In this thesis, two algorithms that detect cross-contamination in tumor samples sequenced with next-generation sequencing (NGS) were evaluated. In genomic medicine, NGS is commonly used to sequence tumor DNA to detect disease-associated genetic variants and determine the most suitable treatment option. Targeted NGS panels are often employed to screen for genetic variations in a selection of specific tumor-associated genes. NGS handles samples from multiple patients in parallel, which poses a risk of cross-contamination between samples. Contamination is a significant issue in the interpretation of NGS results, as it can lead to the incorrect identification of genetic variants and, consequently, incorrect treatment. Therefore, contamination detection is a crucial quality control steps in the analysis of NGS data. Numerous algorithms for detection of cross-contamination have been developed, but many of these algorithms are not suited for small, targeted NGS panels, and several are not developed for tumor data. In this thesis, GATK's CalculateContamination and a self-created algorithm called ContaCheck were evaluated on simulated tumor NGS data. NGS samples were generated in silico with a Python script called BAMSynth and mixed to simulate cross-contamination with contamination rates between 1% and 50%. ContaCheck accurately detected contaminations ranging from 3% to 50% and identified the correct contaminant with an accuracy of 94%. CalculateContamination, on the other hand, detected contaminations ranging from 1% to 15% relatively accurately, but consistently failed to detect high level contaminations. The study showed that ContaCheck outperformed CalculateContamination on simulated NGS data, but to determine which algorithm is the best on real data and determine ContaCheck's applicability in a clinical setting, the algorithms need to be further evaluated on real tumor NGS samples.

Objectifs: Malgré leur grande hétérogénéité histologique et moléculaire, la prise en charge clinique des carcinomes ovariens de haut-grade (COHG) reste peu variable. Le pronostic sombre de cette pathologie implique un réel besoin des nouvelles thérapies. Au-delà des marqueurs pronostiques histologiques classiques et des enquêtes oncogénétiques, l’objectif de cette étude a consisté à rechercher des cibles moléculaires pharmacologiquement recrutables afin de pouvoir proposer aux patientes un accès à la thérapie innovante et personnalisée. Méthodes: Cette étude a été réalisée chez 53 patientes (pts) (âge moyen 58,9 ans, intervalle 25-87) de COHG histologiquement prouvés dont 45 pts de sous-type séreux. 19 pts ont fait l’objet d’une consultation et d’un test oncogénétique sur la base d’antécédents familiaux / personnel de cancer de sein/ovaire. chez. L’expression de P53 et de PTEN a été évaluée sur des tissus fixés au formol et inclus en paraffine par immunohistochimie. Les mutations somatiques de KRAS, NRAS, BRAF, PIK3CA et MET ont été recherchées par PCR-HRM (Polymerase Chain Reaction High Resolution Melting) puis vérifiées par NGS (Next Generation Sequencing) sur des extraits d'ADN préparés à partir d'échantillons de tumeurs congelés, prélevés au moment du diagnostic. Résultats: Des mutations germinales de BRCA1 / 2 ont été identifiées chez 7 pts, toutes atteintes des carcinomes séreux. Une mutation du gène KRAS (exon 2), 2 mutations du gène NRAS (exon 3), 6 mutations du gène PIK3CA (exon 5, 10 et 21) et 5 mutations du gène MET (exon 14 et 18) ont été identifiées chez les 53 tumeurs par NGS, dont deux mutations du gène NRAS et 2 mutations du gène PIK3CA détectées précédemment par PCR-HRM. Aucun profil mutationnel multiple n’a été retrouvé. La surexpression de P53 et la perte d’expression de PTEN ont été constatées chez 32 sur 53 (60%) et 19 sur 46 (41%) des tumeurs. L’analyse statistique n’a été réalisée que chez le sous-groupe de pts atteintes des carcinomes séreux à cause de l’effectif de l’étude. Avec un suivi médian de 38 mois (intervalle de 6-93), 35 pts ont eu une rechute de la maladie et 25 pts sont décédées. La survie sans progression à 2 ans est 28%, et la survie globale à 5 ans est 37%. La surexpression de P53 a été trouvée associée à une meilleure chimiosensibilité, une meilleure survie sans progression et une meilleure survie globale. Conclusion: Pour des COHG, au-delà des altérations de P53 et PTEN, des anomalies génétiques somatiques concernant les voies de signalisation PI3K et MAPK ne sont pas rares et peuvent être détectées par NGS. L’identification de ces anomalies somatiques pourrait offrir une possibilité des thérapies ciblées innovantes pour les patientes