Setup, Validation, and Quality Control of a Centralized Whole-Genome-Sequencing Laboratory: Lessons Learned.

Routine use of whole-genome analysis for infectious diseases can be used to enlighten various scenarios pertaining to public health, including identification of microbial pathogens, relating individual cases to an outbreak of infectious disease, establishing an association between an outbreak of food poisoning and a specific food vehicle, inferring drug susceptibility, source tracing of contaminants, and study of variations in the genome that affect pathogenicity/virulence. We describe the setup, validation, and ongoing verification of a centralized whole-genome-sequencing (WGS) laboratory to carry out sequencing for these public health functions for the National Infection Services, Public Health England, in the United Kingdom. The performance characteristics and quality control metrics measured during validation and verification of the entire end-to-end process (accuracy, precision, reproducibility, and repeatability) are described and include information regarding the automated pass and release of data to service users without intervention.

A Rapid and Sensitive Diagnostic Screening Assay for Detection of Mycobacteria Including Mycobacterium tuberculosis Directly from Sputum without Extraction.

We report a novel approach utilising a real-time PCR screening assay targeting a 53 bp tandemly repeated element present at various loci within the Mycobacterium tuberculosis (Mtb) genome. Positive samples were identified within a discriminatory melting curve range of 90-94°C, with results obtained in under one hour directly from decontaminated sputum samples without extraction. A panel of 89 smear-positive sputa were used for analytical validation of the assay with 100% concordance, with sensitivity matching that of culture. Cross reactivity was detected within a narrow range of mycobacteria other than tuberculosis (MOTT) (five sputa, three in silico), with the highest sensitivity within M. avium complex (MAC). A year-long head to head evaluation of the test with the GeneXpert platform was carried out with 104 consecutive samples at the Royal Free Hospital, UK. Receiver operating characteristics (ROC) analysis of the data revealed that the two tests are approximately equivalent in sensitivity, with the area under the curve being 0.85 and 0.80 for the GeneXpert and our assay, respectively, indicating that the test would be a cost effective screen prior to GeneXpert testing.

IS6110-based global phylogeny of Mycobacterium tuberculosis.

IS6110, a Mycobacterium tuberculosis complex species-specific insertion element, is targeted primarily for DNA fingerprinting of M. tuberculosis strains. The number and chromosomal positions of copies of this element have been found to be highly variable between unrelated strains and have been exploited for molecular epidemiological purpose but the utility of IS6110 as an informative marker of strain phylogeny has yet to be demonstrated. In the current study, a recently proposed IS6110-targetting PCR based typing methodology, fluorescent amplified fragment length polymorphism (fAFLP) was applied to a global panel of 166 of the clinically more predominant 'modern' strains characterised by spoligotype and, where available, Variable Number Tandem Repeat (VNTR) to identify potentially evolutionarily informative common fragments that could define strains as belonging to established genetic lineages. These common fragments are hereby proposed to be ancestral insertion sites present in common ancestors of these strains rather than preferential insertion sites or 'hot spots'. Results indicate that the exact same spoligotype and VNTR-defined lineages are reflected in the fragment patterns but with greater resolution and are able to clearly define the very distinct Haarlem, LAM, X and also the currently ill-defined T and S and lineages and spoligotypes designated as U, or unknown, without ambiguity. The biogeographical patterns generated reflect the migration of mankind across the globe and indicate that only four successful clones (or individual bacteria) gave rise to virtually all of the tuberculosis in Europe and the Americas. Potential lies in the application of the data to determine IS6110 evolutionary events that have occurred during the evolution of these lineages.

