward Ryder, Daniel M Barrett, Michaela Bruntraeger, Sanger Mouse Genetics, James Bussell, Jonathan Burvill, Radka Platte, Debarati Sethi, Diane Gleeson |
| المصدر: | Methods (San Diego, Calif.) |
| بيانات النشر: | Elsevier BV, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | FASTQ format, Genotyping, Genotype, Genotyping Techniques, Mouse, Computational biology, Biology, Polymerase Chain Reaction, Genome, Article, General Biochemistry, Genetics and Molecular Biology, DNA sequencing, Mice, 03 medical and health sciences, PCR, polymerase chain reaction, Animals, CRISPR, CRISPR, clustered regularly interspaced short palindromic repeat, hom, homozygous, Allele, Molecular Biology, Alleles, QC, 030304 developmental biology, 0303 health sciences, fungi, 030302 biochemistry & molecular biology, sgRNA, single guide RNA, Mutant, food and beverages, High-Throughput Nucleotide Sequencing, NGS, next generation sequencing, het, heterozygous, Amplicon, WT, wild-type, Mice, Mutant Strains, QC, quality control, NHEJ, non-homologous end joining, NGS |
| الوصف: | Highlights • Next generation sequencing is a scalable solution to genotyping mutant mice. • Ratios of wild type and mutant sequence counts are used to call the genotype. • Hundreds of samples can be multiplexed into one sequencing experiment. • Amplification of high-homology genes can be easily filtered out during analysis. 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. |
| تدمد: | 1046-2023 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ef79b367f901763da5e833510b51bcdcTest https://doi.org/10.1016/j.ymeth.2020.10.011Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....ef79b367f901763da5e833510b51bcdc |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10462023 |
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