sur la base d’éléments diagnostics moléculaires. / Objectives: Despite the great histological and molecular heterogeneity, the clinical management of high-grade ovarian carcinoma remains univo-cal. As a major subgroup of ovarian carcinoma, high-grade ovarian carci-nomas (HGOC) need novel therapy. Additionally to conventional histolog-ical prognostic markers and oncogenetic investigations, molecular diag-nostic was performed using PCR-HRM (Polymerase Chain Reaction High Resolution Melting) and NGS (Next Generation Sequencing) to identify "druggable" targets that could provide access to innovative personalized therapy. Methods: This study was performed in 53 patients (pts) (mean age 58.9 years, range 25-87) with histologically proven HGOC of which 45 pts with serous carcinoma. BRCA1/2 germline mutations had been screened in 19 pts with familial/personal history of breast/ovarian cancer justifying on-cogenetic investigations. P53 and PTEN expression was assessed on for-malin fixed paraffin-embedded tissues using immunohistochemistry. So-matic mutations of KRAS, NRAS, BRAF, PIK3CA and MET were screened using PCR-HRM and then confirmed using NGS on DNA extracts from frozen tumor specimens taken at diagnosis. Results: Seven pts had BRCA1 / 2 germline mutations, all had serous carcinomas. One mutation of KRAS (exon 2), 2 mutations of NRAS (exon 3), 6 mutations of PIK3CA (exon 5, 10 and 21) and 5 mutations of MET (exon 14 and 18) were identified using NGS, of which 2 mutations of NRAS and 2 mutations de PIK3CA detected previously by PCR-HRM, no multiple mutation was detected. P53 overexpression and PTEN loss of expression was detected respectively in 32 of 53 (60%) and 19 of 46 (41%) of all the tumors. Because of the efffective of the study, statistical analyses were restricted to pts with serous carcinoma. With a median follow-up of 38 months (range 6-93), 35 pts had disease progression and 25 pts died during the follow-up. The 2-year progression-free survival (PFS) rate was 28% and 5-year overall survival (OS) rate was 37%. Overexpression of mutant P53 was found to be associated with chemosensitivity and longer PFS and OS. Conclusion: In HGOC, beside P53 and PTEN alterations, somatic genetic abnormalities of PI3K and MAPK signaling pathways can be detected us-ing NGS and provide molecular rationale for targeted therapies, potential-ly offering new therapeutic opportunities to the patients.

Apesar de altamente estável, o DNA sofre milhares de alterações em sua estrutura diariamente, sejam essas espontâneas ou pela exposição a agentes mutagênicos. A maior parte dessas alterações é prontamente removida por um conjunto de eventos de reparo de DNA. A via de reparo por excisão de nucleotídeos (NER) é a mais versátil e flexível lidando com uma variedade de lesões que podem gerar distorções das hélices do DNA. Esses danos resultam em alterações características que, caso não reparadas, podem gerar mutações ou morte celular e, consequentemente, câncer e envelhecimento. Algumas síndromes, nas quais os pacientes são sensíveis à luz solar, estão relacionadas à deficiência no processo de NER, como a Xeroderma Pigmentosum (XP), síndrome de Cockayne (CS) e Tricotiodistrofia (TTD). Indivíduos brasileiros, incluindo pacientes com diagnóstico clínico de XP e membros das famílias, passaram por um processo in silico para a identificação variantes em genes relacionados aos processos de reparo do DNA após o sequenciamento do DNA por plataformas de nova geração (NGS: plataforma ABI 5500XL SOLiD e MiSeq Illumina) e análises de Bioinformática. Para cada paciente, foram selecionados os melhores valores de parâmetros para se realizar a busca por variantes considerando a qualidade de alinhamento e a taxa de cobertura das bases alvo. SNPs já depositados no banco de dados do projeto 1000genomes foram removidos de nossos dados. O restante das variantes foi analisado para encontrar potenciais candidatos que poderiam explicar o diagnóstico clínico do paciente. Em muitas amostras foi possível determinar pelo menos uma variante (mutação) com uma elevada possibilidade de ser responsável pelos sintomas XP. Para alguns pacientes, a má qualidade do sequenciamento ou eventos não esclarecidos durante este, dificultou a identificação de candidatos à mutação patogênica. Potenciais mutações não sinônimas foram analisadas com os programas SIFT e PROVEAN, que identificaram a potencial capacidade deletéria da alteração de aminoácido na proteína. Finalmente, foi desenvolvida uma interface de domínio público amigável, a Human Variantes do Finder Interface (http://www.varfinderhg.com.br), que visa facilitar