Technical Validation of a Hepatitis C Virus Whole Genome Sequencing Assay for Detection of Genotype and Antiviral Resistance in the Clinical Pathway.

Choice of direct acting antiviral (DAA) therapy for Hepatitis C Virus (HCV) in the United Kingdom and similar settings usually requires knowledge of the genotype and, in some cases, antiviral resistance (AVR) profile of the infecting virus. To determine these, most laboratories currently use Sanger technology, but next-generation sequencing (NGS) offers potential advantages in throughput and accuracy. However, NGS poses unique technical challenges, which require idiosyncratic development and technical validation approaches.

Sequencing of human genomes with nanopore technology.

Whole-genome sequencing (WGS) is becoming widely used in clinical medicine in diagnostic contexts and to inform treatment choice. Here we evaluate the potential of the Oxford Nanopore Technologies (ONT) MinION long-read sequencer for routine WGS by sequencing the reference sample NA12878 and the genome of an individual with ataxia-pancytopenia syndrome and severe immune dysregulation. We develop and apply a novel reference panel-free analytical method to infer and then exploit phase information which improves single-nucleotide variant (SNV) calling performance from otherwise modest levels.

Resistance analysis of genotype 3 hepatitis C virus indicates subtypes inherently resistant to nonstructural protein 5A inhibitors.

Hepatitis C virus (HCV) genotype (gt) 3 is highly prevalent globally, with non-gt3a subtypes common in Southeast Asia. Resistance-associated substitutions (RASs) have been shown to play a role in treatment failure. However, the role of RASs in gt3 is not well understood.

MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry.

Background: Long-read sequencing is rapidly evolving and reshaping the suite of opportunities for genomic analysis. For the MinION in particular, as both the platform and chemistry develop, the user community requires reference data to set performance expectations and maximally exploit third-generation sequencing. We performed an analysis of MinION data derived from whole genome sequencing of K-12 using the R9.

Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus.

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism.

Evolutionary dynamics of reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance.

, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing.

Virus genomics and evolution: the transformative effect of new technologies and multidisciplinary collaboration on virus research and outbreak management.

The first Virus Genomics and Evolution Conference was held at the Wellcome Genome Campus in Hinxton, UK, 8-10 June 2016.

Comparison of Next-Generation Sequencing Technologies for Comprehensive Assessment of Full-Length Hepatitis C Viral Genomes.

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared.

Evaluation of Viremia Frequencies of a Novel Human Pegivirus by Using Bioinformatic Screening and PCR.

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%).

MinION Analysis and Reference Consortium: Phase 1 data release and analysis.

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community.

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study.

Background: Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis.

Mobile elements drive recombination hotspots in the core genome of Staphylococcus aureus.

Horizontal gene transfer is an important driver of bacterial evolution, but genetic exchange in the core genome of clonal species, including the major pathogen Staphylococcus aureus, is incompletely understood. Here we reveal widespread homologous recombination in S. aureus at the species level, in contrast to its near-complete absence between closely related strains.

Diverse sources of C. difficile infection identified on whole-genome sequencing.

Background: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions.

Methods: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C.

A modified RNA-Seq approach for whole genome sequencing of RNA viruses from faecal and blood samples.

To date, very large scale sequencing of many clinically important RNA viruses has been complicated by their high population molecular variation, which creates challenges for polymerase chain reaction and sequencing primer design. Many RNA viruses are also difficult or currently not possible to culture, severely limiting the amount and purity of available starting material. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA shotgun sequencing (also known as RNA-Seq).

Within-host evolution of Staphylococcus aureus during asymptomatic carriage.

Background: Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S.

Microevolutionary analysis of Clostridium difficile genomes to investigate transmission.

Background: The control of Clostridium difficile infection is a major international healthcare priority, hindered by a limited understanding of transmission epidemiology for these bacteria. However, transmission studies of bacterial pathogens are rapidly being transformed by the advent of next generation sequencing.

Results: Here we sequence whole C.

Recombinational switching of the Clostridium difficile S-layer and a novel glycosylation gene cluster revealed by large-scale whole-genome sequencing.

Background: Clostridium difficile is a major cause of nosocomial diarrhea, with 30-day mortality reaching 30%. The cell surface comprises a paracrystalline proteinaceous S-layer encoded by the slpA gene within the cell wall protein (cwp) gene cluster. Our purpose was to understand the diversity and evolution of slpA and nearby genes also encoding immunodominant cell surface antigens.

Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study.

Background: Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains.

A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance.

Objectives: To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management.

Design: The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile.

Evolutionary dynamics of Staphylococcus aureus during progression from carriage to disease.

Whole-genome sequencing offers new insights into the evolution of bacterial pathogens and the etiology of bacterial disease. Staphylococcus aureus is a major cause of bacteria-associated mortality and invasive disease and is carried asymptomatically by 27% of adults. Eighty percent of bacteremias match the carried strain.

Are prorocentroid dinoflagellates monophyletic? A study of 25 species based on nuclear and mitochondrial genes.

The dinoflagellate sub-class Prorocentrophycidae has a distinct morphology, lacking the typical dinoflagellate cell structure of a clear cingulum and sulcus. It includes species that produce the toxin okadaic acid. Despite its uniqueness, the group has been found polyphyletic in some previous molecular phylogenetic studies.