Elucidation of the outer membrane proteome of Salmonella enterica serovar Typhimurium utilising a lipid-based protein immobilization technique.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a major cause of human gastroenteritis worldwide. The outer membrane proteins expressed by S. Typhimurium mediate the process of adhesion and internalisation within the intestinal epithelium of the host thus influencing the progression of disease. Since the outer membrane proteins are surface-exposed, they provide attractive targets for the development of improved antimicrobial agents and vaccines. Various techniques have been developed for their characterisation, but issues such as carryover of cytosolic proteins still remain a problem. In this study we attempted to characterise the surface proteome of S. Typhimurium using Lipid-based Protein Immobilisation technology in the form of LPI FlowCells. No detergents are required and no sample clean up is needed prior to downstream analysis. The immobilised proteins can be digested with proteases in multiple steps to increase sequence coverage, and the peptides eluted can be characterised directly by liquid chromatography - tandem mass spectrometry (LC-MS/MS) and identified from mass spectral database searches. RESULTS: In this study, 54 outer membrane proteins, were identified with two or more peptide hits using a multi-step digest approach. Out of these 28 were lipoproteins, nine were involved in transport and three with enzyme activity These included the transporters BtuB which is responsible for the uptake of vitamin B12, LamB which is involved in the uptake of maltose and maltodextrins and LolB which is involved in the incorporation of lipoproteins in the outer membrane. Other proteins identified included the enzymes MltC which may play a role in cell elongation and division and NlpD which is involved in catabolic processes in cell wall formation as well as proteins involved in virulence such as Lpp1, Lpp2 and OmpX. CONCLUSION: Using a multi-step digest approach the LPI technique enables the incorporation of a multi-step protease work flow ensuring enough sequence coverage of membrane proteins subsequently leading to the identification of more membrane proteins with higher confidence. Compared to current sub-cellular fractionation procedures and previous published work, the LPI technique currently provides the widest coverage of outer membrane proteins identified as demonstrated here for Salmonella Typhimurium.

Evolutionary clues from comparative analysis of Mycobacterium tuberculosis variable-number tandem repeat sequences within genetic families.

Mycobacterium tuberculosis is one of the most successful bacterial pathogens in the history of mankind, and the study of the evolutionary and genetic behaviour of this organism is ongoing. The potential of variable-number tandem repeat (VNTR) regions to provide insight into the evolutionary past of this organism has yet to be evaluated. The aim of this study was to investigate diversity occurring within VNTR loci within established evolutionary lineages. Mycobacterial interspersed repetitive unit (MIRU) 4 and 26 nucleotide sequencing was undertaken revealing significant differences in VNTR discriminatory power and distribution of allelic types within defined sub-lineages, within a moderately discriminant and a highly discriminant locus, respectively. These findings indicate potential allele bias at these loci within the defined groups dependent on the genotype of their progenitor strain, and the patterns of distribution suggest that a step-wise contraction/expansion process is responsible for VNTR repeat evolution. The results also identified a possible incidence of genetic drift within major genetic group 2 (MGG2), which has a point mutation at locus MIRU26 in a clonal subgroup.

Evolution of short sequence repeats in Mycobacterium tuberculosis.

Whole genome comparison has revealed the presence of short sequence repeats (also called mycobacterial interspersed repeat units and variable number tandem repeat units) used for genotyping schemes. In this study, we have used deletion analysis, single nucleotide polymorphism data and spoligotype taken from published data from others to investigate the evolution of selected repeats that form the common denominators of the majority of established schemes. Analysis of the number of repeats per locus from over 400 isolates revealed that the general trend globally appears to be loss of repeats in modern strains compared with ancestral strains.

Disease progression in heterosexual patients infected with closely related subtype B strains of HIV type 1 with differing coreceptor usage properties.

Previously we described a heterosexual outbreak of HIV-1 subtype B in a town in the north of England (Doncaster) where 11 of 13 infections were shown to be linked by phylogenetic analysis of the env gp120 region. The 11 infections were related to a putative index case, Don1, and further divided into two groups based on the patients' disease status, their viral sequences, and other epidemiological information. Here we describe two further findings. First, we found that viral isolates and gp120 recombinant viruses derived from patients from one group used the CCR5 coreceptor, whereas viruses from the other group could use both the CCR5 and CXCR4 coreceptors. Patients with the X4/R5 dual tropic strains were symptomatic when diagnosed and progressed rapidly, in contrast to the other patient group that has remained asymptomatic, implying a link between the tropism of the strains and disease outcome. Second, we present additional sequence data derived from the index case, demonstrating the presence of sequences from both clades, with an average interclade distance of 9.56%, providing direct evidence of a genetic link between these two groups. This new study shows that Don1 harbored both strains, implying he was either dually infected or that over time intrahost diversification from the R5 to R5/X4 phenotype occurred. These events may account for/have led to the spread of two genetically related strains with different pathogenic properties within the same heterosexual community.