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

Our goal was to assess the accuracy of next generation sequencing (NGS) compared to Sanger. We performed single genome amplification (SGA) of HIV-1 gp160 on extracted tissue DNA from two HIV+ individuals. Amplicons (n=30) were sequenced with Sanger, or re-amplified with barcoded primers and pooled before sequencing using Oxford Nanopore Technologies [ONT] and Pacific Bioscience [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 [n=9] or nearly identical [n=14] compared to 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 to Sanger/NGS. In summary, we show that both ONT and PB can produce amplicon consensus sequences with similar or higher accuracy compared to 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.