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    • 3. 发明申请
    • SAFE SEQUENCING SYSTEM
    • 安全排序系统
    • WO2012142213A2
    • 2012-10-18
    • PCT/US2012/033207
    • 2012-04-12
    • THE JOHNS HOPKINS UNIVERSITYVOGELSTEIN, BertKINZLER, Kenneth W.PAPADOPOULOS, NickolasKINDE, Isaac
    • VOGELSTEIN, BertKINZLER, Kenneth W.PAPADOPOULOS, NickolasKINDE, Isaac
    • C12Q1/68C12N15/11
    • C12Q1/6874C12Q1/6806C12Q1/6869C12Q1/6876C12Q2563/179C12Q2600/158C12Q2535/122C12Q2565/514
    • The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though 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. One example of this approach, called "Safe-SeqS" for (Safe-Sequencing System) includes (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 truly mutant ("super-mutants") 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.
    • 存在于DNA模板的一小部分中的突变的鉴定对于在几个生物医学研究领域的进展是必不可少的。 尽管大规模并行测序仪器原则上非常适合于此任务,但是这些仪器中的错误率通常太高,无法确定罕见的变体。 我们在这里描述了一种可以显着增加大规模并行测序仪器对此目的的灵敏度的方法。 这种方法的一个例子是(Safe-Sequencing System),称为“Safe-SeqS”,包括(i)将唯一标识符(UID)分配给每个模板分子; (ii)每个唯一标记的模板分子的扩增以产生UID家族; 和(iii)扩增产物的冗余测序。 具有相同UID的PCR片段是真正的突变体(“超突变体”),如果其中95%含有相同的突变。 我们说明了这种方法用于确定聚合酶的保真度,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流行率的效用。