Validation of the Oxford Nanopore (MinION) sequencing platform for identification of HIV-1 transmitted founder viruses

Abstract

Quick identification and characterization of HIV-1 Transmitted/founder (TF) viruses is critical for guiding HIV-1 TF based prevention, and eradication strategies. Therefore, a high throughput sequencing platform is needed to identify HIV-1 TF viruses. While the Oxford Nanopore MinION sequencing technology (ONT) is an ideal platform, its suitability for TF virus identification has not been validated. In this study, the potential of using ONT for HIV-1 TF virus identification was assessed by benchmarking against the Sanger technique using 195 single genome amplicons (SGAs) from 20 archived samples previously identified and characterized by Sanger sequencing. ONT sequencing primarily involved library preparation which involved target DNA end repair, addition of barcodes labels to enable multiplex sequencing on a single flow cell, addition of sequencing adaptors to the barcoded DNA, conditioning the library (the barcoded DNA adaptor complex) and finally loading and sequencing the library on the MinION device ONT raw reads were first base-called, filtered, error-corrected, and polished to generate consensus sequences for downstream analysis (TF identification). The ONT platform identified TF viruses, from the study samples with 89.7% sensitivity (35 out of 39 sanger positive samples also being positive by ONT). ONT's high sequencing depth revealed minor variants not previously detected by Sanger, further increasing within-patient diversity, and complicating deciphering single virus transmission events in such samples. Importantly, an average sequence similarity of 99.7% was obtained between ONT deduced TF sequences and Sanger TFs from the same samples, this further validating ONT’s ability to identify TFs with high accuracy and reliability. Overall, this study advances our ability to diagnose and understand HIV-1 genetic diversity by using a primer-free high throughput long-read sequencing method as an alternative to Sanger sequencing and paving the path for future advances in HIV-1 research sequencing technologies.