Bronwyn M. Kirby

Next generation sequencing improves detection of drug resistance mutations in infants after PMTCT failure.

BACKGROUND Next generation sequencing (NGS) allows the detection of minor variant HIV drug resistance mutations (DRMs). However data from new NGS platforms after Prevention-of-Mother-to-Child-Transmission (PMTCT) regimen failure are limited. OBJECTIVE To compare major and minor variant HIV DRMs with Illumina MiSeq and Life Technologies Ion Personal Genome Machine (PGM) in infants infected despite a PMTCT regimen. STUDY DESIGN We conducted a cross-sectional study of NGS for detecting DRMs in infants infected despite a zidovudine (AZT) and Nevirapine (NVP) regimen, before initiation of combination antiretroviral therapy. Sequencing was performed on PCR products from plasma samples on PGM and MiSeq platforms. Bioinformatic analyses were undertaken using a codon-aware version of the Smith-Waterman mapping algorithm and a mixture multinomial error filtering statistical model. RESULTS Of 15 infants, tested at a median age of 3.4 months after birth, 2 (13%) had non-nucleoside reverse transcriptase inhibitor (NNRTI) DRMs (K103N and Y181C) by bulk sequencing, whereas PGM detected 4 (26%) and MiSeq 5 (30%). NGS enabled the detection of additional minor variant DRMs in the infant with K103N. Coverage and instrument quality scores were higher with MiSeq, increasing the confidence of minor variant calls. CONCLUSIONS NGS followed by bioinformatic analyses detected multiple minor variant DRMs in HIV-1 RT among infants where PMTCT failed. The high coverage of MiSeq and high read quality improved the confidence of identified DRMs and may make this platform ideal for minor variant detection.

Diversity of dsDNA Viruses in a South African Hot Spring Assessed by Metagenomics and Microscopy

The current view of virus diversity in terrestrial hot springs is limited to a few sampling sites. To expand our current understanding of hot spring viral community diversity, this study aimed to investigate the first African hot spring (Brandvlei hot spring; 60 °C, pH 5.7) by means of electron microscopy and sequencing of the virus fraction. Microscopy analysis revealed a mixture of regular- and ‘jumbo’-sized tailed morphotypes (Caudovirales), lemon-shaped virions (Fuselloviridae-like; salterprovirus-like) and pleiomorphic virus-like particles. Metavirome analysis corroborated the presence of His1-like viruses and has expanded the current clade of salterproviruses using a polymerase B gene phylogeny. The most represented viral contig was to a cyanophage genome fragment, which may underline basic ecosystem functioning provided by these viruses. Furthermore, a putative Gemmata-related phage was assembled with high coverage, a previously undocumented phage-host association. This study demonstrated that a moderately thermophilic spring environment contained a highly novel pool of viruses and should encourage future characterization of a wider temperature range of hot springs throughout the world.

Virome assembly and annotation: A surprise in the Namib Desert

Sequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ≥50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies.

Draft Genome Sequences of Three Bacillus Species from South African Marine Sponges

ABSTRACT The rise in antibiotic-resistant bacteria has spurred efforts to identify novel compounds with antimicrobial activity. This brief report describes the genome sequence of three Bacillus species isolates from South African marine sponges, which produce compounds with antimicrobial activity. A search for secondary metabolite clusters revealed several secondary metabolite pathways in these genomes, which may hold promise as novel antibiotics.

Correction: Zablocki, O.; et al. Diversity of dsDNA Viruses in a South African Hot Spring Assessed by Metagenomics and Microscopy. Viruses 2017, 9, 348

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