High-resolution deep sequencing reveals biodiversity, population structure, and persistence of HIV-1 quasispecies within host ecosystems.

BACKGROUND: Deep sequencing provides the basis for analysis of biodiversity of taxonomically similar organisms in an environment. While extensively applied to microbiome studies, population genetics studies of viruses are limited. To define the scope of HIV-1 population biodiversity within infected individuals, a suite of phylogenetic and population genetic algorithms was applied to HIV-1 envelope hypervariable domain 3 (Env V3) within peripheral blood mononuclear cells from a group of perinatally HIV-1 subtype B infected, therapy-naïve children. RESULTS: Biodiversity of HIV-1 Env V3 quasispecies ranged from about 70 to 270 unique sequence clusters across individuals. Viral population structure was organized into a limited number of clusters that included the dominant variants combined with multiple clusters of low frequency variants. Next generation viral quasispecies evolved from low frequency variants at earlier time points through multiple non-synonymous changes in lineages within the evolutionary landscape. Minor V3 variants detected as long as four years after infection co-localized in phylogenetic reconstructions with early transmitting viruses or with subsequent plasma virus circulating two years later. CONCLUSIONS: Deep sequencing defines HIV-1 population complexity and structure, reveals the ebb and flow of dominant and rare viral variants in the host ecosystem, and identifies an evolutionary record of low-frequency cell-associated viral V3 variants that persist for years. Bioinformatics pipeline developed for HIV-1 can be applied for biodiversity studies of virome populations in human, animal, or plant ecosystems.

HPV Population Profiling in Healthy Men by Next-Generation Deep Sequencing Coupled with HPV-QUEST.

Multiple-type human papillomaviruses (HPV) infection presents a greater risk for persistence in asymptomatic individuals and may accelerate cancer development. To extend the scope of HPV types defined by probe-based assays, multiplexing deep sequencing of HPV L1, coupled with an HPV-QUEST genotyping server and a bioinformatic pipeline, was established and applied to survey the diversity of HPV genotypes among a subset of healthy men from the HPV in Men (HIM) Multinational Study. Twenty-one HPV genotypes (12 high-risk and 9 low-risk) were detected in the genital area from 18 asymptomatic individuals. A single HPV type, either HPV16, HPV6b or HPV83, was detected in 7 individuals, while coinfection by 2 to 5 high-risk and/or low-risk genotypes was identified in the other 11 participants. In two individuals studied for over one year, HPV16 persisted, while fluctuations of coinfecting genotypes occurred. HPV L1 regions were generally identical between query and reference sequences, although nonsynonymous and synonymous nucleotide polymorphisms of HPV16, 18, 31, 35h, 59, 70, 73, cand85, 6b, 62, 81, 83, cand89 or JEB2 L1 genotypes, mostly unidentified by linear array, were evident. Deep sequencing coupled with HPV-QUEST provides efficient and unambiguous classification of HPV genotypes in multiple-type HPV infection in host ecosystems.

HPV-QUEST: A highly customized system for automated HPV sequence analysis capable of processing Next Generation sequencing data set.

Next Generation sequencing (NGS) applied to human papilloma viruses (HPV) can provide sensitive methods to investigate the molecular epidemiology of multiple type HPV infection. Currently a genotyping system with a comprehensive collection of updated HPV reference sequences and a capacity to handle NGS data sets is lacking. HPV-QUEST was developed as an automated and rapid HPV genotyping system. The web-based HPV-QUEST subtyping algorithm was developed using HTML, PHP, Perl scripting language, and MYSQL as the database backend. HPV-QUEST includes a database of annotated HPV reference sequences with updated nomenclature covering 5 genuses, 14 species and 150 mucosal and cutaneous types to genotype blasted query sequences. HPV-QUEST processes up to 10 megabases of sequences within 1 to 2 minutes. Results are reported in html, text and excel formats and display e-value, blast score, and local and coverage identities; provide genus, species, type, infection site and risk for the best matched reference HPV sequence; and produce results ready for additional analyses.