High-accuracy, long-range sequencing for HIV-1 genotyping

项目摘要/摘要 在过去的十年中，测序技术的创新是背后的关键驱动力 医学和生命科学领域正在进行的革命。纳米孔测序平台(牛津纳米孔 技术)是具有几个未来主义特征的较新的、所谓的第三代测序技术， 包括超长的读取长度容量、实时数据输出和袖珍移动性(得益于 附属物测序仪)。这项新兴技术更广泛应用的一个关键障碍是它的 令人失望的高测序错误率。本研究的总体目标是开发一种新型的条形码 从根本上提高单分子靶DNA测序准确性的技术 纳米孔测序平台。远距离、高精度的测序将是描绘 在癌症或病毒感染等疾病中细胞或病毒种群的异质性。在这项提案中，我们 将重点放在HIV-1的基因分型上，作为原则的证明。HIV-1是一个很好的模型系统，因为它 相对较小的基因组(约9kbp)，患者内部高度的遗传多样性，以及 提供足够的NGS和Sanger序列数据，以便进行比较。我们将发展和 建立生成新一代条形码库和使用该条形码库的标准实验程序 文库执行测序纠错过程(目标1和2)；然后我们将测试我们的 方法使用HIV-1(AIMS 3)实验室毒株。

PROJECT SUMMARY / ABSTRACT Innovations in sequencing technologies over the past decade have been critical driving forces behind the ongoing revolution in medicine and the life sciences. The Nanopore sequencing platform (Oxford Nanopore Technologies) is a newer, so-called third generation sequencing technology with several futuristic features, including a tremendously long-read length capacity, real-time data output, and pocket-size mobility (thanks to the MinION sequencer). A critical barrier to broader applications of this emerging technology has been its disappointingly high sequencing error rates. The overall goal of this study is to develop a novel barcoding technology with which to radically improve the accuracy of single-molecule, target DNA sequencing for the Nanopore sequencing platform. Long-range, high-accuracy sequencing will be essential in delineating the heterogeneity of cellular or viral populations in diseases such as cancer or viral infection. In this proposal, we will focus on the genotyping of HIV-1 as a proof of principle. HIV-1 is an excellent model system because of its relatively small genome (approximately 9 Kbp), the high degree of intra-patient genetic diversity, and the availability of sufficient NGS and Sanger sequence data for the purpose of comparison. We will develop and establish standard experimental procedures for generating a new-generation barcode library and for using this library to perform sequencing error correction processes (Aims 1 and 2); We will then test the utility of our method using laboratory strains of HIV-1 (Aims 3).