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通过降低试剂成本、增加读取长度和大幅降低输入DNA要求，有可能显著降低批量方法的测序成本。当从测序技术中检索到的信息揭示了表观遗传修饰和无法扩增的受损DNA碱基时，后一个优势至关重要。Pacific Biosciences开发并商业化了一种单分子真实的时间（SMRT）测序仪器，该仪器利用零模波导（ZMW）阵列或穿过不透明金属膜的纳米级孔作为基底。ZMW阵列通过采用单一DNA聚合酶进行合成测序来实现多重DNA测序，并使用4色荧光显微镜和染料标记的核苷酸实现检测。然而，目前两个主要挑战限制了该技术的成本降低和能力。第一个是功能性ZMW的低产量， 一个DNA聚合酶分子，如测序所需。第二个是依赖于聚合酶/模板复合物扩散到ZMW中，这导致次优加载到阵列中并增加DNA输入要求。尽管已证明SMRT测序鉴定DNA碱基修饰的能力，但所需的高DNA输入排除了从天然DNA收集表观遗传序列数据，这对于理解DNA修饰在衰老和各种疾病中的作用很重要。在该提案中，我们提出了一种创新的方法，用于降低SMRT DNA测序的成本，以及使基因组和表观基因组测序从皮克水平的DNA，而无需事先扩增。我们的方法是用含有单个纳米孔的薄膜代替每个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，极大地提高了产率和受控的化学计量; 2）要测序到ZMW中的输入DNA的加载速率的数量级增强。为了实现我们的目标，我们组建了一个多学科的研究团队，他们拥有尖端制造技术，纳米科学和分子生物学的专业知识。我们将我们的目标分为目标，包括ZMW-孔装置的制造，ZMW-孔阵列中单个DNA聚合酶的锚定，以及通过将活性DNA加载到ZMW-孔装置中来减少输入DNA需求。最后，我们将展示使用我们的ZMW孔阵列测序的控制DNA库，以及直接表观基因组测序从皮克水平的人脑线粒体DNA。如果成功，我们的方法将大大降低DNA测序的成本，并为大规模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.