High-throughput technology that enables sequencing depth for colorectal CA

描述(申请人提供)：随着第二代测序(SGS)的出现，首次有可能在较短的时间内以相对较低的成本对单个癌症患者的大部分基因组进行测序，从而检测出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，在提供患者肿瘤样本中真正可靠的突变指纹识别方面仍然缺少一个环节，因为具有异质性、间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，并且仍然可能被错过。然而，识别这些低水平突变指纹的临床意义在几种情况下是主要的，因为这些突变经常是赋予耐药性、提供预后和预测信息的突变，这将对治疗后续有用。不幸的是，新的测序技术在检测低水平突变方面失去了动力，目前对SGS来说，要么是深度测序，要么是高通量能力，但不是两者兼而有之。因此，SGS与临床实践的结合不能被有效地利用。我们开发了低变性温度共扩增(COLD-PCR)，这是一种新的PCR形式，它优先从野生型和含有突变的序列的混合物中扩增少数等位基因，无论突变位于哪里，在聚合酶链式反应过程中提供了对突变序列的强烈浓缩。我们建议建立大规模并行冷-PCR，在通过SGA(Illumina)筛选突变序列之前对其进行丰富，从而在保持高通量能力的同时实现“深度”测序。为了实现大规模并行的冷-聚合酶链式反应，在聚合酶链式反应之前在单个纳米液滴中分配DNA和聚合酶链式反应试剂的微流控装置(RainDance)将被改装成同时在数百万个不同的纳米反应中进行冷-聚合酶链式反应。这一新的技术组合将被用于识别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。拟议使用的新型变革性新兴技术也适用于其他类型的癌症，并提供了一种解决方案，缩小了技术差距，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.