RESUMEN
There is growing evidence for the value of bacterial whole-genome sequencing in hospital outbreak investigations. Our aim was to develop methods that support efficient and accurate low-throughput clinical sequencing of methicillin-resistant Staphylococcus aureus (MRSA) isolates. Using a test panel of 25 MRSA isolates previously associated with outbreak investigations, we devised modifications to library preparation that reduced the processing time by 1 hour. We determined the maximum number of isolates that could be sequenced per run using an Illumina MiniSeq platform and a 13-hour (overnight) run time, which equated to 21 MRSA isolates and 3 controls (no template, positive, and negative). Repeatability and reproducibility assays based on this sequencing methodology demonstrated 100% accuracy in assigning species and sequence type (ST) and in detecting mecA Established genetic relatedness between isolates was recapitulated. Quality control (QC) metrics were evaluated over nine sequencing runs. Of the test panel MRSA genomes, 168/173 (97%) passed QC metrics based on the correct species assigned, detection of mecA and ST, and depth/coverage metrics. An evaluation of contamination in these 9 runs showed that positive and negative controls and test MRSA sequence files contained <0.14% and <0.48% of fragments that matched another species, respectively. Deliberate contamination experiments confirmed that this was insufficient to impact data interpretation. These methods support reliable and reproducible clinical MRSA sequencing with a turnaround time (from DNA extraction to availability of data files) of 24 hours.
Asunto(s)
Genoma Bacteriano , Staphylococcus aureus Resistente a Meticilina/genética , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Secuenciación Completa del Genoma , Pruebas Diagnósticas de Rutina , Humanos , Laboratorios de Hospital , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Técnicas Microbiológicas , Tipificación de Secuencias Multilocus , Secuenciación Completa del Genoma/métodosRESUMEN
All cancers carry somatic mutations. A subset of these somatic alterations, termed driver mutations, confer selective growth advantage and are implicated in cancer development, whereas the remainder are passengers. Here we have sequenced the genomes of a malignant melanoma and a lymphoblastoid cell line from the same person, providing the first comprehensive catalogue of somatic mutations from an individual cancer. The catalogue provides remarkable insights into the forces that have shaped this cancer genome. The dominant mutational signature reflects DNA damage due to ultraviolet light exposure, a known risk factor for malignant melanoma, whereas the uneven distribution of mutations across the genome, with a lower prevalence in gene footprints, indicates that DNA repair has been preferentially deployed towards transcribed regions. The results illustrate the power of a cancer genome sequence to reveal traces of the DNA damage, repair, mutation and selection processes that were operative years before the cancer became symptomatic.
Asunto(s)
Genes Relacionados con las Neoplasias/genética , Genoma Humano/genética , Mutación/genética , Neoplasias/genética , Adulto , Línea Celular Tumoral , Daño del ADN/genética , Análisis Mutacional de ADN , Reparación del ADN/genética , Dosificación de Gen/genética , Humanos , Pérdida de Heterocigocidad/genética , Masculino , Melanoma/etiología , Melanoma/genética , MicroARNs/genética , Mutagénesis Insercional/genética , Neoplasias/etiología , Polimorfismo de Nucleótido Simple/genética , Medicina de Precisión , Eliminación de Secuencia/genética , Rayos UltravioletaRESUMEN
BACKGROUND: Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients. METHODOLOGY/PRINCIPAL FINDINGS: Here we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci. We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. CONCLUSIONS: Our results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs.