Tesis sobre el tema "MinION sequencing"

In-field whole genome sequencing has been made possible by the introduction of the MinION™ DNA sequencer by Oxford Nanopore Technologies. Current forensic DNA analysis requires samples to be sent to a dedicated lab offsite. Depending on the location this process can take time. The evaluation of alternatives such as field sequencing using the MinION™, with the possibility of providing access to results in a shorter time with lower costs, should be researched and validated. The potential applications of the MinION™ in forensic scenarios have not been studied partly due to the recent release of the product. This study focused on evaluating the utility of the MinION™ for shotgun sequencing of forensically relevant samples without targeted amplification.

The requirement for DNA evidence in forensics has increased, meaning the demand for DNA typing has also increased. Current analytical processes for DNA evidence are known to be costly and time-consuming and traditionally occur at a centralised laboratory which can impact on the amount of time from sample collection to DNA profile generation. Therefore, research has focused on creating technologies that are capable of in-field analysis. Oxford Nanopore Technologies developed MinION™, a portable, cost-effective nanopore sequencer that is capable of in-field analysis. The development of in-field sequencing technologies is favourable for isolated and remote communities where traditional laboratory environments are not feasible. Furthermore, the development of these processes is favourable as backlogs and costs with traditional methods can be reduced. In-field sequencing also has the potential to be used in a range of disciplines including personal healthcare, pathogen identification and disaster victim identification. With the advancement of sequencing technologies research has also focused on how to increase the discriminatory power of DNA typing with the selection of alternative markers specific for human identification. This review will investigate current sequencing technologies and techniques as well as evaluating current targets for DNA analysis.

Bien que les sexes séparés (dioecie) soient plus rares que chez les animaux, ∼15 600 espèces dioiques ont évolué chez les angiospermes (∼6% de l'ensemble des espèces). La manière dont le sexe de ces plantes est contrôlé est une question centrale de la biologie végétale, mais également de l'agronomie car de nombreuses plantes cultivées sont des plantes dioiques (∼20% des espèces cultivées) mais dont un seul sexe (généralement les femelles) présente un intérêt agronomique. Pourtant, seulement trois gènes du déterminisme du sexe ont été identifiés à ce jour chez les plantes dioiques, chez le kaki, l'asperge et la fraise. La dioecie a vraisemblablement évolué plusieurs fois chez les angiospermes et il est possible que les gènes du déterminisme du sexe soient divers. Deux voies principales d'évolution vers la dioecie ont été identifiées. Les deux partent d'une espèce dont les fleurs sont hermaphrodites, le régime de reproduction ancestral chez les angiospermes, puis passent soit par un intermédiaire monoique (espèce avec des fleurs unisexuées mâles et femelles sur le même individu), soit par un intermédiaire gynodioique (espèce avec des femelles et des individus avec des fleurs hermaphrodites). Cette thèse a pour objet la comparaison de deux systèmes de plantes représentant ces deux voies. Chez Coccinia grandis, une cucurbitacée ayant également des chromosomes XY, l'évolution de la dioecie est passée par la monoecie. Chez Silene latifolia, une plante dioique bien étudiée avec des chromosomes sexuels XY, l'évolution de la dioecie s'est faite à partir de la gynodioecie. Trois gènes contrôlant la monoecie ont été identifiés chez le melon et il a été proposé que ces gènes soient les gènes du déterminisme dans les espèces dioiques proches du melon comme C. grandis. Nous avons donc opté pour une approche gène candidat dans cette espèce. Très peu de ressources génétiques et génomiques sont disponibles chez C. grandis, et nous avons choisi d'utiliser SEXDETector, une méthode probabiliste qui utilise des données RNA-seq pour génotyper des parents et leurs descendants, et qui infère les gènes lies au sexe sans génome de référence. Cette méthode m'a permis d'identifier 1 364 gènes présents sur les chromosomes sexuels de C. grandis. J'ai établi que les gènes differentiellement exprimés entre les sexes étaient plus abondants sur chromosomes sexuels que sur les autosomes. J'ai également observé des marques de la dégénérescence du chromosome Y chez cette plante, comme des diminutions d'expression ou des pertes de gènes. Enfin, mes résultats démontrent la présence de compensation de dosage chez C. grandis. Le test des gènes candidats est en cours. Chez S. latifolia, 3 grandes régions liées au déterminisme ont déjà été identifiées sur le chromosome Y. Pour identifier les gènes du déterminisme, nous avons choisi de séquencer ce chromosome. Le séquençage des chromosomes Y est encore un défi pour la génomique. La phase d'assemblage est très difficile à cause des répétitions présentes en grand nombre sur ces chromosomes. En conséquence, les séquences complètes de chromosome Y sont très rares, et principalement disponibles chez les animaux. Afin de minimiser les problèmes d'assemblage dus aux répétitions, nous avons utilisé des techniques dites de 3eme génération (avec de grandes lectures). J'ai moi-même généré des données MinION (Oxford Nanopore) à partir d'ADN de chromosome Y. L'assemblage a été réalisé en combinant des données Illumina, PacBio et MinION. Notre assemblage final fait une taille de 563 Mb pour un N50 de 6 114 pb, et contient 16 219 gènes annotés de novo
Although rarer than in animals, separate sexes (dioecy) have evolved in ∼15,600 angiosperm species (∼6% of all angiosperm species). How sex is controlled is a central question in plant sciences and also in agronomy as many crops are dioecious (∼20% of crops) with only one useful sex (usually female). Only three master sex-determining genes have been identified in dioecious plants so far, namely in persimmons, asparagus and strawberry. Dioecy likely evolved several times independently in angiosperms, suggesting that sex-determining genes are of diverse origins. Hermaphroditism is the predicted ancestral state of the angiosperm flower. Two main pathways have been identified that explain the evolution of hermaphroditism towards dioecy: either through a monoecious state (with both unisexual male and female flowers on the same individual) or a gynodioecious state (with females and individuals having hermaphroditic flowers). My aim is to compare two plant systems representing each one of these two pathways. In Coccinia grandis, a Cucurbitaceae with an XY chromosome system, dioecy evolved through monoecy. In Silene latifolia, a well-studied dioecious plant with XY sex chromosomes, dioecy evolved through gynodioecy. Three genes controlling monoecy have been identified in melon, and it was suggested that these genes act as sex-determining genes in closely related dioecious species such as C. grandis. I therefore chose a candidate gene approach in this species. Very few genetic and genomic data are available in C. grandis, and we chose to use SEX-DETector, a probabilistic method that uses RNA-seq data to genotype parents and their offspring, and infers sex-linked genes with no need for a reference genome. This method allowed me to identify 1,364 genes that are present on the sex chromosomes of C. grandis. I found that the sex chromosomes are enriched in sex-biasedgenes when compared to autosomes and I characterized Y chromosome degeneration in terms of decreased expression and gene loss. Finally, I showed that dosage compensation occurs in C. grandis. Testing for the three candidates genes is ongoing. In S. latifolia 3 regions involved in sex determination have already been identified on the Y chromosome. We chose to sequence this chromosome to identify sex-determining genes. The sequencing of Y chromosomes remains one of the greatest challenges of current genomics. The assembly step is very difficult because of their highly repeated content. Consequently, fully sequenced Y chromosomes are rare and mainly available for research in animals. To overcome the difficulty of assembling reads with many repeats, I used third generation sequencing (TGS, producing long reads). I produced a dataset using the Oxford Nanopore MinION sequencer with Y chromosome DNA. Assembling was performed using a combination of Illumina, MinION and PacBio sequencing data. The final assembly had a total length of 563 Mb with a scaffold N50 of 6,114 bp, and contained 16,219 de novo annotated genes

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.

This thesis reviews, analyses and classifies extraction methods and sequences used in steep, tabular orebodies in Ontario hardrock mines and how new bulk mining techniques have affected them.
The thesis examines, as a case study, the complexities of sequencing the extraction of the Hemlo orebody through three adjacent mines and details the planning and monitoring of extracting one section of the David Bell orebody. It examines the use of numerical modelling as a tool in extraction sequence planning and demonstrates how, in conjunction with an instrumentation program, the results assist successful completion of the plan.
The thesis concludes that bulk mining sequences have increased resource extraction and productivity, lowered costs and improved ground control aspects of extraction planning; but have compromised grade control. This aspect should be improved to maximize the economic benefits. In addition, as automated or continuous mining methods are developed, their benefits should be analyzed in the context of overall sequence planning objectives.

Magíster en Minería
Ingeniero Civil de Minas
Currently, mine plans are optimized by using many criteria, such as profit, life of mine, concentration of some pollutants, mining costs, confidence level or mineral resources, while attending constraints related to production rates, plant capacities and grades. Whilst this approach is successful in terms of producing high value production schedules, it uses a static sequence of production units (for open pit and underground mine) and therefore the optimization is performed within the level of freedom left by the original opening schedule and is far from the optimal value of the project according to the objective function. This approach is often used in the industry and therefore the value addition that is involved when optimizing the mining sequence is disregarded. This thesis summarizes a research that includes applying a model to optimize the NPV value, as the objective function, in a panel cave mine and evaluating this model with different mining systems, to study the drawpoints opening sequence and the NPV variations. The emphasis is in the precedence, geometrical and production constraints that are required to produce meaningful operational drawpoints opening sequences considering the exploitation method (panel caving), physical considerations and logical rules. Further on, while it applies the standard approach of maximizing NPV, other targets for optimization, such as the mining material handling system, are considered. The idea is to consider the drawpoints opening sequence as an output of the problem and to select the best sequence considering different mining systems. The results indicate that the selection of the mining system is important when comparing the results of the objective function or the grade. The results can vary up to 18%. En la actualidad, los planes de producción mineros son optimizados usando diferentes criterios como el beneficio económico, la vida de la mina, la concentración de contaminantes, los costos mina, el nivel de confiabilidad o las reservas mineras, atendiendo a restricciones relacionadas a la producción, capacidades de planta y leyes. Si bien esta aproximación es eficaz en términos de producir planes de producción de alto valor agregado, usa una secuencia de las unidades de producción estática (tanto para minería a cielo abierto como para minería subterránea) y por lo tanto la optimización es realizada con un grado de libertad menos, debido al uso de la secuencia predefinida (secuencia original) y esto está lejos de ser el valor óptimo del proyecto, de acuerdo con la función objetivo utilizada. Esta aproximación se usa a menudo en la industria y por lo tanto, cuando se optimiza la secuencia de explotación de las unidades de producción, el valor agregado involucrado no se percibe. Esta tesis resume una investigación que incluye la aplicación de un modelo para optimizar el VAN como función objetivo, en una mina de Panel Caving y evaluando este modelo para distintos sistemas mineros, de tal forma de estudiar la secuencia de apertura de los puntos de extracción y las variaciones del VAN asociadas. El énfasis se encuentra en las restricciones de precedencia, geométricas y de producción, que son requeridas para producir secuencias de apertura de puntos de extracción significativas considerando el método de explotación (Panel Caving), consideraciones físicas y reglas lógicas. Entonces, mientras se aplica la aproximación estándar para maximizar el VAN, se consideran otros inputs para la optimización como por ejemplo el sistema minero. La idea es considerar la secuencia de apertura de puntos de extracción como un output del problema y seleccionar la mejor secuencia dados distintos sistemas mineros. Los resultados indican que la selección del sistema minero es importante, ya que los resultados de la función objetivo son muy distintos (así como la ley media) para cada sistema considerado. Los resultados pueden variar hasta un 18%.

This study presents a comprehensive mathematical model for open pit mine block sequencing problem which considers technical aspects of real-life mine operations. As the open pit block sequencing problem is an NP-hard, state-of-the-art heuristics algorithms, including constructive heuristic, local search, simulated annealing, and tabu search are developed and coded using MATLAB programming language. Computational experiments show that the proposed algorithms are satisfactory to solve industrial-scale instances. Numerical investigation and sensitivity analysis based on real-world data are also conducted to provide insightful and quantitative recommendations for mine schedulers and planners.

