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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Detection and quantification of rare mutations with massively parallel sequencing
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Detection and quantification of rare mutations with massively parallel sequencing

机译：大规模并行测序检测和量化罕见突变

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摘要

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of bio-medical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if >95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.
机译：一小部分DNA模板中存在的突变的鉴定对于生物医学研究的多个领域的进展至关重要。尽管大规模并行测序仪器原则上非常适合此任务，但此类仪器中的错误率通常过高，无法可靠地鉴定稀有变体。为此，我们在这里描述了一种可以大大提高大规模并行测序仪器灵敏度的方法。这种方法的关键，称为安全测序系统（Safe-SeqS），是（i）为每个模板分子分配唯一标识符（UID），（ii）扩增每个唯一标记的模板分子以创建UID家族，以及（iii）扩增产物的冗余测序。具有相同UID的PCR片段仅在> 95％包含相同突变的情况下才视为突变体（“超突变体”）。我们说明了这种方法在确定聚合酶保真度，在体外合成的寡核苷酸的准确性以及正常细胞的核和线粒体基因组中突变的普遍性方面的实用性。

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  • 作者

    Isaac Kinde; Jian Wu; Nick Papadopoulos; Kenneth W. Kinzler; Bert Vogelstein;

  • 作者单位

    The Luewig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical. Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231;

    The Luewig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical. Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231;

    The Luewig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical. Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231;

    The Luewig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical. Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231;

    The Luewig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical. Institute, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21231;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    diagnostics; early diagnosis; biomarkers; genetics; cancer;

    机译：诊断;早期诊断;生物标志物遗传学癌症;

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