Picogram-level DNA sequencing using Nanopores and Zero-Mode Waveguides

描述(由申请人提供)：基于单分子阵列的基因组测序方法有可能通过降低试剂成本、增加读取长度和大幅减少输入DNA要求而显著降低测序成本。当从测序技术中检索到的信息揭示了表观遗传修饰和受损的DNA碱基时，后一种优势是至关重要的，而这些DNA碱基是无法放大的。太平洋生物科学公司开发了一种单分子实时(SMRT)测序仪并将其商业化，该测序仪使用零模波导(ZMW)阵列作为衬底，或通过不透明金属膜的纳米级孔。ZMW阵列通过使用单个DNA聚合酶进行合成测序来实现多路DNA测序，并使用四色荧光显微镜和染料标记的核苷酸来实现检测。然而，目前有两大挑战限制了这项技术的成本降低和能力。第一个是功能性ZMW的低成品率，这些ZMW完全包含 测序所需的一个DNA聚合酶分子。第二种是依赖于聚合酶/模板复合体扩散到ZMW中，这导致次优的负载到阵列中，并增加了DNA输入需求。尽管SMRT测序证实了识别DNA碱基修饰的能力，但需要高DNA输入排除了从天然DNA中收集表观遗传序列数据的可能性，这对于理解DNA修饰在衰老和各种疾病中的作用非常重要。在这项提案中，我们提出了一种创新的方法来降低SMRT DNA测序的成本，并使基因组和表观基因组测序能够从皮卡水平的DNA中进行，而不需要事先进行扩增。我们的方法是用含有单个纳米孔的薄膜取代每个ZMW的玻璃底部。纳米孔将用于两个主要目的：1)在ZMW阵列中精确定位单个DNA聚合酶，大大提高产量和受控的化学计量比；2)将输入DNA的加载速度提高数量级，这些DNA将被测序到ZMW中。为了实现我们的目标，我们组建了一支拥有尖端制造技术、纳米科学和分子生物学专业知识的多学科研究团队。我们将我们的目标分为几个目标，包括ZMW孔器件的制造，单个DNA聚合酶在ZMW孔阵列中的锚定，以及通过将活性DNA负载到ZMW孔器件中来减少输入DNA的需求。最后，我们将演示使用我们的ZMW孔阵列对对照DNA文库进行测序，以及从皮卡水平的人脑线粒体DNA直接进行表观基因组测序。如果我们的方法成功，将极大地降低DNA测序的成本，并为获得海量的高通量基因组和表观基因组序列数据铺平道路。

DESCRIPTION (provided by applicant): Single-molecule array-based approaches to genome sequencing have the potential to deliver a dramatic reduction in sequencing costs over bulk methods by reducing reagent costs, increasing read lengths, and substantially decreasing the input DNA requirements. The latter advantage is critical when information retrieved from the sequencing technique sheds light on epigenetic modifications and damaged DNA bases, which cannot be amplified. Pacific Biosciences has developed and commercialized a single-molecule real- time (SMRT) sequencing instrument that utilizes as substrates an array of zero-mode waveguides (ZMWs), or nanoscale holes through an opaque metallic film. The ZMW array enables multiplexed DNA sequencing by employing single DNA polymerases for sequencing by synthesis, and detection is achieved using 4-color fluorescence microscopy and dye-labeled nucleotides. However, two major challenges currently limit the cost reduction and the capabilities of this technology. The first is the low yield of functional ZMWs that contain exactly one DNA polymerase molecule, as required for sequencing. The second is the reliance on diffusion of polymerase/template complexes into ZMWs, which results in sub-optimal loading into the array and increases the DNA input requirement. Despite the demonstrated ability of SMRT sequencing to identify DNA base modifications, high DNA inputs required preclude the gathering of epigenetic sequence data from native DNA, important for understanding the role of DNA modifications in aging and various diseases. In this proposal we present an innovative approach for reducing the cost of SMRT DNA sequencing, as well as enabling genomic and epigenomic sequencing from picogram levels of DNA without prior amplification. Our approach is to replace the glass bottom of each ZMW with a thin membrane that contains a single nanopore. The nanopore will serve two major purposes: 1) Precise positioning of individual DNA polymerases in ZMW arrays with greatly improved yields and controlled stoichiometry; 2) Orders of magnitude enhancement of the loading rates of input DNA that is to be sequenced into the ZMWs. To achieve our goals we have assembled a multidisciplinary team of investigators with expertise in cutting edge fabrication techniques, nanoscale science, and molecular biology. We divide our goals into aims that include the fabrication of the ZMW-pore devices, anchoring of individual DNA polymerases in ZMW- pore arrays, and the reduction of input DNA requirements by active DNA loading into the ZMW-pore devices. Finally, we will demonstrate using our ZMW-pore arrays sequencing of control DNA libraries, as well as direct epigenomic sequencing from picogram levels of human brain mitochondrial DNA. Our approach, if successful, will dramatically reduce the cost of DNA sequencing and pave way to massive high-throughput genomic and epigenomic sequence data.