a identificação de variantes em dados gerados por NGS. / Although highly stable, DNA molecule undergoes thousands of damage in its structure every day, due to spontaneous lesions or exposure to various mutagens. Most of these lesions are readily removed by a number of cellular DNA repair processes. The process of nucleotide excision repair (NER) is the most versatile and flexible dealing with a variety of lesions that can lead to distortions of the DNA strands. Ultraviolet irradiation induced DNA damage are the main substrates for NER. These DNA damage, if not repaired, can generate mutations or cell death causing several diseases, including cancer and aging. Some syndromes, sensitive to sunlight, are related to deficiencies in the NER process, such as Xeroderma Pigmentosum (XP), Cockayne syndrome (CS) and Trichothiodystrophy (TTD). Brazilian individuals, including patients with clinical diagnosis of XP and family members, went through in silico process for the identification of variants in genes related to DNA repair processes after DNA sequencing by next generation sequencing (NGS in the platforms ABI 5500XL SOLiD and MiSeq Illumina) and dedicated Bioinformatics pipelines. For each patient the best search pattern of variant calling was used considering the alignment quality and coverage rate of bases in target. SNPs already deposited at the 1000genomes project database were removed from the data. The remaining variants were analyzed to find potential candidates that could explain the clinical diagnosis. In many samples, it was possible to determine at least one variant (mutation) with a high possibility of being responsible for the clinical XP. For some patients, the poor quality of the sequencing or unclear events during sequencing hampered the identification of clear mutation candidates. Potential nonsynonymous mutations were analyzed with SIFT and PROVEAN softwares, which identified the potential deleterious capacity of the amino acid change in the protein. Finally, we developed a user-friendly public domain interface, the Human Variants Finder Interface (http://www.varfinderhg.com.br), which, we expect, will facilitate the identification of variants in data generated by NGS.

Dans cette thèse, j'ai étudié le rôle des sRNAs dérivés de l'hôte et du virus lors de l'infection du colza (Brassica napus, Canola) par la souche UK1 du virus de la mosaïque du navet (TuMV-UK1). En utilisant un dérivé de TuMV fusionné avec un gène codant pour la protéine fluorescente verte (TuMV-GFP), deux cultivars de colza (‘Drakkar’ et ‘Tanto’) qui diffèrent par leur susceptibilité à ce virus ont été identifiés. Le profil transcriptionnel des foyers d'infection locale, dans les feuilles de Drakkar et de Tanto, par séquençage nouvelle génération (NGS) a révélé de nombreux gènes exprimés de manière différentielle. Les mêmes échantillons d'ARN provenant de feuilles de Drakkar et de Tanto, traitées par des virus ou utilisées en contrôle, ont également servi à établir le profil NGS des sRNAs (sRNAseq) et de leurs cibles potentielles d'ARN (PAREseq). Les analyses bioinformatiques et leur validation in vivo, ont permis d’identifier les événements de clivage de transcrits impliquant des micro ARN (miRNA) connus et encore inconnus. Fait important, les résultats indiquent que TuMV détourne la voie du RNA silencing de l’hôte avec des siRNAs issus de son propre génome (vsiRNA) pour cibler les gènes de l’hôtes. Le virus déclenche également le ciblage à grande échelle des ARN messagers (ARNm) de l’hôte par l’activation de la production de siRNAs secondaires en phase, à partir de locus PHAS. À leur tour, les vsiRNAs et les siRNAs dérivés de l'hôte (hsRNAs) ciblent et clivent l'ARN viral par le complexe RISC. Ces observations éclairent le rôle des siRNAs dérivés de l'hôte et du virus dans la coordination de l'infection virale. Un autre chapitre de cette thèse est consacré à l'analyse des maladies induites par des virus en utilisant comme modèle de plante Arabidopsis, infectée par un tobamovirus, le virus de la mosaïque du colza (ORMV). De plus, ces observations ont permis de proposer un modèle dans lequel cette guérison dépend d’un adressage important de vsiRNAs secondaires antiviraux depuis leur source de production jusqu’à leurs tissus de destination, et l'établissement d'un apport en vsiRNAs capable de bloquer l'activité VSR impliquée dans la formation des feuilles symptomatiques. / In this thesis, I investigated the role of host- and virus-derived sRNAs during infection of Rapeseed (Brassica napus, Canola) by the UK1 strain of Turnip mosaic virus (TuMV-UK1). By using a TuMV derivative