A Universal Front End to Improve Assembly Outcomes for Next-Gen Sequencing and Re

通用前端可改善下一代测序和重新组装的结果

基本信息

批准号：
7945357
负责人：
Annelise Emily Barron
金额：
$ 73.96万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2012-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): DNA sequencing is currently in the midst of disruptive technological shifts, with 454, Illumina, and Solid providing us with enormous throughput increases and large reductions in cost per base. Massively parallel technologies deliver a few Gbp of sequence per week as short fragments, or reads. New applications of sequencing only recently considered impractical are enabled: personal genome sequencing, "metagenomics" analysis of 'soups' containing several, to hundreds of unique organisms, and finally, de novo sequencing of novel genomes of complex organisms. No matter how the sequencing is done, reads must be assembled computationally, if they are to be useful. Given the read length and read quality limitations of new instruments and the massive volume of data generated, the computational assembly problem is becoming critical, with the cost of computational infrastructure and personnel exceeding reagent and instrument-related costs. Moreover, the results of assembly are currently far from ideal; for example, much of the human genome remains invisible due to high percentage of repeats. We propose to develop a new "front end" to next-gen sequencers for DNA preparation, the "Read-Cloud Method", which can reduce computational cost of genome assembly by 2-3 orders of magnitude, produce more complete and accurate genomes, and make metagenomics tractable. We propose a hierarchical sequencing approach, without any need for bacterial cloning. We will achieve this by handling single DNA molecules, tiled across the genome with high redundancy, on microfluidic devices. We will design, prototype, and thoroughly test technology to (i) shear genomic DNA into 200- kbp fragments with narrow size distributions; (ii) randomly amplify each individual, 200-kbp DNA in isolation, within a porous gel microcontainer that will be formed around the dsDNA molecule within a microdevice; (iii) digest micro-encapsulated DNA into small fragments, of tunable size; (iv) bar-code the progeny of each 200-kbp DNA with a 12mer oligonucleotide, to identify each read as associated with a particular 200-kbp DNA. A planar microfluidic device will be fabricated to allow one unique bar- code sequence to be blunt-end-ligated to both DNA termini. Bar-coded DNA is pooled, and next-gen sequencing is done. The results are a highly reducible data set. The method and algorithm are applicable universally, to next-generation platforms. The PIs (Batzoglou, Barron, Shaqfeh, Quake) will collaborate to make an efficient approach to hierarchical sequencing in microfluidic devices. PUBLIC HEALTH RELEVANCE: Project Narrative Gene sequencing is important to medicine. Our DNA sequencing method has the potential for reducing computational cost by orders of magnitude while making the assembled genomes significantly more complete and accurate. The key to this step is using microfluidic handling technologies to subdivide genomic DNA into 200kbp fragments, which are then amplified in isolation from each other and uniquely-labeled to form a highly reducible dataset for genomic assembly.

描述（由申请人提供）：DNA 测序目前正处于颠覆性技术变革之中，454、Illumina 和 Solid 为我们提供了巨大的通量增加和每个碱基成本的大幅降低。大规模并行技术每周以短片段或读取形式提供几 Gbp 的序列。直到最近才被认为不切实际的测序新应用才得以实现：个人基因组测序、对包含数个到数百个独特生物体的“汤”进行“宏基因组学”分析，以及最后对复杂生物体的新基因组进行从头测序。无论测序如何完成，如果要发挥作用，读数就必须通过计算进行组装。考虑到新仪器的读取长度和读取质量限制以及生成的大量数据，计算组装问题变得至关重要，计算基础设施和人员的成本超过了试剂和仪器相关的成本。而且，目前的组装效果还很不理想；例如，由于重复率很高，人类基因组的大部分内容仍然不可见。我们建议为下一代测序仪开发用于DNA制备的新“前端”，即“读取云方法”，它可以将基因组组装的计算成本降低2-3个数量级，产生更完整和准确的基因组，并使宏基因组学变得易于处理。我们提出了一种分层测序方法，无需任何细菌克隆。我们将通过在微流体装置上处理单个 DNA 分子来实现这一目标，这些分子以高冗余度分布在基因组中。我们将设计、原型化并彻底测试技术，以 (i) 将基因组 DNA 剪切成具有窄尺寸分布的 200-kbp 片段； (ii) 在多孔凝胶微容器内随机扩增每个单独的 200-kbp DNA，该微容器将在微装置内的 dsDNA 分子周围形成； (iii) 将微封装的 DNA 消化成大小可调的小片段； (iv) 用 12mer 寡核苷酸对每个 200-kbp DNA 的后代进行条形码编码，以识别与特定 200-kbp DNA 相关的每个读数。将制造一种平面微流体装置，以允许将一个独特的条形码序列平端连接到两个 DNA 末端。汇集条形码 DNA，并完成下一代测序。结果是一个高度可简化的数据集。该方法和算法普遍适用于下一代平台。 PI（Batzoglou、Barron、Shaqfeh、Quake）将合作开发一种在微流体设备中进行分层测序的有效方法。公共卫生相关性：项目叙述基因测序对医学很重要。我们的 DNA 测序方法有可能将计算成本降低几个数量级，同时使组装的基因组更加完整和准确。此步骤的关键是使用微流体处理技术将基因组 DNA 细分为 200kbp 的片段，然后将这些片段彼此隔离地扩增并进行独特标记，以形成用于基因组组装的高度可简化的数据集。