In this thesis, I propose new algorithms and models to address biological problems. Computer science in fact plays a key role in proteomics and genetics research due to the advent of big datasets. In the context of protein study, I developed new methods for protein function prediction based on information retrieval principles. By using heterogeneous source of knowledge, like graph search and sequence similarity, I designed a tool called INGA that can be used to annotate entire genomes. It has been benchmarked during the Critical Assessment of Function Annotation challenge, and it proved to be one of the most effective approach for function inference. To better characterize proteins from the structural point of view, I proposed a protein conformers detection strategy based on residue interaction network (RIN) data. RIN graphs were extended to deal with the time-dependent protein coordinate fluctuations, and were generated by clustering algorithms. An implementation called RING MD highlighted effectively the key amino acids known to be functionally relevant in Ubiquitin. These amino acids in fact are very important to explain the protein three-dimensional dynamics. With the same rationale, RIN graphs were used also to predict the impact of mutations within a protein structure. By combining information about a mutant node in the network and its features, an artificial neural network was trained to estimate the free Gibbs energy change of a protein. Extreme changes in the internal energy might lead to the protein unfolding, and possibly to disease. The reduction of a protein flexibility may hamper its function as well. As an example, the extreme fluctuations observed in intrinsically disordered proteins (IDPs) are fundamental for their activities. To better understand IDPs, I contributed in the collection of the largest dataset of disordered regions. In the following analysis, it was shown what are the typical functions of these sequences and the biological processes where they are involved. Due to the importance of their detection, a comprehensive assessment of disorder predictors was performed to show what are the state-of-the-art methods and their limitations. In the context of genetics, I focused on phenotype prediction. During the Critical Assessment of Genome Interpretation (CAGI), I proposed new approaches for the analysis of exome data to prioritize the risk of Crohn's disease and abnormal cholesterol levels. These are often defined as complex disease, since the mechanism behind their insurgence is still unknown. In my study, human samples with an enrichment of mutations in critical genes were predicted to have an high genetic risk. In addition to disease associated genes, protein interaction networks were considered to better account for variants accumulation in biological pathways. Such strategy was shown to be among the best approaches by CAGI organizers. In the simpler case of Mendelian traits, with BOOGIE I designed a method for human blood groups prediction based on exome data. It uses a specialized version of nearest neighbor algorithm in order to match the gene variants in an unannotated exome with the ones available in a reference knowledge base. The most similar hit is used to transfer the blood group. With an accuracy above 90%, BOOGIE is a proof-of-concept that shows the potential applications of genetic prediction, and can be easily extended to any Mendelian trait. To summarize, this thesis is a partial answer to the exponential growth of sequences available that need further experiments. By integrating heterogeneous information and designing new predictive models based on machine learning, I developed novel tools for biological data analysis and classification. All implementations are freely available for the community and might be helpful during future investigations like in drug design and disease studies.
In questa tesi, vengono proposti nuovi algoritmi e modelli per affrontare problemi biologici. L'informatica svolge un ruolo chiave nella proteomica e nella ricerca genetica dovuto alla gestione delle grandi moli di dati biologici. Nel contesto dello studio di proteine, ho sviluppato nuovi metodi per la predizione delle loro funzioni basati su principi di reperimento dell'informazione. Utilizzando fonti eterogenee di conoscenza, come la ricerca su grafi e la similarità di sequenze, ho progettato uno strumento chiamato INGA che può essere utilizzato per annotare interi genomi. Questo è stato valutato imparzialmente dal Critical Assessment of Function Annotation, e ha dimostrato di essere uno degli approcci più efficaci per l'inferenza di funzione. Per meglio caratterizzare le proteine dal punto di vista strutturale, ho proposto una strategia di rilevamento delle conformazioni delle proteine basata su rete di interazione di residui (RIN). Le reti RIN sono state quindi estese per gestire le fluttuazioni temporali delle coordinate atomiche. Tali grafi sono stati infine generati automaticamente da algoritmi di clustering. Un'implementazione chiamata RING MD ha evidenziato efficacemente i principali amminoacidi noti per essere funzionalmente rilevanti nell'Ubiquitina. Questi aminoacidi sono infatti molto importanti per spiegare la dinamica strutturale della proteina. Con la stessa logica, sono stati usati i grafi RIN anche per prevedere l'impatto delle mutazioni all'interno di una struttura proteica. Combinando informazioni sul nodo mutante in una rete e le sue caratteristiche, una rete neurale artificiale è stata addestrata per stimare la variazione di energia libera di Gibbs all'interno di una proteina. Cambiamenti estremi nell'energia interna potrebbe portare all'unfolding della proteina, ed eventualmente ad una malattia. D'altro canto, anche la riduzione della flessibilità proteica può ostacolare la sua funzione. Ad esempio, le fluttuazioni estreme osservate nelle proteine intrinsecamente disordinate (IDP) sono fondamentali per le loro attività. Per studiare le IDP, ho contribuito alla raccolta del più grandi dataset di regioni disordinate mai esistito. Nella seguente analisi è stato dimostrato quali sono le funzioni tipiche di queste sequenze e i processi biologici in cui sono coinvolte. Data l'importanza della loro identificazione, una valutazione globale di predittori del disordine è stata eseguita per mostrare quali sono i metodi più efficaci e le loro limitazioni. Nel contesto della genetica, mi sono concentrato sulla previsione di fenotipi. Durante il Critical Assessment of Genome Interpretation (CAGI), ho proposto nuovi approcci per l'analisi dei dati dell'esoma progettati per valutare il rischio di morbo di Crohn e di ipercolesterolemia. Queste sono spesso definite come malattie complesse, dal momento che il meccanismo alla base della loro insorgenza è ancora sconosciuto. Nel mio studio, i campioni umani con un arricchimento di mutazioni in geni critici sono stati predetti come soggetti a rischio genetico elevato. Oltre ai geni associati alla malattia, le reti di interazione proteiche sono state considerate per valutare l'accumulo di varianti in pathway biologici. Tale strategia ha dimostrato di essere tra le migliori secondo gli organizzatori del CAGI. Nel caso più semplice dei tratti mendeliani, con BOOGIE ho progettato un metodo per la predizione dei gruppi sanguigni umani basata su dati di esoma. Esso utilizza una versione specializzata dell'algoritmo nearest neighbour al fine di far corrispondere le varianti genetiche in un esoma non annotato con quelle disponibili in una base di conoscenza di riferimento. L'esempio più simile è usato per trasferire il gruppo sanguigno. Con una precisione superiore al 90%, BOOGIE è un prototipo che mostra le potenziali applicazioni della predizione genetica, e può essere facilmente esteso a qualsiasi tratto mendeliano. Riassumendo, questa tesi è una risposta parziale alla crescita esponenziale di sequenze disponibili che necessitano ulteriori esperimenti. Integrando informazioni eterogenee e la progettazione di nuovi modelli predittivi basati su apprendimento automatico, ho sviluppato nuovi strumenti per l'analisi di dati biologici e per la loro classificazione. Tutte le implementazioni sono liberamente disponibili per la comunità e potrebbero essere utili durante indagini future come in studi di malattie e nella progettazione di farmaci.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Aeronautics and Astronautics, 2005.
Includes bibliographical references (p. 99-101).
(cont.) 65.6% and 67.0% and the average NAS delay by 24.3% and 24.7% relative to the FIFO and Serve-the-Longest-Queue algorithms respectively.
Two methods to improve runway throughput are evaluated in this thesis. The first, increasing runway capacity during periods of bad weather by reducing IFR arrival-arrival wake vortex separations. The second, increasing runway efficiency in all weather conditions using event sequencing algorithms. Two algorithms were studied: a Serve-the-Longest-Queue algorithm for flight sequencing coupled with a greedy heuristic algorithm for runway assignment and a mixed integer programming optimization algorithm for simultaneous flight sequencing and runway assignment. The MIT Extensible Air Network Simulation (MEANS) was used to simulate NAS op- erations to determine the potential benefits in terms of delay reduction for both methods. For the case where reduced IFR arrival-arrival wake vortex separations was studied, the Airport Runway Capacity Calculator (ARCC), developed in support of this work, was used to determine the increased capacity at eleven congested US airports. Results indicate that the total delay in the National Airspace System (NAS) could have been reduced by 31.8% over the month of January, 1999 (a reduction of 243,672 minutes) representing a benefit of 116 minutes per IFR hour. For the cases where the event sequencing algorithms were studied, the algorithms were only implemented at Newark Airport (EWR) and the resulting delay values were compared to the performance of a FIFO algorithm that is representative of existing operations. The flight delay for the Serve-the-Longest-Queue algorithm and the FIFO algorithm were similar, though relative performance depended on the airline schedule. The integer programming optimization algorithm out performed the other two algorithms significantly reducing the average delay at EWR by
by Elizabeth Bly.
S.M.

Over the last two decades the advancement in DNA sequencing technologies has enormously increased the amount of sequencing data available to researchers and geneticists. This has been accompanied by the development of tools for sequencing data analysis, including the human reference genome, that is undoubtedly an indispensable resource. It is known that the reference genome does not always represent the real consensus sequence of the human population, due to the inclusion of rare alleles and sequencing errors. Moreover, genomic duplications are often misassembled and, as a result, they may be found in the reference genome as a collapsed consensus, thus generating false variants. In this work I performed a thorough search for conflicting information between the human reference genome (GRCh37 and GRCh38) and some of the most popular human genetic resources such as the 1000 Genomes Project, to disclose minor alleles and to mine genetic inconsistencies. To search for unreported genomic duplications, I performed a genome wide screening for unbalanced heterozygosity. I found that inaccuracies and errors are much higher than expected. Minor alleles occurring with a frequency <10% are found on average every ~7,000 bases and include many rare variants that are never found elsewhere, producing high numbers of false positives as well as possible false negatives. The systematic screening for unbalanced heterozygosity revealed ~86,000 variants that are likely the result of unreported genomic duplications, involving functionally relevant genes such as MAP2K3 and KCNJ12. My findings may help the ongoing quest to obtain a highly accurate human genome reference sequence. Moreover, the results presented in this thesis will be useful to human geneticists in the process of filtering and selecting causative variants. The advancement in DNA sequencing technologies also accounts for the increasing usage of Whole Genome Sequencing approaches both in the research and clinical fields, thus revealing that the large majority of disease-associated SNPs are located in non-coding regions of the human genome. However, the functional interpretation of non-coding variants is still challenging. Part of my work also addressed this problem, aiming to develop a method for non-coding variant prioritization. The method, presented in the last chapter of this thesis, is based on a comparative genomics approach for the identification of functional constraints in primate orthologous genes. The first steps of my approach have proved to be powerful in identifying orthologous genes, but further work is necessary to optimize the multiple sequence alignment step and the identification of conserved domains.
Nel corso dell’ultimo ventennio l’avanzamento tecnologico nel campo del sequenziamento del DNA ha portato a un enorme aumento della quantità di dati di sequenziamento accessibili a ricercatori e genetisti. Questa crescita è stata accompagnata dallo sviluppo di strumenti necessari all’analisi dei dati; tra questi il genoma umano di riferimento è senza dubbio una risorsa indispensabile. È noto che il genoma di riferimento non sempre rappresenta la reale sequenza consenso della popolazione umana, poiché alleli rari ed errori di sequenziamento sono stati inclusi in essa. Inoltre, duplicazioni genomiche sono spesso mal assemblate e, di conseguenza, possono essere trovate nel genoma di riferimento come collassate, generando così false varianti. In questa tesi è descritta la ricerca approfondita di incongruenze tra il genoma umano di riferimento (GRCh37 e GRCh38) e alcune delle più popolari risorse di genetica umana, come il 1000 Genomes Project, per scovare alleli minori e inconsistenze genetiche. Per identificare duplicazioni genomiche non riportate nel genoma, è stata poi condotta un’ampia ricerca di eterozigosità sbilanciata. Questa analisi ha dimostrato che incongruenze ed errori sono molto più frequenti di quanto atteso. Infatti, alleli minori con una frequenza <10% sono stati trovati in media ogni ~7,000 basi e tra essi sono presenti molte varianti rare mai riportate nei database. Lo screening sistematico per l’eterozigosità sbilanciata ha mostrato inoltre che ~86,000 varianti possono derivare da duplicazioni genomiche non riportate nella sequenza di riferimento e che alcune di esse coinvolgono geni importanti come MAP2K3 e KCNJ12. I risultati descritti in questo lavoro possono contribuire alla definizione di una sequenza di riferimento del genoma umano altamente accurata. Inoltre, questi stessi risultati potranno essere utili ai genetisti umani nel processo di filtraggio e selezione delle varianti potenzialmente associate a malattie. L’avanzamento nel settore del sequenziamento del DNA ha condotto inoltre dell’utilizzo sempre maggiore degli approcci di sequenziamento dell’intero genoma, sia nel campo della ricerca sia nella diagnosi clinica, rivelando così che la gran parte degli SNP associati a malattia è localizzata nelle regioni non codificanti del genoma umano. Tuttavia, l’interpretazione funzionale delle varianti non codificanti è ancora una questione problematica. Parte del mio lavoro ha riguardato anche questo aspetto, con lo scopo di sviluppare un metodo per la prioritizzazione delle varianti non codificanti. Questo metodo, descritto nell’ultimo capitolo della tesi, si basa su un approccio di genomica comparata per l’identificazione di domini funzionali in geni ortologhi di organismi primati. I primi passaggi di questo approccio hanno dimostrato essere molto buoni per l’identificazione dei geni ortologhi, ma ulteriore lavoro è necessario per ottimizzare il processo di allineamento multiplo delle sequenze e l’identificazione dei domini conservati.

