Accessible high-throughput single-cell genome sequencing

可获得的高通量单细胞基因组测序

基本信息

批准号：
10612457
负责人：
Andrew Adey
金额：
$ 37.3万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-21 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612457
关键词：
ATAC-seq Acoustics Area Atlases Bar Codes Basic Science Benchmarking Biological Assay Cancer Biology Catalogs Cell Line Cell Nucleus Cells Chemistry Chromium Clinical Collaborations Communities Complex Computer software Cost Effectiveness Analysis Cost Savings Custom DNA Sequence Rearrangement Data Detection Evolution Genes Genetic Genome Genomics High-Throughput Nucleotide Sequencing Human Hybrids In Situ Libraries Liquid substance Metastatic breast cancer Methodology Methods Microfluidics Mus Nucleosomes Pancreatic Ductal Adenocarcinoma Point Mutation Population Primary Brain Neoplasms Property Protocols documentation Publishing Reagent Reproducibility Research Resistance Sampling Single Nucleotide Polymorphism Solid Neoplasm Structure Technology Therapeutic Validation analysis pipeline anticancer research cost cost effective density design exome exome sequencing experimental study gene panel genome sequencing genomic profiles improved in situ sequencing indexing instrument instrumentation interest nano novel single cell sequencing therapy resistant tool tumor tumor initiation tumor progression variant detection whole genome

项目摘要

PROJECT SUMMARY Initiation and evolution of most solid tumors involve complex genomic rearrangements in addition to point mutations – all of which can contribute to cancer progression and to the evolution of resistance to therapeutics. Single-cell whole genome sequencing (scWGS) has proven invaluable for the identification of tumor subpopulations and in advancing our understanding of clonal dynamics and tumor evolution. Despite the value of scWGS, there is a dearth of commercially-available options that provide enough cell throughput (i.e. power) and/or genomic coverage to fully catalogue and characterize clonal populations within a tumor sample. This can be particularly problematic when rare, possible therapy-resistant, subpopulations are present within a tumor that may elude detection using existing methodologies. Here we will solve the accessibility problem by developing technologies that leverage widely-available instrumentation and off-the-shelf reagents. We previously described proof-of-principle technologies to construct scWGS libraries contained within the nucleus (in situ) of each cell that are then carried through cell barcoding using our custom workflow. We will extend, develop, and adapt these technologies to provide three workflows that will meet the needs of the cancer research community by providing readily-accessible order-of-magnitude improvements to cell throughput, cell coverage and cost savings for scWGS analysis over current options. The first is focused on high cell throughput using widely available instrumentation; the second leverages our high- content chemistry to produce high-coverage single cell genomic profiles with reduced reagents and improved throughput; and finally, the third approach implements target capture to target exonic sequence or other regions of interest to reduce sequencing costs. Each of these is targeted to meet the needs of specific areas of cancer research, from basic science to clinical, and share the theme of accessibility.

项目概要大多数实体瘤的发生和进化除了点突变外还涉及复杂的基因组重排。突变——所有这些都可能导致癌症进展和治疗耐药性的进化。单细胞全基因组测序（scWGS）已被证明对于肿瘤的识别具有无价的价值尽管有价值，但它有助于促进我们对克隆动力学和肿瘤进化的理解。对于 scWGS，缺乏提供足够细胞吞吐量（即功率）的商业可用选项和/或基因组覆盖率，以全面编目和表征肿瘤样本中的克隆群体。当一个群体内存在罕见的、可能对治疗有抵抗力的亚群时，问题可能会特别严重。使用现有方法可能无法检测到的肿瘤。在这里，我们将通过开发利用广泛可用的技术来解决可访问性问题我们之前描述了构建原理验证技术。 scWGS 文库包含在每个细胞的细胞核（原位）内，然后通过细胞条形码进行携带使用我们的自定义工作流程，我们将扩展、开发和调整这些技术以提供三种工作流程。这将通过提供易于访问的数量级来满足癌症研究界的需求与当前选项相比，scWGS 分析的细胞吞吐量、细胞覆盖范围和成本得到了改善。第一个重点是使用广泛使用的仪器来实现高细胞通量；第二个利用我们的高通量内容化学，以减少试剂和改进的方法产生高覆盖率的单细胞基因组图谱最后，第三种方法实现对目标外显子序列或其他的目标捕获。其中每一个都是针对特定领域的需求来降低测序成本的。癌症研究，从基础科学到临床，共享可及性的主题。