Detection of ultra-rare mutations by next-generation sequencing
Detection of ultra-rare mutations by next-generation sequencing
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DOI：
10.1073/pnas.1208715109
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发表时间：
2012-09-04
影响因子：
11.1
通讯作者：
Loeb, Lawrence A.
中科院分区：
文献类型：
--
作者：
Schmitt, Michael W.;Kennedy, Scott R.;Loeb, Lawrence A.
下一代DNA测序有望彻底改变临床医学和基础研究。然而，虽然这项技术有能力在一次实验中产生数千亿个DNA核苷酸序列，但类似于1%的错误率会导致数亿个测序错误。在某些应用中，这些分散的错误是可以容忍的，但当“深度测序”基因异质混合物时，例如肿瘤或混合微生物种群，这些错误就变得非常成问题。为了克服测序精度的限制，我们开发了一种称为双工测序的方法。这种方法通过对DNA双链中的每一条进行独立标记和测序，大大减少了错误。由于两条链是互补的，真正的突变是在两条链的同一位置发现的。相比之下，PCR或测序错误只会导致一条链发生突变，因此可以忽略为技术错误。我们确定双工测序的理论背景错误率小于每十亿核苷酸测序一个人为突变。此外，我们确定检测突变存在于双链DNA的两条链中的一条可以用来识别DNA损伤的位置。我们应用该方法直接评估人类细胞线粒体DNA随机突变的频率和模式。
Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of similar to 1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.