Mutation discovery in the mouse using genetically guided array capture and resequencing
Mutation discovery in the mouse using genetically guided array capture and resequencing
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DOI：
10.1007/s00335-009-9200-y
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发表时间：
2009-07-01
期刊：
影响因子：
2.5
通讯作者：
Reinholdt, Laura
中科院分区：
文献类型：
--
作者：
D'Ascenzo, Mark;Meacham, Carl;Reinholdt, Laura
正向遗传学（表型驱动的方法）仍然是小鼠等位基因变异的主要来源。不幸的是，可观察到的表型和致病基因型之间的差距限制了自发和诱导小鼠突变体的广泛使用。作为传统定位克隆和突变检测方法的替代方案，序列捕获和下一代测序技术可用于快速测序基因组的子集。这些技术在小鼠突变检测工作中的应用有可能通过消除对高分辨率遗传图谱、远程PCR和单个PCR扩增物测序的需要来显著减少突变鉴定所需的时间和资源。作为原理的证明，我们使用基于阵列的序列捕获和焦磷酸测序来对来自五个非互补Kit突变体（一个已知等位基因和四个未知等位基因）中的每一个的经典定义的Kit基因座（类似于200 kb）的等位基因系列进行测序，并且已经成功地鉴定和验证了每个等位基因的非同义编码突变。这些数据代表了这些新技术可用于有效发现致病突变的第一个文件和验证。重要的是，这些数据还为在实验室小鼠中开展大规模突变检测工作提供了具体的方法学基础。
Forward genetics (phenotype-driven approaches) remain the primary source for allelic variants in the mouse. Unfortunately, the gap between observable phenotype and causative genotype limits the widespread use of spontaneous and induced mouse mutants. As alternatives to traditional positional cloning and mutation detection approaches, sequence capture and next-generation sequencing technologies can be used to rapidly sequence subsets of the genome. Application of these technologies to mutation detection efforts in the mouse has the potential to significantly reduce the time and resources required for mutation identification by abrogating the need for high-resolution genetic mapping, long-range PCR, and sequencing of individual PCR amplimers. As proof of principle, we used array-based sequence capture and pyrosequencing to sequence an allelic series from the classically defined Kit locus (similar to 200 kb) from each of five noncomplementing Kit mutants (one known allele and four unknown alleles) and have successfully identified and validated a nonsynonymous coding mutation for each allele. These data represent the first documentation and validation that these new technologies can be used to efficiently discover causative mutations. Importantly, these data also provide a specific methodological foundation for the development of large-scale mutation detection efforts in the laboratory mouse.