Desenvolver o sequenciamento de mina envolve muitos fatores e uma grande quantidade de informações, consequentemente, a rentabilidade do projeto dependerá fortemente do cronograma de produção. Um projeto de mineração pode estar condicionado ao sequenciamento não ideal, o que afeta os resultados econômicos do projeto e também leva a uma utilização inadequada dos recursos minerais. Durante o sequenciamento da mina é necessário tomar decisões para cada bloco durante a extração, tais como: 1) Se um determinado bloco deve ou não ser minerado; 2) Se minerado, quando deveria ser extraído; 3) Uma vez extraído, como e quando deve ser processado. Essas decisões são fatores variáveis do fluxo de caixa que afetam um dado projeto. Via de regra, o limite da cava final é otimizado para um conjunto fixo de parâmetros (preço, custos, modelos de recursos etc.), sabendo que mudanças nesses parâmetros terão impactos sobre a vida do projeto. Incertezas são avaliadas apenas em analises de sensibilidade posteriores. O método convencional de sequenciar uma mina é dividido em três etapas principais: primeiro, delinear a cava final; segundo, dividir a cava final em pushbacks (avanços de lavra) e terceiro, definir os blocos do sequenciamento em cada um desses pushbacks levando em consideração a mina, a planta de processamento e as capacidades de mercado. Contudo, existem aspectos que não são geralmente incorporados no sequenciamento de produção como inclusão de minério abaixo do nível freático. Esse aspecto, por exemplo, impõe algumas limitações de avanços verticais que devem ser consideradas. O objetivo deste estudo é demonstrar a relevância e o impacto do lençol freático como restrição adicional no resultado do VPL (Valor Presente Líquido). Inclui-se como uma condição de contorno relacionada com a necessidade de rebaixamento do nível freático, considerando as variáveis: teor e relação estéril/minério no sequenciamento de uma mina de fosfato. A metodologia adotada consiste em fazer ajustes na determinação dos pushbacks dentro da cava final, respeitando as condições de contorno e perseguindo a rentabilidade global como objetivo principal do processo de otimização.
Developing mine sequencing involves many factors and a large amount of information, consequently the profitability of the project will strongly depend on the production schedule. A mining project may be conditioned to non-optimal sequencing, which may affect the economic results of the project and also lead to an inadequate utilization of mineral resources. During the mine sequencing it is necessary to make decisions for each block during extraction, such as: 1) If a particular block should or should not be mined; 2) If mined, when it should be extracted; 3) Once extracted, how and when it should be processed. These decisions are variable factors of cash flow that affect a given project. Normally, the final pit limit is optimized for a fixed set of parameters (price, costs, resource model, etc.), knowing that changes in these parameters will have impact on the life of mine plan. Uncertainties are held only in subsequent sensitivity analyzes. The conventional method of sequencing a mine is divided into three main steps: First the delineation of the final pit; second, subdividing the final pit in operational pushbacks (mining advances) and third, sequencing blocks in each of these pushbacks, taking into consideration the mine, processing plant and market capacities. However, there are aspects that are not usually incorporated into the production scheduling, including mining ore below the water table, this aspect imposes some vertical mining advancement constraints that must be considered. The objective of this study is to demonstrate the relevance and impact on the NPV result from the water level as a constraint related to the need for water table drawdown, also considering grades and stripping ratio variability during the mining sequence for a phosphate mine. The methodology adopted considers adjustments when determining each pushback within the limit of the final pit, trying to respect the constraints and pursuing global profitability as the main goal of the optimization process.