tagged with a gene encoding green fluorescent protein (TuMV-GFP), two rapeseed cultivars (‘Drakkar’ and ‘Tanto’) that differ in susceptibility to this virus were identified. Transcriptional profiling of local infection foci in Drakkar and Tanto leaves by next generation sequencing (NGS) revealed numerous differentially expressed genes. The same RNA samples from mock- and virus- treated Drakkar and Tanto leaves were also used for the global NGS profiling of sRNAs (sRNAseq) and their potential RNA targets (PAREseq). The bioinformatic analysis and their in vivo validation led to the identification of transcript cleavage events involving known and yet unknown miRNAs. Importantly, the results indicate that TuMV hijacks the host RNA silencing pathway with siRNAs derived from its own genome (vsiRNAs) to target host genes. The virus also triggers the widespread targeting of host messenger RNAs (mRNAs) through activation of phased, secondary siRNA production from PHAS loci. In turn, both vsiRNAs and host-derived siRNAs (hsRNAs) target and cleave the viral RNA by the RISC-mediated pathway. These observations illuminate the role of host and virus-derived sRNAs in the coordination of virus infection. Another chapter of this thesis is dedicated to the analysis of virus-induced diseases by using Arabidopsis plants infected with the Oilseed rape mosaic tobamovirus (ORMV) as a model. Initially, the infected plants develop leaves with strong disease symptoms. However, at a later stage, disease-free, “recovered” leaves start to appear. Analysis of symptoms recovery led to the identification of a mechanism in which the VSR and virus derived-siRNAs play a central role. I used Arabidopsis mutants impaired in transcriptional and post-transcriptional silencing pathways (TGS and PTGS respectively) and a plant line carrying a promoter-driven GFP transgene silenced by PTGS (Arabidopsis line 8z2). Using various techniques able to monitor virus infection, small and long viral RNA molecules, VSR activity, as well as phloem-mediated transport with in these lines, this study led to the identification of genes required for disease symptoms and disease symptom recovery. Moreover, the observations allowed to propose a model in which symptoms recovery occurs upon robust delivery of antiviral secondary vsiRNAs from source to sink tissues, and establishment of a vsiRNA dosage able to block the VSR activity involved in the formation of disease symptoms.

Apesar de altamente estável, o DNA sofre milhares de alterações em sua estrutura diariamente, sejam essas espontâneas ou pela exposição a agentes mutagênicos. A maior parte dessas alterações é prontamente removida por um conjunto de eventos de reparo de DNA. A via de reparo por excisão de nucleotídeos (NER) é a mais versátil e flexível lidando com uma variedade de lesões que podem gerar distorções das hélices do DNA. Esses danos resultam em alterações características que, caso não reparadas, podem gerar mutações ou morte celular e, consequentemente, câncer e envelhecimento. Algumas síndromes, nas quais os pacientes são sensíveis à luz solar, estão relacionadas à deficiência no processo de NER, como a Xeroderma Pigmentosum (XP), síndrome de Cockayne (CS) e Tricotiodistrofia (TTD). Indivíduos brasileiros, incluindo pacientes com diagnóstico clínico de XP e membros das famílias, passaram por um processo in silico para a identificação variantes em genes relacionados aos processos de reparo do DNA após o sequenciamento do DNA por plataformas de nova geração (NGS: plataforma ABI 5500XL SOLiD e MiSeq Illumina) e análises de Bioinformática. Para cada paciente, foram selecionados os melhores valores de parâmetros para se realizar a busca por variantes considerando a qualidade de alinhamento e a taxa de cobertura das bases alvo. SNPs já depositados no banco de dados do projeto 1000genomes foram removidos de nossos dados. O restante das variantes foi analisado para encontrar potenciais candidatos que poderiam explicar o diagnóstico clínico do paciente. Em muitas amostras foi possível determinar pelo menos uma variante (mutação) com uma elevada possibilidade de ser responsável pelos sintomas XP. Para alguns pacientes, a má qualidade do sequenciamento ou eventos não esclarecidos durante este, dificultou a identificação de candidatos à mutação patogênica. Potenciais mutações não sinônimas foram analisadas com os programas SIFT e PROVEAN, que identificaram a potencial capacidade deletéria da alteração de aminoácido na proteína. Finalmente, foi desenvolvida uma interface de domínio público amigável, a Human Variantes do Finder Interface (http://www.varfinderhg.com.br), que visa facilitar a identificação de variantes em dados gerados por NGS. / Although highly stable, DNA molecule undergoes thousands of damage in its structure every day, due to spontaneous lesions or exposure to various mutagens. Most of these lesions are readily removed by a number of cellular DNA repair processes. The process of nucleotide excision repair (NER) is the most versatile and flexible dealing with a variety of lesions that can lead to distortions of the DNA strands. Ultraviolet irradiation induced DNA damage are the main substrates for NER. These DNA damage, if not repaired, can generate mutations or cell death causing several diseases, including cancer and aging. Some syndromes, sensitive to sunlight, are related to deficiencies in the NER process, such as Xeroderma Pigmentosum (XP), Cockayne syndrome (CS) and Trichothiodystrophy (TTD). Brazilian individuals, including patients with clinical diagnosis of XP and family members, went through in silico process for the identification of variants in genes related to DNA repair processes after DNA sequencing by next generation sequencing (NGS in the platforms ABI 5500XL SOLiD and MiSeq Illumina) and dedicated Bioinformatics pipelines. For each patient the best search pattern of variant calling was used considering the alignment quality and coverage rate of bases in target. SNPs already deposited at the 1000genomes project database were removed from the data. The remaining variants were analyzed to find potential candidates that could explain the clinical diagnosis. In many samples, it was possible to determine at least one variant (mutation) with a high possibility of being responsible for the clinical XP. For some patients, the poor quality of the sequencing or unclear events during sequencing hampered the identification of clear mutation candidates. Potential nonsynonymous mutations were analyzed with SIFT and PROVEAN softwares, which identified the potential deleterious capacity of the amino acid change in the protein. Finally, we developed a user-friendly public domain interface, the Human Variants Finder Interface (http://www.varfinderhg.com.br), which, we expect, will facilitate the identification of variants in data generated by NGS.

L'objectif de mon travail de thèse était de développer des outils bio informatiques permettant d'améliorer la traditionnelle gestion de l'information scientifique au sein d'un grand centre de recherche et en particulier au sein d'une plateforme de génomique. Trois outils ont été développés: un cahier de laboratoire électronique, un système de gestion de l'information de laboratoire pour des applications de génomique dont le séquençage de nouvelle génération, ainsi qu'un système de gestion des échantillons pour de grandes bio-banques. Ce travail a été réalisé en étroite collaboration avec des biologistes, épidémiologistes et informaticiens. Il a également inclus la mise en place d'interactions entre les différents outils pour former un système informatique intégré. Les trois outils ont été rapidement adoptés par l'ensemble des scientifiques du centre de recherche et sont désormais utilisés au quotidien pour le suivi de toutes les activités de laboratoire mais aussi plus globalement pour les autres activités scientifiques du centre de recherche. Ces outils sont transposables dans d'autres instituts de recherche / The aim of my thesis work was to develop bioinformatics tools to improve the traditional scientific information management within a large research centre and especially within a genomics platform. Three tools have been developed: an electronic laboratory notebook, a laboratory information management system for genomics applications including next generation sequencing, as well as a sample management system for large biobanks. This work has been conducted in close collaboration with biologists, epidemiologists and IT specialists. It has also included the setup of interactions between the different tools to make an integrated IT system. The three tools have been rapidly adopted by all the scientists of the research centre and are now daily used for the tracking of all the laboratory’s activities but also more globally for the research centre’s other scientific activities. These tools are transposable in other research institutes

