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Title

Applied genomics for tuberculosis diagnosis and surveillance

AuthorsGoig, Galo A.
AdvisorComas, Iñaki
KeywordsTuberculosis
Genomics
Sequencing
Epidemiology
Microbiology
Issue Date24-Jul-2020
PublisherUniversidad de Valencia
CSIC - Instituto de Biomedicina de Valencia (IBV)
CitationApplied genomics for tuberculosis diagnosis and surveillance: 202 p. (2020)
Abstract[EN] Tuberculosis (TB) remains one of the main causes of death worldwide. Over the past years, whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB), its causative agent, has become an invaluable tool in the study, diagnosis and surveillance of the disease. Analyzing the complete genome of the bacteria allows the accurate prediction of drug resistance, detect transmission between patients and study outbreaks with unprecedented resolution, and provides new insights into the evolution and genetic diversity of this deathly pathogen. However, its effective use as a diagnostic tool has been hampered by its dependence on the long and cumbersome process of culturing MTB bacteria to obtain enough biomass for DNA extraction. For this reason, WGS of MTB directly from clinical specimens (dWGS) is considered to be a major breakthrough in TB diagnosis and control. The aim of this thesis is to apply the cutting-edge genomic techniques used in the research laboratory to develop novel tools for the diagnosis and surveillance of TB, including the development of a workflow that allows performing dWGS of MTB. Through the thesis, we performed a large-scale comparative analysis to identify genetic markers that are completely specific to MTB bacteria. We found that markers used up to date in TB tests, even in those endorsed by the World Health Organization, are non-specific. We provide a comprehensive catalog of MTB-specific markers to develop novel molecular assays for TB, and develop a highly specific qPCR that allows to accurately quantify MTB DNA in complex samples such as clinical specimens. We also developed a solid framework for the computational analysis of MTB WGS data, with special emphasis in data obtained directly from clinical specimens. Interestingly, we found that contaminant DNA in WGS analysis is a major pitfall in sequencing studies, introducing many errors that greatly bias the variant analysis. We implemented and validated a methodology to remove such contamination from sequencing data and demonstrated that this methodology is pivotal when performing dWGS of bacterial organisms. Finally, we implemented a complete workflow to perform dWGS of MTB. We were able to sequence and analyze the complete genomes of the infective MTB bacteria directly from respiratory samples of TB patients in less than a week. With our analysis, we were able not only to detect MTB in the samples, but to provide a full report of antibiotic resistance and transmission between patients. We incorporated our data to the transmission network of TB in the Comunidad Valenciana and showed, for the first time, the use of dWGS for high-resolution genomic epidemiology of TB.
[ES] La tuberculosis es una de las principales causas de muerte a nivel mundal. A lo largo de los ultimos años, la secuenciación de genoma comleto de Myobacterium tuberculosis, el agente infeccioso, se ha convertido en una herramienta extraordinaria en el estudio, diagnóstico y vigilancia epidemiológica de la enfermedad. Analizar el genoma completo de la bacteria permite la predicción precisa de resistencias a antibióticos, estudiar brotes con una resolución sin preecdentes, a la vez que permite estudiar la evolución y la diversidad genética del patógeno. Sin embargo el uso de la secuenciación genómica se ha visto dificultado por la dependencia de un proceso largo y costoso de cultivo microbiano, necesiario para obtener suficiente biomasa para extraer el ADN. Por ese motivo, la capacidad de secuenciar el genoma de la bacteria de una manera independiente de cultivo se ha considerado ser uno de los grandes avances que están por venir en el campo. El objetivo de esta tesis es aplicar las técnicas punteras de genómica que usamos en los laboratores de investigación para desarrollar nuevas herramientas para el diagnóstico y la epidemiología de la tuberculosis, incluyendo un método para secuenciar el genoma completo de una manera independiente del cultivo. En esta tesis hacemos un análisis genómico a gran escala para detectar nuevos marcadores diagnósticos para la tubterculosis. También desarrollamos una metodología de análisis computacional de datos de secuenciación independiente de cultivo de mycobacterium tuberculosis. Descubrimos que las contaminaciones con ADN bacteriano de otras especies son comunes en estudios de secuenciación masiva de bacterias,lo que tiene un gran impacto en el análisis, y desarrollamos una metodología eficaz para evitar que afecten al análisis. Finalmente, desarrollamos la metodología, de laboratorio y computacional, que nos permite obtener genomas completos de la bacteria M. tuberculosis de muestras clínicas de pacientes con tuberculosis de manera directa, sin necesitar realizar un cultivo previo. Realizmos un estudio piloto que incluye la transmisión de tuberculosis en la Comunidad Valenciana mostrando, por primera vez, el uso de la secuenciación directa en epidemiología genómica de alta resolución.
DescriptionTesis doctoral, 212 pág, figutas, tablas y material suplementario.
URIhttp://hdl.handle.net/10261/219509
Appears in Collections:(IBV) Tesis
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