Validation and Advanced Development of Duplex Sequencing

描述（由申请人提供）：下一代DNA测序（NGS）有能力彻底改变生物学和医学。确定人类基因组中30亿个核苷酸的序列不再是一项艰巨的任务。然而，在癌症检测和研究领域全面实施该技术的主要障碍是其高风险。 错误率我们已经建立了一种利用双链DNA中固有的互补性的方法。作为对DNA两条链测序的结果，我们可以将真正的突变鉴定为存在于两条链中相同位置的突变。使用这种技术，我们可以忽略由于PCR扩增引起的取代，因为它们将仅存在于两条链中的一条中。此外，我们可以消除大多数由于DNA模板损坏而导致的测序错误。因此，我们已经建立了一种比下一代DNA测序中使用的标准方法准确1000倍的方法。本申请的目标是验证和进一步开发双链体测序方法。将进行实验以优化双链体序列的回收率并探索该方法的异常灵敏度。为了评估双链体DNA测序的实用性，我们将分析难以测序的人类基因组片段，这些片段使用其他方法难以准确测序。我们还将讨论在开始化疗之前，人类癌症中是否存在使细胞对化疗药物产生耐药性的突变。

DESCRIPTION (provided by applicant): Next generation DNA sequencing (NGS) has the power to revolutionize biology and medicine. Determining the sequence of the 3 billion nucleotides in the human genome is no longer a daunting task. However, a major impediment to the full implementation of this technology in the area of cancer detection and research is its high error rate. We have established a method that takes advantage of the complementarity inherent in double-stranded DNA. As a result of sequencing both strands of DNA we can identify true mutations as those present at the same position in both strands. Using this technology we can disregard substitutions due to PCR- amplification since they would only be present in one of the two strands. In addition, we can eliminate most sequencing errors resulting from damage to DNA templates. As a result we have establish a method that is 1000-fold more accurate than standard methods used in next generation DNA sequencing The goal of this application is to validate and further develop the method of Duplex Sequencing. Experiments will be carried out to optimize the recovery of duplex sequences and to probe the exceptional sensitivity of the methodology. To evaluate the utility of duplex DNA sequencing we will analyze hard to sequence segments of the human genome that have been resistant to sequence with accuracy using other approaches. We will also address the question of whether mutations rendering cells resistant to chemotherapeutic agents are preexistent in human cancers prior to the initiation of chemotherapy.