Mattox Austin K, Wang Yuxuan, Springer Simeon, Cohen Joshua D, Yegnasubramanian Srinivasan, Nelson William G, Kinzler Kenneth W, Vogelstein Bert, Papadopoulos Nickolas
Ludwig Center, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.
Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD 21128.
Proc Natl Acad Sci U S A. 2017 May 2;114(18):4733-4738. doi: 10.1073/pnas.1701382114. Epub 2017 Apr 17.
The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.
识别临床样本中低频存在的突变是精准医学的重要组成部分。下一代测序分子条形码技术的发展极大地提高了通过大规模平行测序检测此类突变的灵敏度。然而，特异性的进一步提高对于各种应用将是有用的。我们在此描述了一种技术（BiSeqS），它可以使测序特异性比分子条形码技术提高至少两个数量级以上，并且可以应用于任何大规模平行测序仪器。BiSeqS采用亚硫酸氢盐处理来区分分子条形码DNA的两条链；其特异性源于需要在两条链中都鉴定出相同的突变。由于无需文库制备，该技术允许非常有效地使用模板DNA以及序列读数，这些读数几乎都局限于感兴趣的扩增子。这种效率对于临床样本（如血浆）至关重要，因为血浆中通常只有极少量的DNA。我们在此表明，BiSeqS可用于评估颠换以及小的插入或缺失，并且能够可靠地在超过10,000个野生型分子中检测到一个突变。
