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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. This applies particularly to virology, where sequence diversity is high and the amount of starting genetic material is low, making it difficult to distinguish real data from artifacts. We describe the development and technical validation of a sequence capture-based HCV whole genome sequencing (WGS) assay to determine viral genotype and AVR profile. We use clinical samples of known subtypes and viral loads, and simulated FASTQ datasets to validate the analytical performances of both the wet laboratory and bioinformatic pipeline procedures. We show high concordance of the WGS assay compared to current "gold standard" Sanger assays. Specificity was 92.3 and 96.1% for AVR and genotyping, respectively. Discordances were due to the inability of Sanger assays to assign the correct subtype or accurately call mixed drug-resistant variants. We show high repeatability and reproducibility with >99.8% sequence similarity between sequence runs as well as high precision for variant frequency detection at >98.8% in the 95th percentile. Post-sequencing bioinformatics quality control workflows allow the accurate distinction between mixed infections, cross-contaminants and recombinant viruses at a threshold of >5% for the minority population. The sequence capture-based HCV WGS assay is more accurate than legacy AVR and genotyping assays. The assay has now been implemented in the clinical pathway of England's National Health Service HCV treatment programs, representing the first validated HCV WGS pipeline in clinical service. The data generated will additionally provide granular national-level genomic information for public health policy making and support the WHO HCV elimination strategy., (Copyright © 2020 Manso, Bibby, Lythgow, Mohamed, Myers, Williams, Piorkowska, Chan, Bowden, Ansari, Ip, Barnes, Bradshaw and Mbisa.)

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. We report the prevalence of RASs in a cohort of direct-acting antiviral treatment-naive, gt3-infected patients, including those with rarer subtypes, and evaluate the effect of these RASs on direct-acting antivirals in vitro. Baseline samples from 496 gt3 patients enrolled in the BOSON clinical trial were analyzed by next-generation sequencing after probe-based enrichment for HCV. Whole viral genomes were analyzed for the presence of RASs to approved direct-acting antivirals. The resistance phenotype of RASs in combination with daclatasvir, velpatasvir, pibrentasvir, elbasvir, and sofosbuvir was measured using the S52 ΔN gt3a replicon model. The nonstructural protein 5A A30K and Y93H substitutions were the most common at 8.9% (n = 44) and 12.3% (n = 61), respectively, and showed a 10-fold and 11-fold increase in 50% effect concentration for daclatasvir compared to the unmodified replicon. Paired RASs (A30K + L31M and A30K + Y93H) were identified in 18 patients (9 of each pair); these combinations were shown to be highly resistant to daclatasvir, velpatasvir, elbasvir, and pibrentasvir. The A30K + L31M combination was found in all gt3b and gt3g samples. Conclusion: Our study reveals high frequencies of RASs to nonstructural protein 5A inhibitors in gt3 HCV; the paired A30K + L31M substitutions occur in all patients with gt3b and gt3g virus, and in vitro analysis suggests that these subtypes may be inherently resistant to all approved nonstructural protein 5A inhibitors for gt3 HCV. (Hepatology 2018)., (© 2018 The Authors. Hepatology published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

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. In the clinical sample, we identify and directly phase two non-synonymous de novo variants in SAMD9L, (OMIM #159550) inferring that they lie on the same paternal haplotype. Whilst consensus SNV-calling error rates from ONT data remain substantially higher than those from short-read methods, we demonstrate the substantial benefits of analytical innovation. Ongoing improvements to base-calling and SNV-calling methodology must continue for nanopore sequencing to establish itself as a primary method for clinical WGS.

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 Escherichia coli K-12 using the R9.0 chemistry, comparing the results with the older R7.3 chemistry., Methods: We computed the error-rate estimates for insertions, deletions, and mismatches in MinION reads., Results: Run-time characteristics of the flow cell and run scripts for R9.0 were similar to those observed for R7.3 chemistry, but with an 8-fold increase in bases per second (from 30 bps in R7.3 and SQK-MAP005 library preparation, to 250 bps in R9.0) processed by individual nanopores, and less drop-off in yield over time. The 2-dimensional ("2D") N50 read length was unchanged from the prior chemistry. Using the proportion of alignable reads as a measure of base-call accuracy, 99.9% of "pass" template reads from 1-dimensional ("1D")  experiments were mappable and ~97% from 2D experiments. The median identity of reads was ~89% for 1D and ~94% for 2D experiments. The total error rate (miscall + insertion + deletion ) decreased for 2D "pass" reads from 9.1% in R7.3 to 7.5% in R9.0 and for template "pass" reads from 26.7% in R7.3 to 14.5% in R9.0., Conclusions: These Phase 2 MinION experiments serve as a baseline by providing estimates for read quality, throughput, and mappability. The datasets further enable the development of bioinformatic tools tailored to the new R9.0 chemistry and the design of novel biological applications for this technology., Abbreviations: K: thousand, Kb: kilobase (one thousand base pairs), M: million, Mb: megabase (one million base pairs), Gb: gigabase (one billion base pairs)., Competing Interests: Competing interests: All flow cells and library preparation kits were provided by ONT free of charge. Ewan Birney is a paid consultant of ONT. MJ, HEO, JT, ML, CI, HJ, JOG and BB have accepted reimbursement for conference travel expenses from ONT. VZ was funded for his work on this project from Oxford Nanopore through an agreement with EMBL.

Enterococcus faecium , 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 E. faecium 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. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn 1549 -like element- vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.

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. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance.

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., Methods: Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance., Findings: We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7-93·7) and 98·4% specificity (98·1-98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes., Interpretation: A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early., Funding: Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union., (Copyright © 2015 Walker et al. Open Access article distributed under the terms of CC-BY. Published by Elsevier Ltd.. All rights reserved.)

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. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location., Results: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile., Conclusions: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).