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

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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).