Au cours de la dernière décennie, la recherche biomédicale et la pratique médicale ont été révolutionné par l'ère post-génomique et l'émergence des « Big Data » en biologie. Il existe toutefois, le cas particulier des maladies rares caractérisées par la rareté, allant de l’effectif des patients jusqu'aux connaissances sur le domaine. Néanmoins, les maladies rares représentent un réel intérêt, car les connaissances fondamentales accumulées en temps que modèle d'études et les solutions thérapeutique qui en découlent peuvent également bénéficier à des maladies plus communes. Cette thèse porte sur le développement de nouvelles solutions bioinformatiques, intégrant des données Big Data et des approches guidées par la connaissance pour améliorer l'étude des maladies rares. En particulier, mon travail a permis (i) la création de PubAthena, un outil de criblage de la littérature pour la recommandation de nouvelles publications pertinentes, (ii) le développement d'un outil pour l'analyse de données exomique, VarScrut, qui combine des connaissance multiniveaux pour améliorer le taux de résolution
Over the last decade, biomedical research and medical practice have been revolutionized by the post-genomic era and the emergence of Big Data in biology. The field of rare diseases, are characterized by scarcity from the patient to the domain knowledge. Nevertheless, rare diseases represent a real interest as the fundamental knowledge accumulated as well as the developed therapeutic solutions can also benefit to common underlying disorders. This thesis focuses on the development of new bioinformatics solutions, integrating Big Data and Big Data associated approaches to improve the study of rare diseases. In particular, my work resulted in (i) the creation of PubAthena, a tool for the recommendation of relevant literature updates, (ii) the development of a tool for the analysis of exome datasets, VarScrut, which combines multi-level knowledge to improve the resolution rate

This dissertation presents the applications of state-of-the-art computation techniques and data analysis algorithms in three physical and biological problems: assembling DNA pieces, optimizing self-assembly yield, and identifying correlations from large multivariate datasets. In the first topic, in-depth analysis of using Sequencing by Hybridization (SBH) to reconstruct target DNA sequences shows that a modified reconstruction algorithm can overcome the theoretical boundary without the need for different types of biochemical assays and is robust to error. In the second topic, consistent with theoretical predictions, simulations using Graphics Processing Unit (GPU) demonstrate how controlling the short-ranged interactions between particles and controlling the concentrations optimize the self-assembly yield of a desired structure, and nonequilibrium behavior when optimizing concentrations is also unveiled by leveraging the computation capacity of GPUs. In the last topic, a methodology to incorporate existing categorization information into the search process to efficiently reconstruct the optimal true correlation matrix for multivariate datasets is introduced. Simulations on both synthetic and real financial datasets show that the algorithm is able to detect signals below the Random Matrix Theory (RMT) threshold. These three problems are representatives of using massive computation techniques and data analysis algorithms to tackle optimization problems, and outperform theoretical boundary when incorporating prior information into the computation.
Engineering and Applied Sciences

A indústria de mineração investiga continuamente processos de reconciliação e técnicas adequadas para mapear possíveis riscos na recuperação do minério e no planejamento de lavra. Um estudo de caso em uma mina de cobre brasileira investiga a adequação do uso de teores simulados para a definição de áreas de risco que afetam o planejamento mineiro e as reservas minerais definidas. Simulações condicionais foram usadas para derivar múltiplos modelos de teores de cobre dentro de um típico corpo de minério do depósito e esses modelos foram comparados com os dados reais de produção (reconciliação). A comparação permitiu uma melhor compreensão sobre a variabilidade da qualidade e ajudou na definição de um plano de produção mais consistente. Com os intervalos de valores provenientes das simulações foi possível mapear importantes áreas de incerteza que afetam o plano de lavra. Análises de risco foram conduzidas para a definição da cava final através da comparação do limite previsto pela estimativa por krigagem ordinária e as cavas resultantes dos modelos simulados de teor de cobre, sendo possível avaliar os impactos operacionais nos principais fatores econômicos (valor presente líquido e fluxo de caixa descontado). Várias cavas finais foram geradas para vários modelos de blocos simulados, comparando-se com a cava anteriormente planejada através da utilização do modelo de blocos produzido pela krigagem ordinária. Além disso, foi definida uma escala de risco utilizando os modelos condicionalmente simulados e os valores estimados pela krigagem (comumente utilizados como referência para definição do orçamento e da alimentação de metal no moinho) a fim de minimizar as variações dos teores. Estas medidas se propõem a reduzir o risco no cumprimento das metas de produção de metal no planejamento de lavra. Esta abordagem, considerando o risco durante o sequenciamento de lavra, foi utilizada no plano de produção anual da mina e comparada com o realizado no período e com a abordagem tradicionalmente aplicada. Os resultados evidenciaram possíveis riscos associados com o limite da cava final operacional e demonstraram os benefícios do uso de análise de risco como uma ferramenta para visualizar e analisar os limites de cava final e planejamento de lavra, ajudando a tomar melhores decisões estratégicas no gerenciamento da mina referente aos fatores técnicos e indicadores econômicos.
Mining industry continuously investigates proper reconciliation procedures and techniques for mapping possible risks in ore recovery and mine planning. A case study at a Brazilian copper mine investigates the adequacy of using simulated grades for defining high risk areas affecting mine planning and the defined mineral reserves. Conditional simulations were used to derive multiple copper grade models within a typical ore-body of the deposit and compared these models against the real production data (reconciliation). The comparison allowed a better understanding of grade variability and helped in defining a more consistent mine plan. The range of values derived from the simulations mapped areas of significant uncertainty affecting the pushback’s plan. Risk analysis were conducted for the final pit definition by comparing the projected final pit limit and mining sequence against the simulated models of copper grade, assessing the operational impacts on key economical factors (mine net present value, discounted cash flow). Several final pit limits were generated for various simulated grade block models and compared to the previously pit planned using the ordinary kriging grade block model. Also, it was defined a risk scale using the conditional simulated models and the ordinary kriged values (commonly used for budget and mill feed reference) in order to minimize grade variations ensuring less risk on completion of the metal production goals on mine planning. This approach considering the risk for the mining sequencing was used on the annual mining plan and compared to the real production and to the traditional approach. The results highlighted possible risks associated with the operational pit limit and demonstrated the benefits of using risk analysis as a tool to preview and review the final pit limits and mine planning, helping to make better strategic decisions by the copper mine management related to the technical factors and economical indicators.

