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体细胞改变的超灵敏检测对于影响患者护理具有巨大潜力，例如用于早期发现、建立诊断、改善预后、指导治疗和监测复发。然而，当前的技术不太适合对频率非常低的体细胞突变进行稳健检测。大规模并行测序代表了一种前进的道路，但其检测非常罕见事件的灵敏度从根本上受到测序错误率的限制。我们的目标是开发一种新的实验范式来克服这一限制。在我们的方法中，样品中存在的目标序列的每个副本在多重捕获反应的第一个循环期间用独特的条形码序列进行分子标记。扩增后，目标扩增子及其相应的条形码进行大规模并行测序。在分析过程中，条形码用于关联具有共同来源的序列读取。通过过采样，条形码相关的读数相互纠错，为每个祖分子产生独立的单倍体共识，即“分子计数”。此外，通常衍生的读数的崩溃本质上纠正了扩增过程中任何等位基因特异性偏差，使得突变等位基因频率的估计可以伴随着精确的置信界限。在我们的第一个目标中，我们将开发实验方法和分析工具，通过分子计数，在 100,000 个未突变拷贝的背景下，以低至 1 个突变拷贝的频率，可靠地检测目标体细胞突变。在我们的第二个目标中，我们将开发三种超灵敏的多重分子计数测定法，专门针对临床相关的癌症突变或基因组，并严格评估它们的重现性。用于对罕见体细胞事件进行超灵敏、多重检测的强大、经济有效、普遍适用的工具的可用性将是将癌症遗传学的发现转化为临床环境的革命性的一步。