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，由于读取长度和准确性的限制，许多重要的问题仍然难以解决。例如，人类转录组中95%的基因被认为是选择性剪接的，平均每个基因有7个剪接。然而，使用当前测序仪获得的短读数不能跨越多个连接，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癌症中肿瘤特异性染色体重排的编目中，准确的长读取也是一项关键的使能技术。为了解决这些重要的生物学问题，需要新的方法来提供更准确、更长的读长测序。我们的研究目标是开发一种突破性的技术，利用基于液滴的微流体实现的单分子条形码，显著提高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分离，扩增，片段化，并使用液滴和分子特有的序列进行条形码。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识别——这些技术可以形成、分裂、注射和培养液滴。这将使我们能够在几分钟内对数百万个分子进行条形码识别，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：开发微流控硬件来分离，扩增，片段和条形码DNA测序目标2：开发用于DNA重建的生物信息学软件；影响：我们的技术将短读深度测序的多余深度转化为高度精确的长读深度。这一核心能力将产生许多影响：1)通过提高准确性和读取长度，它将极大地简化基因组组装，使目前“难以接近”的基因组部分得以测序。2)它将允许更大比例的reads被映射到支架上，减少获得所需覆盖序列所需的测序深度，从而降低测序成本。3)无论剪接结构如何，它将允许在同工异构体水平上对转录组中的剪接变异进行完整的询问，从而允许转录本进行具有多重覆盖的完整测序。4)它将通过提高序列准确性和读取长度，从而实现精确的从头组装，从而使癌症中的染色体重排鉴定具有高可信度。5)通过对每种微生物的“热点”区域进行单独测序，可以对持续性感染患者的细菌和病毒的超进化进行调查。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，并且由于测序已经对生物科学产生了变革性的影响，我们预计在基础和临床研究领域会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.