In recent years, it has become established the idea of a novel medicine where a patient is the center around which multidisciplinary teams (made up of physicians, statisticians and bioinformaticians) sew targeted treatments. Precision medicine involves the use of detailed patient-specific molecular information for diagnosing, categorizing and guiding treatment of a disease, with the main purpose of improving the clinical outcome compared to a more classical approach. In precision medicine it is supposed that the cause of a disease is at least partially attributable to specific genetic or epigenetic characteristics of a patient. Therefore, identifying these specificities helps building the best treatment for each individual. Next-generation sequencing techniques are massively employed, giving the ability to quickly and at relatively low cost analyze whole genomes, epigenomes and transcriptomes. This ability is clinically important since the prediction of treatment effectiveness is usually affected by many factors. A fundamental function in this new medicine is played by bioinformatics. It has a crucial role in every aspect of precision medicine, such as the accurate classification of patients, the prediction of new therapies based on current knowledge, the identification of possible outcomes of a disease or therapy, and the enrichment of current knowledge on pathogenic processes or on pharmaceuticals. The aim of this thesis is the development of an integrated framework, based on synergistically operating tools, models and algorithms, which help to fill some of the major gaps in each step of the production of highly customized therapies, overcoming, if possible, the limitations of currently employed techniques, defining a new standard for precision medicine informatics.

O Sistema Cananéia-Iguape sofreu importantes alterações ambientais devido à abertura do canal do Valo Grande que o conectou ao rio Ribeira de Iguape. Com a intensa atividade de mineração ocorrida no Vale do Ribeira, a bacia do rio Ribeira foi afetada, atingindo o Sistema através desse canal. Diante desse cenário, o principal objetivo deste estudo foi avaliar a disponibilidade de Cu, Pb e Zn no Sistema Cananéia-Iguape, a partir do teor associado aos principais componentes dos sedimentos. Para estimar a contaminação química, o teor desses elementos foi comparado aos limites canadenses (ISQG e PEL), ao teor na região em período que antecedeu à intensa atividade de mineração e a valores de background (composição média dos folhelhos). Considerando-se os resultados de Cu e Zn, estima-se que em caso de remobilização, possivelmente, não representariam perigo às comunidades bióticas. Entretanto, o teor disponível de Pb excedeu os valores de comparação em várias estações, sugerindo atenção quanto à sua biodisponibilidade. Em geral, a região que mais favoreceu o acúmulo de metais foi o Mar Pequeno, provavelmente, devido às condições oceanográficas. Dados de geoquímica e de geocronologia de testemunhos possibilitaram reconhecer a inserção antrópica do Pb no Sistema durante o período de mineração.
The Cananéia-Iguape System suffered important changes due to the opening of Valo Grande Channel, which connect this System to the Ribeira de Iguape River. The mining activities in the Ribeira Valley affected the river basin, reaching this System through Valo Grande. The main goal of this study was to evaluate the availability of Cu, Pb and Zn in the Cananéia-Iguape System, based on the content associated with the main components of the sediments. In order to estimate chemical contamination, the content of these elements was compared to Canadians limits (ISQG and PEL), to the content of these metals in the region in period preceding to the intense mining activity and to the background values (shale average composition). The results for Cu and Zn indicated that in case of remobilization, they possibly would not represent a hazard to the biotic communities. However, the available content for Pb exceeded the comparative values at various stations, suggesting attention regarding its bioavailability. In general, the region that favored the most the accumulation of metals was Mar Pequeno, possibly due to oceanographic conditions. Data regarding geochemistry and geochronology of cores enable the observation of the insertion the Pb anthropogenic in the System during the mining period.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geociências-Geoquímica. Niterói, RJ
Este estudo buscou avaliar a contaminação, a mobilidade e a bioacessibilidade do As na água subterrânea e em solos e sedimentos superficiais, em uma área de mineração de ouro no município de Paracatu-MG. O As apresentou valores abaixo do limite estabelecido pela legislação brasileira (< 10 g.L-1) em todas as amostras de águas subterrâneas. As avaliações realizadas indicam que as condições físicoquímicas encontradas, juntamente com as características geológicas da região são fatores preponderantes para estes baixos teores. Nas amostras de solos e sedimentos, a concentração de As total variou de 32,9 a 1280 g.g-1 e de não detectável a 4727 g.g-1, respectivamente. Os maiores teores de As nos sedimentos e solos foram encontrados na bacia do córrego Rico, mostrando a influência direta dos processos oriundos da área de mineração em sua nascente. As demais áreas apresentaram teores inferiores a esta área, mas acima da média mundial e do folhelho médio, podendo estar sob a influência de alguns processos, tais como, a ciclagem biogeoquímica natural deste elemento, deposição de sedimentos por erosão hídrica, descarga de águas subterrâneas, e dispersão atmosférica de material particulado enriquecido em As, oriundo da área de mineração. Todos estes processos proporcionam em maior ou menor proporção o enriquecimento de As na região. De acordo com os ensaios de extração sequencial utilizados, em todas as amostras de solos e sedimentos, as frações solúvel e trocável exibiram valores extremamente baixos, correspondendo a menos de 2 % do teor total de As contido nestas amostras, mostrando baixa disponibilidade do As para o ambiente nos solos e sedimentos em Paracatu. A mobilidade do As trocável nos solos de Paracatu foi avaliada através de experimentos de lixiviação em coluna. Os resultados mostraram uma associação do As com os óxidos e hidróxidos de Fe que favorecem a retenção deste metaloide nos solos mesmo na fração trocável. Segundo os resultados deste e de outros estudos, a adição de Ca a solos contaminados favorece o processo de retenção do As, mostrando grande potencial na utilização de compostos deste elemento em estratégias que visem a remediação de áreas contaminadas.
This study aims to evaluate the mobility and inaccessibility of As in groundwater and in surface soils and sediments in a mining area of gold in the Paracatu municipality- MG. The Arsenic concentrations in all the groundwater samples showed below the established limits by the Brazilian legislation (< 10 μg.L-1). The evaluations were indicating that the physical-chemical conditions encountered together with the geological characteristics of the region are predominant factors responsible to these lower values. In the soil and sediment samples, the concentration of As varied from 32,9 to 1280 μg.g-1 and to below detection limit to 4727 μg.g-1 respectively. The higher values of As in the sediments and soils were found in the Rico stream, showing the direct influence of the processes coming from its source of the mining area. The other areas showed inferior values to this area, but above world average and of the shale values, which may be under the influence of some processes, such as the natural biogeochemical cycling of this element, deposition of sediment by hydrological erosion, groundwater discharge and atmospheric dispersion of particulate material originated by mining. All these processes provide in proportions more or less the As enrichment in the region. According to the sequential extraction tests utilized, in all the soils and sediments samples, the soluble and exchangeable fractions exhibited extremely lower values corresponding less than 2% of the total As in these samples. These results showed low availability of As to the environment by the soils and sediments in Paracatu. The mobility of the exchangeable As in the soils of Paracatu was assessed through column leaching experiments. The results showed an association of the As with the Fe oxides and hydroxides which favors to retain this metalloid even in the exchangeable fraction in the soils. According to the results of this and other studies, an addition of Ca to the contaminated soils favors the process of the As retention, showing great potential in the use of compounds of this element in strategies that aimed at the remediation of contaminated areas

