1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2].
1] Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands. [2] Cergentis B.V., Utrecht, the Netherlands. [3].
Nat Biotechnol. 2014 Oct;32(10):1019-25. doi: 10.1038/nbt.2959. Epub 2014 Aug 17.
Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.
尽管靶向基因测序和全基因组分析技术取得了进展，但仍然难以稳健地检测到感兴趣基因及其周围所有的遗传变异，包括结构变异，且具有等位基因特异性。在这里，我们提出了靶向基因扩增（TLA），这是一种基于物理邻近序列交联来选择性扩增和测序整个基因的策略。我们表明，与其他靶向重测序方法不同，TLA 无需详细的基因座信息即可工作，因为只需一个或几个引物对即可对数十到数百千碱基的周围 DNA 进行测序。这使得能够稳健地检测到临床相关基因（包括 BRCA1 和 BRCA2）中的单核苷酸变异、结构变异和基因融合，并实现单倍型分析。我们还表明，TLA 也可用于揭示整合转基因和病毒的插入位点和序列。因此，当需要全面或等位基因特异性遗传信息时，TLA 有望成为遗传研究和诊断中的一种有用方法。
