用于检测与髓系恶性肿瘤相关体细胞突变的多重PCR测序的设计与应用

Design and Application of Multiplex PCR Seq for the Detection of Somatic Mutations Associated with Myeloid Malignancies.

作者信息

Park Naomi, Vassiliou George

机构信息

DNA Pipelines Research and Development, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.

Haematological Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, CB10 1SA, UK.

出版信息

Methods Mol Biol. 2017;1633:87-99. doi: 10.1007/978-1-4939-7142-8_6.

DOI:10.1007/978-1-4939-7142-8_6

PMID:28735482

Abstract

Targeted sequencing, in which only a selected set of genomic loci are sequenced, enables a much higher coverage of each target than what is obtained using whole genome or exome sequencing. Multiplex PCR offers a simple and affordable technique for specific capture of target regions and can be easily adapted to generate next-generation sequencing (NGS)-ready amplicons. Here we describe a multiplex PCR (MxPCR) approach for capturing 13 leukemia-associated mutation hotspots followed by MiSeq sequencing that enables robust detection of mutations with a variant allele fraction (VAF) as low as 0.8% (0.008) in blood DNA.

摘要

靶向测序仅对选定的一组基因组位点进行测序，与全基因组或外显子组测序相比，能使每个靶点获得更高的覆盖度。多重PCR为特异性捕获靶区域提供了一种简单且经济的技术，并且可以很容易地用于生成适用于新一代测序（NGS）的扩增子。在此，我们描述了一种多重PCR（MxPCR）方法，用于捕获13个白血病相关突变热点，随后进行MiSeq测序，该方法能够在血液DNA中可靠地检测到变异等位基因分数（VAF）低至0.8%（0.008）的突变。