Accurate, long-read sequencing with droplet-microfluidic barcoding

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，由于阅读长度和准确性的限制，许多重要问题仍然是这一强大方法的难题。例如，人类转录组中95%的基因被认为是交替剪接的，平均每个基因有7个剪接接头。然而，用目前的测序仪获得的短读数不能跨越多个结点，因此不能完全表征这种变异。在细菌和病毒进化的研究以及对癌症中肿瘤特异性染色体重排的编目中，准确的长阅读也是一项关键的使能技术。对于这些需要解决的重要生物学问题，需要新的方法来提供更准确、更长阅读长度的测序。我们的研究目标是开发一项突破性技术，通过在基于液滴的微流体中实现单分子条形码来显著提高NGS平台的准确性和读取长度。每个几千碱基的长分子将在液滴微反应器中分离，扩增、碎片化，并用液滴和分子独有的序列进行条形码编码。使用基于液滴的微流体，我们将每秒对数千个分子进行条形码编码--这些技术可以形成、分解、注入和孵化液滴的速度。这将使我们能够在几分钟内对数百万个分子进行条形码编码，远远超过其他微流体系统的可能，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。目的1：开发微流控硬件以分离、扩增、片段DNA并对其进行条形码测序目标2：开发用于DNA重建的生物信息学软件；验证方法的影响：我们的技术将短读深度测序的过量深度转化为高精度的长读序。这一核心能力将产生许多影响：1)它将通过增加准确性和读取长度来极大地简化基因组组装，使目前无法访问的基因组部分能够被测序。2)它将允许更大比例的读物映射到支架上，从而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)它将允许在异构体水平上对转录本中的剪接变异进行完整的询问，从而允许对转录本进行多重覆盖的完整测序，而不考虑剪接结构。4)它将通过增加序列准确性和读取长度，实现准确的从头组装，从而实现对癌症中染色体重排的高置信度识别。5)它将允许对每种微生物的“热点”区域进行单独测序，从而能够研究持续感染人群中的细菌和病毒的超进化。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，由于我们的技术显著增加了测序平台的读取长度和准确性，而且测序已经对生物科学产生了革命性的影响，我们预计将在基础和临床研究领域产生广泛和持续的影响。

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.