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，许多重要问题仍然难以解决。例如，人类转录组中95%的基因被认为是可变剪接的，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段不能跨越多个接合点，因此不能完全表征这种变化。在细菌和病毒进化的研究中，以及在对癌症中的肿瘤特异性染色体重排进行编目时，准确的长读段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，更长的读取长度测序。我们研究的目标是开发一种突破性技术，使用在基于液滴的微流体中实现的单分子条形码来显著提高NGS平台的准确性和读取长度。每个多聚腺苷酸酶长分子将在液滴微反应器中分离，扩增，片段化，并用液滴和分子特有的序列进行条形码化。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化这些技术可以形成，分裂，注射和孵育液滴的速度。这将使我们能够在几分钟内对数百万个分子进行条形码化，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：开发微流控硬件以分离、扩增、片段化和条形码化DNA用于测序  目标二：开发用于DNA重建的生物信息学软件;验证方法影响：我们的技术将短读段深度测序的过度深度转化为高度准确的长读段。这种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大大简化基因组组装，允许对基因组的当前“不可访问”部分进行测序。2)它将允许更大部分的读数被映射到支架，减少获得所需覆盖度的序列所需的测序深度，从而降低测序成本。3)它将允许在同种型水平y下完全询问转录组中的剪接变异，从而允许转录物以多倍覆盖率整体测序，而不管剪接结构如何。4)它将允许通过增加序列准确性和读取长度来高置信度地鉴定癌症中的染色体重排，从而实现准确的从头组装。5)它将允许调查细菌和病毒的超进化的人与持续感染的允许“热点”地区的测序为每一个微生物单独。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，我们预计将在基础和临床研究领域产生广泛而持续的影响。

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.