A5 Near full-length HIV-1 genome sequencing in newly diagnosed individuals in Sweden

The Swedish HIV-1 epidemic is characterized by a high diversity in HIV subtypes and recombinants as a result of migration. To study the time from infection through viral diversification, transmission patterns, and drug resistance in minor quasispecies, a robust protocol for pan-genotypic near full-length HIV-1 genome (HIV-NFLG) next-generation sequencing (NGS) is key. Our group has established two protocols for HIV-NFLG on the Illumina platform that we aim to compare and, if necessary, modify to find a method with optimized coverage, depth, and subtype inclusivity. Zanini et al. (https://doi.org/10.7554/eLife.11282.001) have developed a method with one-step RT-PCR with six overlapping primer sets, followed by NGS and quality filtering and assembly with in-house methods. Aralaguppe et al. (https://doi.org/10.1016/j.jviromet.2016.07.010) have designed amplification in two fragments, followed by multiplexed NGS and quality control and assembly with Iterative Virus Assembler and VICUNA. Both methods have high coverage per nucleotide and low error rates in amplification and sequencing and can reliably identify SNPs at 1 per cent of the viral population with linkage within the quasispecies. Subtype inclusivity remains a challenge even though both methods show success in amplifying and sequencing subtypes B, C, and the common recombinants 01_AE and 02_AG. Therefore, we aim to evaluate and optimize our NFLG NGS methods on a panel of patient samples that more completely reflects HIV-1 diversity in Sweden. Patient samples from fifty treatment-naïve viremic individuals representing the genotypic HIV-1 panorama in Sweden, including CRFs, are being amplified and sequenced by both protocols. Coverage of the genome, error rate, and possible depth of quasispecies analysis is being evaluated. We will compare number of reads, coverage across the HIV genome, and representation of minor single nucleotide variants as well as subtype inclusivity and impact of plasma RNA levels. To do this we will use an in-house bioinformatic pipeline. The NFLG sequences will also be analyzed with phylogenetic tools for determination of subtypes including CRFs and URFs.