Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping
Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping
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DOI：
10.1038/nbt.2959
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发表时间：
2014-10-01
影响因子：
46.9
通讯作者：
de Laat, Wouter
中科院分区：
文献类型：
--
作者：
de Vree, Paula J. P.;de Wit, Elzo;de Laat, Wouter
尽管靶向基因测序和全基因组分析技术取得了进展，但以特定等位基因的方式对感兴趣基因内部和周围的所有遗传变异(包括结构变异)进行强有力的检测仍然是一个挑战。在这里，我们提出了靶点扩增(TLA)，这是一种基于物理上邻近序列的交联性选择性地扩增和测序整个基因的策略。我们表明，与其他靶向重新测序方法不同，TLA在没有详细的先前基因座信息的情况下工作，因为一个或几个引物对足以对周围数十到数百个碱基的DNA进行测序。这使得能够可靠地检测包括BRCA1和BRCA2在内的临床相关基因中的单核苷酸变异、结构变异和基因融合，并使单倍型成为可能。我们发现TLA也可以用来发现整合的转基因和病毒的插入位点和序列。因此，当需要全面的或特定等位基因的遗传信息时，TLA有望成为遗传学研究和诊断中的一种有用的方法。
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.