Nanopore Sequencing for Characterization of HIV-1 Recombinant Forms

Abstract

HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases.High genetic diversity, including the emergence of recombinant forms (RFs), is one of the most prominent features of human immunodeficiency virus type 1 (HIV-1). Conventional detection of HIV-1 RFs requires pretreatments, i.e., cloning or single-genome amplification, to distinguish them from dual- or multiple-infection variants. However, these processes are time-consuming and labor-intensive. Here, we constructed a new nanopore sequencing-based platform that enables us to obtain distinctive genetic information for intersubtype RFs and dual-infection HIV-1 variants by using amplicons of HIV-1 near-full-length genomes or two overlapping half-length genome fragments. Repeated benchmark tests of HIV-1 proviral DNA revealed consensus sequence inference with a reduced error rate, allowing us to obtain sufficiently accurate sequence data. In addition, we applied the platform for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection according to Sanger sequencing-based genotyping tests for HIV-1 drug resistance. For each RF infection case, replicated analyses involving our nanopore sequencing-based platform consistently produced long consecutive analogous consensus sequences with mosaic genomic structures consisting of two different subtypes. In contrast, we detected multiple heterologous sequences in each dual-infection case. These results demonstrate that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Since the genetic diversity of HIV-1 continues to gradually increase, this system will help accelerate full-length genome analysis and molecular epidemiological surveillance for HIV-1. IMPORTANCE HIV-1 is characterized by large genetic differences, including HIV-1 recombinant forms (RFs). Conventional genetic analyses require time-consuming pretreatments, i.e., cloning or single-genome amplification, to distinguish RFs from dual- or multiple-infection cases. In this study, we developed a new analytical system for HIV-1 sequence data obtained by nanopore sequencing. The error rate of this method was reduced to ~0.06%. We applied this system for sequence analyses of 9 clinical samples with suspected HIV-1 RF infection or dual infection, which were extracted from 373 cases of HIV patients based on our retrospective analysis of HIV-1 drug resistance genotyping test results. We found that our new nanopore sequencing platform is applicable to identify the full-length HIV-1 genome structure of intersubtype RFs as well as dual-infection heterologous HIV-1. Our protocol will be useful for epidemiological surveillance to examine HIV-1 transmission as well as for genotypic tests of HIV-1 drug resistance in clinical settings.