Validation and Advanced Development of Duplex Sequencing

描述(申请人提供)：下一代DNA测序(NGS)将给生物学和医学带来革命性的变化。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，这项技术在癌症检测和研究领域全面实施的一个主要障碍是它的高 错误率。我们已经建立了一种利用双链DNA固有的互补性的方法。作为对两条DNA链进行测序的结果，我们可以识别真正的突变，即存在于两条链中相同位置的突变。使用这项技术，我们可以忽略由于PCR扩增引起的替换，因为它们只存在于两条链中的一条中。此外，我们还可以消除由于破坏DNA模板而导致的大多数测序错误。因此，我们建立了一种比用于下一代DNA测序的标准方法准确1000倍的方法。本应用程序的目标是验证和进一步发展双链测序方法。将进行实验，以优化双链序列的回收，并探索该方法的异常敏感性。为了评估双重DNA测序的实用性，我们将使用其他方法准确地分析难以测序的人类基因组片段。我们还将解决这样一个问题，即在化疗开始之前，使细胞对化疗药物产生抗药性的突变是否在人类癌症中存在。

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.