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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(申请人提供)：DNA测序目前正处于颠覆性的技术变革之中，454、Illumina和Solid为我们提供了巨大的产能增加和每个碱基成本的大幅降低。大规模并行技术每周提供几个英镑的序列作为短片段或读取。直到最近才被认为不切实际的测序新应用得以实现：个人基因组测序，对数百种独特生物的包含几个的“汤”的“元基因组”分析，以及对复杂生物的新基因组的从头测序。无论排序是如何进行的，如果要使读取有用，就必须通过计算将其组合起来。鉴于新仪器的读取长度和读取质量限制以及产生的大量数据，计算组装问题正变得至关重要，计算基础设施和人员的成本超过了试剂和仪器相关成本。此外，目前组装的结果远不理想；例如，由于高比例的重复，大部分人类基因组仍然不可见。我们建议开发一种新的用于DNA制备的下一代测序仪的“前端”--“读云方法”，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容易处理。我们提出了一种分层测序的方法，不需要任何细菌克隆。我们将通过在微流体设备上处理单个DNA分子来实现这一点，这些DNA分子以高冗余度跨基因组拼接。我们将设计、制作原型和彻底测试技术，以(1)将基因组DNA剪切成尺寸分布窄的200-KBP片段；(2)在一个多孔的凝胶微容器中随机扩增分离的每个200-KBP DNA，该微容器将在微型设备中的dsDNA分子周围形成；(3)将微囊化的DNA消化成大小可调的小片段；(4)用12聚体寡核苷酸对每个200-KBP DNA的后代进行条形码编码，以确定每个读取的片段都与特定的200-KBP DNA相关。将制造一个平面微流控装置，允许一个独特的条形码序列钝端连接到两个DNA末端。条形码DNA被汇集在一起，完成了下一代测序。结果是一个高度可还原的数据集。该方法和算法具有普适性，适用于下一代平台。PI(Batzoglou，Barron，Shaqfeh，Quake)将合作制定一种有效的微流控设备分层测序方法。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数量级，同时使组装的基因组更加完整和准确。这一步的关键是使用微流控技术将基因组DNA细分为200kbp的片段，然后相互分离地扩增并唯一标记，形成一个高度可还原的数据集，用于基因组组装。