Accurate, long-read sequencing with droplet-microfluidic barcoding

使用液滴微流控条形码进行准确的长读长测序

• 批准号: 8491721
• 负责人: Adam R. Abate
• 金额: $ 23.56万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8491721
• 关键词: Address; Area; Bioinformatics; Biological; Biological Sciences; Buffers; Cataloging; Catalogs; Cells; Chromosomal Rearrangement; Clinical; Computer software; DNA; DNA Sequence; Data; Dependence; Drops; Embryo; Evolution; Gene Expression Profile; Generations; Genes; Genetic; Genome; Genomics; Goals; Hot Spot; Human; Incubated; Individual; Infection; Injection of therapeutic agent; Investigation; Kidney; Length; Malignant Neoplasms; Maps; Measures; Methods; Microbe; Microfluidics; Organism; Output; Persons; Positioning Attribute; Protein Isoforms; RNA Sequences; RNA Splicing; Reaction; Reading; Reagent; Research; Software Tools; Spottings; Structure; System; Systems Biology; Techniques; Technology; Transcript; Variant; Viral; Work; base; cost; deep sequencing; infectious disease treatment; microbial; next generation sequencing; operation; prototype; public health relevance; reconstruction; scaffold; single molecule; tumor

DESCRIPTION (provided by applicant): Next Generation Sequencing (NGS) has fundamentally changed the way we study living systems; however, many important questions remain intractable to this powerful approach, due to limitations in read length and accuracy. For example, 95% of all genes in the human transcriptome are thought to be alternatively spliced, with an average of 7 splice junctions per gene. The short reads obtained with current sequencers, however, are unable to span multiple junctions and thus cannot fully characterize this variation. In the study of bacterial and viral evolution and in cataloging tumor-specific chromosomal rearrangements in cancer, accurate long reads are also a key enabling technology. For important biological questions like these to be addressed, new methods are needed that provide more accurate, longer read-length sequencing. The objective of our research is to develop a breakthrough technology to significantly enhance the accuracy and read length of NGS platforms using single-molecule barcoding implemented in droplet-based microfluidics. Each multi-kilobase long molecule will be isolated in a droplet microreactor, amplified, fragmented, and barcoded with a sequence unique to the drop and, thus, to the molecule. Using droplet-based microfluidics, we will barcode thousands of molecules per second-the rate at which these techniques can form, split, inject, and incubate drops. This will allow us to barcode millions of molecules in minutes, far exceeding what is possible with other microfluidic systems and the scale needed to utilize the full capacity of NGS platforms and maximally exploit the barcoding concept.  Aim 1: Develop microfluidic hardware to isolate, amplify, fragment, and barcode DNA for sequencing  Aim 2: Develop bioinformatics software for DNA reconstruction; validate the approach Impact: Our technology converts the excess depth of short-read deep sequencing into highly accurate long reads. This core capability will have numerous impacts: 1) It will greatly simplify genome assembly by increasing accuracy and read length, allowing currently "inaccessible" portions of the genome to be sequenced. 2) It will allow a greater fraction of reads to be mapped to scaffolds, reducing the depth of sequencing required to obtain a sequence of a desired coverage, thereby reducing the cost of sequencing. 3) It will allow complete interrogation of splice variation in transcriptomes at the isoform level y allowing transcripts to be sequenced in their entirety with multifold coverage, irrespective of splice structure. 4) It will allow high-confidence identification of chromosomal rearrangements in cancer by increasing sequence accuracy and read length enabling accurate de novo assembly. 5) It will allow investigation of bacterial and viral hyper- evolution in persons with persistent infection by allowing "hot spot" regions to be sequenced for each microbe individually. Thus, our work will have impacts in genomics, systems biology, cancer, and microbial evolution. Indeed, since our technology markedly increases the read length and accuracy of sequencing platforms, and since sequencing has already had a transformative impact on the biological sciences, we anticipate broad and sustained impacts in basic and clinical areas of research.

描述（由申请人提供）：下一代测序（NGS）从根本上改变了我们研究生命系统的方式；然而，由于读取长度和准确性的限制，许多重要的问题仍然无法用这种强大的方法解决。例如，人类转录组中 95% 的基因被认为是选择性剪接的，每个基因平均有 7 个剪接点。然而，用当前测序仪获得的短读长无法跨越多个连接点，因此无法完全表征这种变异。在细菌和病毒进化的研究以及癌症中肿瘤特异性染色体重排的研究中，准确的长读数也是一项关键的支持技术。为了解决诸如此类的重要生物学问题，需要新的方法来提供更准确、更长读长的测序。我们研究的目标是开发一种突破性技术，使用基于液滴的微流控技术实现单分子条形码，显着提高 NGS 平台的准确性和读取长度。每个数千碱基长的分子将在液滴微反应器中被分离、扩增、片段化，并使用液滴乃至分子特有的序列进行条形码标记。使用基于液滴的微流体技术，我们将每秒对数千个分子进行条形码——这些技术可以形成、分裂、注射和孵化液滴的速率。这将使我们能够在几分钟内对数百万个分子进行条形码，远远超过其他微流体系统的可能性以及利用 NGS 平台的全部容量和最大限度地利用条形码概念所需的规模。目标 1：开发微流体硬件来分离、扩增、片段化和条形码 DNA 以进行测序目标 2：开发用于 DNA 重建的生物信息学软件；验证该方法影响：我们的技术将短读长深度测序的多余深度转换为高度准确的长读长。这一核心功能将产生诸多影响：1）它将通过提高准确性和读取长度来极大地简化基因组组装，从而允许对基因组中当前“无法访问”的部分进行测序。 2) 它将允许更大比例的读数映射到支架，减少获得所需覆盖范围的序列所需的测序深度，从而降低测序成本。 3) 它将允许在亚型水平 y 上对转录组中的剪接变异进行完全询问，从而允许转录本以多重覆盖进行完整测序，而不管剪接结构如何。 4) 通过提高序列准确性和读取长度，实现准确的从头组装，可以高置信度地鉴定癌症中的染色体重排。 5) 它将允许对每种微生物的“热点”区域进行单独测序，从而研究持续感染者的细菌和病毒超进化。因此，我们的工作将对基因组学、系统生物学、癌症和微生物进化产生影响。事实上，由于我们的技术显着增加了测序平台的读取长度和准确性，并且由于测序已经对生物科学产生了变革性影响，因此我们预计会对基础和临床研究领域产生广泛和持续的影响。