NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology

Abstract

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms

“…Recently, Wright et al reported a canonical nanopore sequencing approach after limiting dilution ( 26 ), which may be applicable for sequence determination of heterogeneous viral variants. However, the method contains a time-consuming and labor-intensive process of limiting dilution.…”

“…Nanopore sequencing technologies have already been applied in pathogen surveillance for Ebola, Zika, coronavirus disease 2019 (COVID-19), and other outbreaks ( 21 – 25 ). In fact, a recent study by Wright et al showed a new application protocol of nanopore sequencing to analyze HIV-1 proviral DNA variants ( 26 ). However, a pretreatment process of limiting dilution is required for this method.…”

An assessment of Nano-RECall: Interpretation of Oxford Nanopore sequence data for HIV-1 drug resistance testing

“…We obtained the most recently generated residual nested-PCR amplicons tested and since amplicon volumes were limited, selected 54 with the highest DNA yield after PCR purification with AMPure XP paramagnetic beads from a total of 65. Similarly, we obtained 35 of the most recent nested-PCR amplicons that had integrase (IN) sequencing, using a published in-house method [8] of which 24 with the best DNA yield after PCR purification with the QIAquick PCR purification kit, were included.…”

“…However, when dealing with highly diverse genomes such as HIV, obtaining accurate alignments have been challenging. Recently we showed an overall agreement of 99.4% between ONT and Illumina when combining a relative low accuracy basecaller with novel iterative alignment pipeline for sequencing HIV proviral PCR products at limiting dilution, with each PCR product expected to represent a single original template[8]. However, in this instance the aim was to obtain a single consensus derived from a single template; such single genome sequencing is however costly and cumbersome.…”

New Insights in the Fight against HIV

“…As an alternative to lifelong pharmacological therapy to keep patients in a status of virologic suppression, novel interventions to purge the HIV reservoir have been pursued in recent years with scarce success due to our poor understanding of the composition and mechanisms regulating latent infection. A research article by Wright et al reports on the optimization and validation of novel analytical tools to characterize integrated HIV sequences with a single genome near full-length amplicon resolution [ 7 ]. Castelli and coauthors discuss the clinical impact of an effective class of anti-cancer drugs, i.e., immune checkpoint inhibitors, in HIV-infected subjects, also as a potential virus reservoir eradication strategy [ 8 ].…”