Novel Amplicons based Method for Near-Full-Length Genome (NFLG) Sequencing of HIV-1 Group M Recombinant Forms

Over the years the spread of HIV-1 across the globe resulted in the creation of multiple subtypes and new recombinant forms (RFs). While the pol gene region of the HIV-1 genome is used for resistance mutations analysis and initial detection of recombinants, whole genome sequencing analysis is required to determine recombination events across the viral genome. Here, we present a novel robust near-full length genome (NFLG) sequencing approach for the sequencing of HIV-1 genomes out of clinical whole blood samples. This method has been successfully tested for various HIV-1 subtypes and RFs.

The method is based on an in-house developed set of 32 pan-genotypic primer pairs, divided into two pools, each containing 16 primer pairs covering the entire HIV-1 genome. Two parallel multiplex PCR reactions were used to generate 32 overlapping DNA fragments spanning the HIV-1 genome. Nextera XT protocol was used to obtain barcoded libraries which were run on the Illumina Miseq platform using a V3 kit. A consensus sequence was determined for each sample and was used to define recombination events across the genome. For that aim, a combined analysis of several computational tools including HIV BLAST, phylogenetic analysis, RIP, SimPlot++ and jpHMM was employed. Overall, plasma samples from 33 patients suspected to carry RFs and 2 different, known pure subtypes controls, were included in this study.

Overall coverage varied between different RFs. While the gag and pol genes were highly covered in all samples analyzed, the highly variable env gene region was not fully covered. Yet, these NFLG analyses enabled the identification of recombination events across the viral genome, with the identification of CRFs and RFs such as CRF01_AE, CRF02_AG, BF1, BC, A6B, CA1 and some potential URFs.

In summary, we describe a methodology for HIV-1 NFLG sequencing, which is based on multiple and partially overlapping PCR fragments spanning the genome of HIV-1. This method enables sequencing of 20 different patient samples in a single MiSeq run and was used for the characterization of different HIV-1 RFs and pure subtypes circulating in Israel. Additionally, this newly refined method was compared to HIV-1 NFLG results of PCR-free metagenomic sequencing approach and proved to obtain higher number of covered bases of the HXB2 reference genome.