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.
Highlights
As of 2019, 38 million people are living with HIV [1]
To validate our NanoHIV pipeline, we compared the HIV-1 consensus sequences generated from the 7 intact and 2 defective proviral genomes collected from four children in the Children with HIV Early Antiretroviral Therapy (CHER) cohort using Oxford Nanopore Technologies (ONT) and NanoHIV against consensus sequences generated from the same samples using an Illumina® sequencer and the SHIVER pipeline
98.9%, while the mean similarity of mapped ONT reads to the relevant consensus sequence was 92.1%
Summary
As of 2019, 38 million people are living with HIV [1]. the introduction of early and effective antiretroviral therapy (ART) has led to significant declines in transmissions, morbidity, and mortality, HIV remains incurable in the large majority of individuals. HIV-1, has regions such as the variable loops in the envelope gene (Env) that are very poorly conserved across subtypes and even within subtypes and within donors [38,39] In these regions, it is difficult or impossible to produce a high-quality alignment of an ONT read to a reference sequence using tools such as minimap, because true variation, including insertions and deletions, is indistinguishable from sequencing errors, especially for homopolymer regions [40]. It is difficult or impossible to produce a high-quality alignment of an ONT read to a reference sequence using tools such as minimap, because true variation, including insertions and deletions, is indistinguishable from sequencing errors, especially for homopolymer regions [40] This problem can potentially be addressed by a de novo assembly approach using tools such as Canu [41]. NanoHIV uses a bootstrap approach to refine a consensus sequence, including the refinement of variable regions, by first constructing a consensus sequence built from only highly conserved regions and refining it by including variable regions from long reads as insertions
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