Single Molecule Sequencing by Nanopore induced Photon Emission (SM-SNIPE)

描述（由申请人提供）：我们的团队已经为开发一种独特的，基于纳米孔诱导光子发射（SNIPE）的DNA测序方法奠定了基础，该方法利用光学检测而不是更普遍的电检测。我们的方法优于其他纳米孔方法，因为读出不涉及酶，并行化简单，读出是非破坏性的。在这项资助中，我们提出了三个不同的目标（并行发展），当它们结合在一起时，将使DNA测序在速度（bbbb10 ^6个碱基/秒）和极低成本方面达到前所未有的规模。我们的第一个目标是大幅提高狙击的吞吐量，速度和准确性。为了实现这一目标，我们将集中精力通过纳米孔阵列（高达100 × 100）实现系统的并行化，将读出从2个颜色转换为4个颜色，并增加读出的S/B。我们的第二个目标是开发和优化我们专有的DNA转化方法，环状DNA转化（CDC）。我们计划首先通过使用商用台式系统实现CDC的自动化和优化。CDC优化后，我们计划开发一种能够转化整个人类基因组的微流体装置。我们的第三个目标是开发基础调用、共识构建、序列组装和防错所需的数据分析算法。在完成这三个目标后，我们将开发出一种全新的、具有成本效益的DNA测序平台，具有长读取长度、高速度和高准确性。预计这将对基础和应用生物医学研究以及个性化医疗保健产生广泛影响。

DESCRIPTION (provided by applicant): Our group has laid the groundwork in developing a unique, nanopore based method for DNA sequencing by nanopore induced photon emission (SNIPE), which utilizes optical detection rather than the more ubiquitous electrical detection. Our approach is superior to other nanopore approaches as the readout does not involve enzymes, parallelization is straightforward, and the readout is non-destructive. In this grant we propose three distinct aims (developed in parallel), which when brought together, will enable DNA sequencing at an unprecedented scale in terms of speed (>2 10^6 bases/s,) and extremely low cost. Our first aim is to dramatically increase the throughput, speed and accuracy of SNIPE. In order to achieve this, we will concentrate our efforts on parallelization of the system through arrays of nanopores (up to 100x100), transformation of the readout from 2 to 4 colors, and increasing the S/B of the readout. Our second Aim is to develop and optimize our proprietary DNA conversion approach, Circular DNA conversion (CDC). We plan on achieving this first though automation and optimization of CDC using a commercially available benchtop system. Post CDC optimization, we plan on developing a microfluidic device capable of converting an entire human genome. Our third Aim is the development of data analysis algorithms needed for base calling, consensus building, sequence assembly, and error proofing. In completing these three aims we will have achieved in developing a radically new, cost-effective DNA sequencing platform, capable of long read lengths, high speed, and high accuracy. This is expected to have a wide-ranging impact on both basic and applied biomedical research and personalized healthcare. PUBLIC HEALTH RELEVANCE: The extraordinary broad impact of ultra-low cost sequencing on biomedical research, comparative genomics and cancer biology, is driving the development of a plurality of DNA sequencing methods. Our group has been developing a nanopore DNA sequencing method that utilizes optical detection from hundreds of nanopores, as the molecules are pulled electrophoretically driven through the pores. This proposal will allow us to develop this method to address the $1,000 genome challenge.