6 research outputs found
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. 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
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. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control
Interferon lambda 4 impacts the genetic diversity of hepatitis C virus
Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism
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 drugsusceptibility
testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining
mutations. We used whole-genome sequencing to characterise common and rare mutations predicting
drug resistance, or consistency with susceptibility, for all fi rst-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 identifi ed from the drug-resistance scientifi c 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% specifi city (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 identifi ed 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 workfl ows, phasing out phenotypic drugsusceptibility
testing while reporting drug resistance early.Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.http://www.thelancet.com/infectionhb201