Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR ‐based single‐molecule sequencing approach
Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR ‐based single‐molecule sequencing approach
复制标题
通过改进的基于乳液 PCR 的单分子测序方法对二倍体个体的单倍型进行直接测序
DOI：
10.1111/1755-0998.12034
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发表时间：
2013
影响因子：
7.7
通讯作者：
Lee, Carol Eunmi
中科院分区：
文献类型：
--
作者：
Metzger, Brian Patrick;Gelembiuk, Gregory William;Lee, Carol Eunmi
虽然标准的DNA测序方法很容易产生基因型序列数据，但单倍型信息通常对群体遗传分析更有用。然而，获得单个单倍型序列可能是昂贵和耗时的，并且有时需要受到偏差和错误影响的统计重建方法。最近在大规模基因组研究中确定个体染色体序列方面取得了进展，但很少有选择以具有成本效益的方式从大量高度多态性个体中获得此信息。作为解决方案，我们开发了一种简单的基于PCR的方法，使用标准实验室设备从单个DNA链中获得序列信息。该方法采用油包水乳液将PCR混合物分离到数千个单独的微反应器中。在这些小囊泡内的PCR导致仅从单个起始DNA模板分子扩增，从而产生单个单倍型。我们通过在PCR中加入SYBR绿色I和熔融琼脂糖溶液，改进了以前的方法，使得单独扩增的DNA分子易于鉴定和分离。我们证明了使用这种方法对一个高度多态性的河口人口的桡足类Eurytemora affinis，目前的分子和计算方法的单倍型测定已经不足。
While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepodEurytemora affinisfor which current molecular and computational methods for haplotype determination have been inadequate.