Maximum efficiency sequencing using nuclease-based mutation enrichment and digital barcodes

项目摘要 低水平的肿瘤体细胞DNA突变可能对转移的发展具有深远的意义， 预后、治疗选择、随访或早期癌症检测策略。除非他们有效地 一旦检测到，这些低水平的突变可能会误导患者的管理决策或被遗漏 个性化医疗的机会。下一代测序（NGS）技术可以有效地揭示 普遍的体细胞突变，但他们'失去蒸汽'，当涉及到检测低水平的DNA突变， 具有克隆异质性的肿瘤或体液中的肿瘤，以及它们与临床实践的整合是有问题的。 对于等位基因比例约为2-5%或更低的突变，NGS会产生过多的假阳性（“噪音”）， 测序深度和阻碍基于突变谱的个性化临床决策。最近 使用随机条形码化策略对NGS进行调整以检测罕见突变可以克服噪声， 不可避免地降低了其高生产能力并增加了成本。 我们最近开发了NaMe-PrO，这是一种简单而强大的消除野生型序列的技术。 基因组DNA中的大量目标。NaME-PrO利用探针引导的核酸酶， 使WT序列不可扩增，从而允许含有突变的序列 就像克隆突变一样进行扩增和测序。该R33建议开发定量NaME- PrO（qNaME-PrO），其将NaME-PrO与分子条形码的新用途组合，以提供严格的 所有突变体序列在其富集后的原始突变丰度的计数。从而 将罕见突变转化为高丰度突变，提高检测的信心， 避免了在NGS期间重复和浪费的序列读取的需要。该方法创建 在测序之前进行大规模平行突变富集的潜力，并产生新的范例 从而罕见突变不需要深度测序来检测。R33（目标1&2）将优化 开发qNaME-PrO面板，以覆盖肿瘤中所有已知的突变热点和全长外显子 与肺癌相关的抑癌基因和癌基因。在目标3中，该方法将在一个 从接受放射-化学疗法的患者纵向收集的血浆样品的汇编。 能够从混合的临床样本中提取“大基因组靶标的突变部分”， 下游分析将转化为测序的速度，准确性和成本的重大提升， 在异质性肿瘤和体液中流行突变，并将加速NGS的临床应用 用于癌症诊断、预后和管理。因此，与公共卫生的相关性很高。

Project Summary Low-level tumor somatic DNA mutations can have profound implications for development of metastasis, prognosis, choice of treatment, follow-up or early cancer detection strategies. Unless they are effectively detected, these low-level mutations can misinform patient management decisions or become missed opportunities for personalized medicine. Next generation sequencing (NGS) technologies can effectively reveal prevalent somatic mutations, yet they 'lose steam' when it comes to detecting low-level DNA mutations in tumors with clonal heterogeneity, or in bodily fluids, and their integration with clinical practice is problematic. For mutations at allelic ratio of ~2-5% or less, NGS generates excessive false positives (‘noise’) independent of sequencing depth and hinders personalized clinical decisions based on mutational profiling. Recent adaptations of NGS to detect rare mutations using random barcoding strategies may overcome the noise but invariably diminish its high throughput capability and increase costs. We recently developed NaMe-PrO, a simple and powerful technology to eliminate wild-type sequences from large numbers of targets in genomic DNA. NaME-PrO utilizes a nuclease guided by probes to thousands of DNA targets, to render WT sequences non-amplifiable thereby allowing mutation–containing sequences to amplify and be sequenced as if they were clonal mutations. This R33 proposes to develop quantitative NaME- PrO (qNaME-PrO), which combines NaME-PrO with a novel use of molecular barcoding, to provide strict enumeration of original mutation abundance for all mutant sequences following their enrichment. Thereby converting rare mutations to high abundance mutations, boosting confidence in their detection and circumventing the need for repeated and wasteful sequence reads during NGS. The method creates the potential for massively parallel mutation enrichment prior to sequencing and engenders a new paradigm whereby rare mutations do not require deep sequencing for their detection. The R33 (Aims 1&2) will optimize and develop qNaME-PrO panels to cover all known mutation hotspots and full length exons in tumor suppressor genes and oncogenes relevant to lung cancer. In Aim 3 the method will be field-tested in a compilation of longitudinally collected plasma samples from patients undergoing radio-chemo-therapy. Being able to extract ‘the mutated portion of a large genomic target’ from a mixed clinical sample prior to downstream analysis will translate to a major boost in the speed, accuracy and cost of sequencing low- prevalence mutations in heterogeneous tumors and bodily fluids and will accelerate clinical application of NGS for cancer diagnosis, prognosis and management. Therefore relevance to Public Health is high.