Ultrasensitive identification and precise quantitation of low frequency somatic m

低频体细胞的超灵敏识别和精确定量

基本信息

批准号：
8517045
负责人：
Jay Ashok Shendure
金额：
$ 17.43万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-16 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8517045
关键词：
Algorithms Alleles Biological Assay Cell Line Clinical Code Consensus Consensus Sequence DNA DNA Sequence Detection Development Diagnosis Diagnostic Early Diagnosis Event Frequencies Gene Frequency Genes Genetic Genetic Heterogeneity Genotype Goals Haploidy Malignant Neoplasms Medicine Methods Molecular Monitor Monitoring for Recurrence Mutate Mutation Mutation Detection Nucleotides Oncogenes Patient Care Patient Monitoring Protocols documentation Public Health Reaction Reading Reagent Reproducibility Sampling Screening for cancer Secondary to Somatic Mutation Surveys Technology Translations Tumor Suppressor Genes analytical tool base cancer genetics cancer genome cancer genomics cancer recurrence clinical application clinically relevant cost effective cost effectiveness digital genome sequencing human disease multiplex detection mutant outcome forecast progenitor research study single molecule tool tumor

项目摘要

DESCRIPTION (provided by applicant): The ultrasensitive detection of clinically relevant somatic alterations in cancer genomes has great potential for impacting patient care, e.g. for early detection, establishing diagnoses, refining prognoses, guiding treatment, and monitoring recurrence. However, current technologies are poorly suited to the robust detection of somatic mutations present at very low frequencies. Massively parallel sequencing represents one path forward, but its sensitivity to detect very rare events is fundamentally constrained by the sequencing error rate. Our goal is to develop a new experimental paradigm that overcomes this limitation. In our approach, each copy of a target sequence that is present in a sample is molecularly tagged during the first cycle of a multiplex capture reaction with a unique barcode sequence. After amplification, target amplicons and their corresponding barcodes are subjected to massively parallel sequencing. During analysis, the barcodes are used to associate sequence reads sharing a common origin. Through oversampling, barcode-associated reads error-correct one another to yield an independent haploid consensus for each progenitor molecule, i.e. "molecular counting". Furthermore, the collapsing of commonly derived reads inherently corrects for any allele-specific bias during amplification, such that estimates of mutant allele frequency can be accompanied by precise confidence bounds. In our first aim, we will develop experimental methods and analytical tools that enable the robust detection of targeted somatic mutations via molecular counting to frequencies as low as 1 mutated copy in a background of 100,000 unmutated copies. In our second aim, we will develop three ultrasensitive, multiplex molecular counting assays that are specifically targeted at panels of clinically relevant cancer mutations or genes, and rigorously evaluate these for reproducibility. The availability of robust, cost-effective, generically applicable tools for the ultrasensitive, multiplex detection of rare somatic events will be a transformative step forward for the translation of discoveries in cancer genetics to a clinical setting.

描述（由申请人提供）：癌症基因组中临床相关的体细胞改变的超敏感检测具有影响患者护理的巨大潜力，例如为了早期检测，建立诊断，精炼预后，指导治疗和监测复发。但是，当前的技术非常适合在非常低的频率下对体细胞突变的可靠检测。大规模平行的测序代表一个前进的路径，但其对检测非常罕见事件的敏感性在根本上受到测序误差率的限制。我们的目标是开发一种克服这一限制的新实验范式。在我们的方法中，在使用独特的条形码序列的多重捕获反应的第一个循环中，在样品中存在的目标序列的每个副本都在分子上进行标记。放大后，目标扩增子及其相应的条形码进行大规模平行测序。在分析过程中，条形码用于将共享共同起源的读数相关联。通过过度采样，条形码相关读取误差 - 校正校正，以对每个祖细胞分子，即“分子计数”产生独立的单倍体共识。此外，在放大过程中，常见衍生的读数的崩溃固有地纠正了任何等位基因特异性偏置，因此突变等位基因频率的估计可以伴随精确的置信度范围。在我们的第一个目标中，我们将开发实验方法和分析工具，以在100,000份未经分配的副本的背景下，通过分子计数到低至1个突变拷贝的频率来实现靶向体细胞突变。在我们的第二个目标中，我们将开发三种超敏感的多重分子计数测定法，这些测定法专门针对临床相关的癌症突变或基因，并严格评估这些测定法以进行可重复性。可用性，具有成本效益，一般适用的工具用于对罕见的体细胞事件的超敏，多重检测将是将癌症遗传学发现中的发现转化为临床环境的变革一步。