Mon projet de doctorat était de créer un pipeline pour taxono-génomique pour la comparaison de plusieurs génomes bactériens. Deuxièmement, je automatisé le processus d'assemblage (NGS) et annotation à l'aide de divers logiciels open source ainsi que la création de scripts de maison pour le laboratoire. Enfin, nous avons intégré le pipeline dans la description de plusieurs espèces bactériennes de laboratoire sur. Cette thèse est divisée principalement en Taxono- génomique et Microbiogenomics. Les avis de la section taxono-génomique, décrit sur les avancées technologiques en génomique et métagénomique pertinentes dans le domaine de la microbiologie médicale et décrit la stratégie taxono-génomique en détail et comment la stratégie polyphasique avec des approches génomiques sont reformatage de la définition de la taxonomie bactérienne. Les articles décrivent les bactéries cliniquement importantes, leur séquençage complet du génome et les études génomiques comparatives, génomiques et taxono-génomique de ces bactéries. Dans cette thèse, j'ai inclus les articles décrivant ces organismes: Megasphaera massiliensis, Corynebacterium ihumii, Collinsella massiliensis, Clostridium dakarense. Bacillus dielmoensis, jeddahense, Occidentia Massiliensis, Necropsobacter rosorum et Pantoea septica. Oceanobacillus / My PhD project was to create a pipeline for taxono-genomics for the comparison of multiple bacterial genomes. Secondly I automated the process of assembly (NGS) and annotation using various open source softwares as well as creating in house scripts for the lab. Finally we incorporated the pipeline in describing several bacterial species from out lab. This thesis is subdivided mainly into Taxono-genomics and Microbiogenomics. The reviews in taxono-genomics section, describes about the technological advances in genomics and metagenomics relevant to the field of medical microbiology and describes the strategy taxono-genomics in detail and how polyphasic strategy along with genomic approaches are reformatting the definition of bacterial taxonomy. The articles describes clinically important bacteria, their whole genome sequencing and the genomic, comparative genomic and taxono-genomic studies of these bacteria.

8 Summary The thesis "The elucidation of the causes of neurogenetic diseases by the MPS data analysis using advanced algorithms" is focused on processing the massively parallel sequencing (MPS) data from a gene panel, whole-exome sequencing (WES) and whole-genome sequencing (WGS). The aim of the study was to develop a suitable pipeline to evaluate at least 250 MPS gene panel data, 150 WES data and 20 WGS data in order to improve molecular genetic testing of rare neurogenetic disorders. Associated data management and database implementation is also described. Targeted gene panel sequencing A custom-designed gene panel consisting of ge- nes previously associated with the disease was used. In the Epileptic Encephalopathy (EE) panel, two prerequisites need to be met for inclusion into the panel: the gene has to have been published in at least two independent publications OR at least in one publication but in multiple independent families. In the case of the EE panel, 112 genes were included. The targeted gene panel sequencing was then performed on 257 patients with EE. Pathogenic or likely pathogenic (according to ACMG criteria) variants have been found in 28% of patients (72 out of 257). Further analysis of the pathogenic or likely pathogenic variants was performed (76 in total); the variants were grouped by...

Deafblindness is a combined impairment of vision and hearing with an incidence of about 1: 8000 children and 1: 5500 adults. The most common genetic causes are the Stickler (STL) and Usher (USH) syndromes. The main goal of this work is to provide an up-to-date overview of STL and USH in the Czech and Slovak Republic (CR and SR), to determine the correlations between the genotype and phenotype in our population and the associated diagnostic criteria. Using sequencing and MLPA we examined 45 patients from 28 families for suspected STL. We found potentially causal variants of STL genes in 39 patients from 22 families. Fifteen different COL2A1 variants (8 being novel) were found in 28 patients from 18 families and 4 novel COL11A1 variants were found in 11 patients from 4 families. We identified the cause of the disease in 79 % of the families. The USH study involved 30 patients from 27 families. The most frequent cause was USH2A pathogenic variants, i.e. 19 variants in 14 families, 9 being novel. Less common were pathogenic variants in MYO7A (6 variants in 3 families, 5 being novel), USH1C and CDH23 (3 variants, 2 being novel, in 2 families both) genes. In 2 families, compound heterozygosity was found for variants in two different USH genes. The deafblindness etiology was clarified for 24 patients from...

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