Targeted single molecule mutation detection with massively parallel sequencing.
Targeted single molecule mutation detection with massively parallel sequencing.
复制标题
靶向单分子突变检测，并进行了大量平行测序。
DOI：
10.1093/nar/gkv915
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发表时间：
2016-02-18
影响因子：
14.9
通讯作者：
Bielas JH
中科院分区：
文献类型：
--
作者：
Gregory MT;Bertout JA;Ericson NG;Taylor SD;Mukherjee R;Robins HS;Drescher CW;Bielas JH
下一代测序（NGS）技术已经改变了基因组研究，并有可能彻底改变临床医学。然而，测序仪器的背景错误率和靶向读段覆盖率的限制已经排除了通过NGS检测罕见DNA序列变体。在这里，我们描述了一种称为CypherSeq的方法，该方法结合了双链条形码纠错和基于滚环扩增（RCA）的靶点富集，大大提高了基于NGS的罕见变异检测。CypherSeq方法涉及将样品DNA连接到环状载体中，所述环状载体含有用于计算错误校正的双链条形码和用于文库制备和测序的衔接子。CypherSeq能够通过基于RCA的富集检测全基因组范围内的罕见突变以及特定靶基因内的突变。我们证明CypherSeq能够纠正文库制备和测序过程中出现的错误，以可重复地检测突变，频率低至2.4 × 10−7/碱基对，并报告酿酒酵母基因组中自发和甲磺酸乙酯诱导突变的频率和光谱。
Next-generation sequencing (NGS) technologies have transformed genomic research and have the potential to revolutionize clinical medicine. However, the background error rates of sequencing instruments and limitations in targeted read coverage have precluded the detection of rare DNA sequence variants by NGS. Here we describe a method, termed CypherSeq, which combines double-stranded barcoding error correction and rolling circle amplification (RCA)-based target enrichment to vastly improve NGS-based rare variant detection. The CypherSeq methodology involves the ligation of sample DNA into circular vectors, which contain double-stranded barcodes for computational error correction and adapters for library preparation and sequencing. CypherSeq is capable of detecting rare mutations genome-wide as well as those within specific target genes via RCA-based enrichment. We demonstrate that CypherSeq is capable of correcting errors incurred during library preparation and sequencing to reproducibly detect mutations down to a frequency of 2.4 × 10−7 per base pair, and report the frequency and spectra of spontaneous and ethyl methanesulfonate-induced mutations across the Saccharomyces cerevisiae genome.