High-throughput technology that enables sequencing depth for colorectal CA

描述（由申请人提供）：随着第二代测序（SGS）的出现，第一次真正有可能在短时间内以相对低的成本对个体癌症患者基因组的大部分进行测序，从而检测可以具有预后或预测价值的突变，或者可以作为特定患者肿瘤随访的指纹。 然而，在提供患者肿瘤样品中突变指纹的真正可靠鉴定方面仍然存在缺失环节，因为具有异质性、基质污染或体液中的肿瘤中的临床相关突变由于与野生型等位基因的混合而存在问题，并且仍然可能被遗漏。然而，在几种情况下，识别这些低水平突变指纹的临床意义是重要的，因为这些突变通常会产生耐药性，提供预后和预测信息，并对治疗随访有用。不幸的是，当涉及到检测低水平突变时，新的测序技术“失去了动力”，对于SGS来说，目前要么是深度测序，要么是高通量能力，但不能两者兼而有之。因此，SGS与临床实践的整合不能得到有效利用。 我们开发了在较低变性温度下的共扩增（COLD-PCR），这是一种新形式的PCR，其优先扩增来自野生型和含突变序列的混合物的少数等位基因，而不管突变位于何处，从而在PCR期间提供突变序列的强烈富集。我们建议建立并行COLD-PCR，以在通过SGA（Illumina）筛选突变体序列之前富集突变体序列，从而实现“深度”测序，同时还保留高通量能力。为了实现大规模并行COLD-PCR，在PCR之前将DNA和PCR试剂分配到单个纳米液滴（RainDance”）中的微流体设备将被改造为同时在数百万个单独的纳米反应中进行COLD-PCR。这种新的技术组合将用于识别20名结肠癌患者肿瘤中的突变指纹，包括低水平突变，然后在放化疗过程中跟踪血浆中的这些指纹，为治疗反应提供分子替代物。新的变革性新兴技术的拟议用途也适用于其他类型的癌症，并提供了一种解决方案，弥合了技术上的差距，使SGS能够应用于临床肿瘤学实践。因此，与公共卫生的相关性很高。

DESCRIPTION (provided by applicant): With the advent of second generation sequencing (SGS), for the first time there is a truly viable possibility of sequencing a substantial portion of an individual cancer patient's genome within a short time period and at relatively low cost, thus detecting mutations that can have prognostic or predictive value, or can serve as a fingerprint for tumor follow-up in a particular patient. However, there is still a missing link in providing a truly reliable identification of mutation fingerprints in patient tumor samples, as clinically-relevant mutations in tumors with heterogeneity, stromal contamination or in bodily fluids is problematic due to admixture with wild type alleles and can still be missed. And yet, the clinical significance of identifying these low-level mutation fingerprints is major in several situations as frequently these are the mutations that confer resistance, offer prognostic and predictive information and that would be useful for treatment follow-up. Unfortunately the new sequencing technologies 'lose steam' when it comes to detecting low-level mutations, and for SGS currently it's either deep sequencing or high-throughput capability, but not both. Thus integration of SGS with clinical practice cannot be effectively exploited. We developed Co-amplification at Lower Denaturation temperature (COLD-PCR), a new form of PCR that amplifies preferentially the minority alleles from mixtures of wild type and mutation-containing sequences, irrespective of where the mutation lies, providing a strong enrichment of the mutated sequences during PCR. We propose to establish massively-parallel COLD-PCR to enrich mutant sequences prior to their screening via SGA (Illumina), thus enabling 'deep' sequencing while also retaining high-throughput capability. To enable massively-parallel COLD-PCR, a micro-fluidic device that dispenses DNA and PCR reagents within individual nano-droplets (RainDance") prior to PCR will be adapted to perform COLD-PCR in millions of separate nano- reactions simultaneously. The novel combination of technologies will be used to identify mutational fingerprints in tumors from 20 colon cancer patients, including low-level mutations, and then follow these fingerprints in plasma in the course of radio-chemo-therapy, to provide a molecular surrogate to therapy response. The proposed use of Novel Transformative Emerging Technologies is also applicable to other types of cancer and provides a solution bridging the gap in technology and enabling SGS to be applied to clinical oncology practice. Therefore relevance to Public Health is high.