Comparing Gold-Standard Sanger Sequencing with Two Next-Generation Sequencing Platforms of HIV-1 gp160 Single Genome Amplicons

• Published in: AIDS research and human retroviruses
• Main Authors: Nolan, David J, DaRoza, Jonathan, Brody, Robin, Ganta, Krishna, Luzuriaga, Katherine, Huston, Chris, Rosenthal, Simon, Lamers, Susanna L, Rose, Rebecca
• Format: Journal Article
• Language: English
• Published: United States 10.07.2024
• Subjects: Oxford Nanopore; single genome sequencing; next-generation sequencing; Pacific Biosciences
• ISSN: 1931-8405
• DOI: 10.1089/aid.2024.0012

Our goal was to assess the accuracy of next generation sequencing (NGS) compared with Sanger. We performed single genome amplification (SGA) of HIV-1 on extracted tissue DNA from two HIV+ individuals. Amplicons ( = 30) were sequenced with Sanger or reamplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB). For each amplicon, a consensus sequence for NGS reads was obtained by (1) mapping reads to the Sanger sequence when available ("reference-based") or (2) mapping reads to a "pseudo-reference" sequence, i.e., a consensus sequence of a subset of NGS reads ("reference-free"). PB reads were clustered based on genetic similarity. A Sanger consensus sequence was obtained for 23/30 amplicons, for which all NGS consensus sequences were identical ( = 9) or nearly identical ( = 14) compared with Sanger. For the nine mismatches between Sanger/NGS, the nucleotide in the NGS sequence matched all other sequences from that patient. Of the 7/30 amplicons without a Sanger sequence, NGS sequences had ≥35 ambiguous calls in five amplicons and 0 ambiguities in two amplicons. Analysis of the electropherograms showed failure of a single sequencing primer for the latter two amplicons (consistent with a single template) and overlapping peaks for the other five (consistent with multiple templates). Clustering results closely followed the Sanger/NGS consensus results, where amplicons derived from a single template also had a single cluster and vice versa (with one exception, which could be the result of barcode misidentification). Representative sequences from the clusters contained 2-13 differences compared with Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared with Sanger and, importantly, without the need for a known reference sequence. Clustering could be useful in some circumstances to predict or confirm the presence of multiple starting templates.