A erosão acelerada resulta em graves consequências para a qualidade do solo e da água, assim como para a sustentabilidade dos sistemas agrários. Para caso de estudo seleccionou-se a bacia hidrográfica do Rio Paraopeba, localizada na região sudoeste do Brasil, na área denominada de Quadrilátero Ferrífero, onde a intensa actividade mineira, se reflecte, não apenas no agravamento dos processos erosivos, mas também, na qualidade da água e do solo. No trabalho desenvolvido, identificaram-se as zonas mais vulneráveis aos processos erosivos, tendo como base metodologias de modelagem matemática computacional e efectuou-se o estudo geoquímico dos solos, tendo em vista a definição das tecnologias mais adequadas para a remediação da bacia. Os trabalhos desenvolvidos permitiram identificar situações de contaminação de solos e apresentar soluções para a sua remediação. Desta forma, foi validada a metodologia de estudo proposta, que se considera viável para ser adaptada ao estudo de outros locais; ABSTRACT: MODELLING OF SOIL EROSION IN A STRONG IMPACT REGION OF MINING AND GEOCHEMICAL STUDY OF THE MOST VULNERABLE AREAS, WITH A VIEW TO REMEDIATION AND REHABILITATION. STUDY OF AN AREA OF THE BASIN OF THE PARAOPEBA RIVER (MINAS GERAIS, BRAZIL) The accelerated erosion results in serious consequences for the quality of soil and water, as well as to the sustainability of agricultural systems. For case study was selected to Paraopeba River Basin, located in the southwestern region of Brazil, in the area known as the Iron Quadrangle, where intense mining activity, reflected not only in the increase of erosion, but also in quality water and soil. In the work, the most vulnerable areas to erosion were identified, based on methodologies of computational mathematical modeling and worked up geochemical study of the soil, with a view to defining the most appropriate technologies for the remediation of the basin. The developed works have identified soil contamination, and provide solutions to their remediation. Thus, it was validated the study proposed methodology, it is considered feasible to be adapted to study other locations.

DNA-barcoding is the process of taxonomic identification based on the sequence of a marker gene. When complex samples are analysed, we refer in particular to meta-barcoding. Barcoding has traditionally been performed with Sanger sequencing platform. The emergence of second-generation sequencing platforms, mainly represented by Illumina, enabled the high-throughput sequencing of hundreds of samples, and allowed the characterization of complex samples through meta-barcoding experiments. However, fragments sequenced with the Illumina platform are shorter than 600 bp, and this greatly limits taxonomic resolution of closely related species. Moreover, both these platforms suffer of long turnaround time, since they require shipping the samples to a sequencing facility, and complex regulations may hamper the export of material out of the country of origin. More recently, Oxford Nanopore Technologies provided the MinION, a portable and cheap third-generation sequencer, which has the potential of overcoming issues of currently available platforms, thanks to the production of long sequencing reads. However, MinION reads suffer of high error rate, therefore suitable analysis pipelines are needed to overcome this issue. In this thesis I describe the development of bioinformatic pipelines for MinION-based DNA barcoding. Starting from the analysis of single samples, I show how improvements both in sequencing chemistry and in software now allow obtaining consensus sequences directly in the field, with accuracy comparable with Sanger. Conversely, when analysing complex samples, sequencing reads cannot be collapsed for reducing the error rate. However, bioinformatic approaches exploiting increased read length largely compensate the higher error rate, resulting in high correlation between MinION and Illumina up to genus level, and a more marked sensitivity of MinION platform to detect spiked-in indicator species. In conclusion, the results presented in this thesis show that bioinformatic pipelines for the analysis of MinION reads can largely mitigate platform issues, paving the way for this platform to become the gold-standard for barcoding in the near future.

碩士
國立臺灣大學
農藝學研究所
100
Simple sequence repeat (SSR) markers have been widely used in many areas of genetic research due to their codominance, ease of use, multiallelic nature, and abundance characteristics in the genome. Though various SSR markers in pepper have been published, it is still not sufficient to create a highly saturated linkage map. Here we utilized the 454 pyrosequencing technique to perform whole genome shotgun sequencing for pepper (Capsicum annuum). Tandem Repeats Finder (TRF) was applied to identify SSRs followed by primer design using Primer3. A Perl script was written to connect these processes. A total of 120 designed primers were verified by 15 varieties of C. annuum and 1 variety of C. baccatum. Verified results show that 104 primer sets were successfully amplified (87%), among them, 65 primer sets possess high polymorphism between the 15 varieties of C. annuum. The success rate and polymorphism of these designed primers were higher than the previously published primers. In order to determine an optimum strategy for SSR marker development, simulated shotgun sequence reads were generated using Flowsim based on rice reference genome sequences to compare the effects of assembly, mining tools, and primer selected methods on SSR primer development. Simulation results revealed that assembling low coverage genomic shotgun sequences not only did not elongate the sequence effectively to facilitate primer design, but also may introduce false sequence information. Compared with commonly used mining tools such as Msatcommander and QDD, we obtained the highest number of SSRs by TRF. We also found that using TRF, coupling with re-PCR or BLAST against original sequence reads may effectively eliminate potentially redundant markers while maintaining higher marker yield. Our findings may provide researchers an efficient SSR marker development strategy and decrease further primer verification cost.

碩士
國立臺灣大學
農藝學研究所
100
Microsatellites or simple sequence repeats (SSRs) are tandem repeats distributed across genomes with 1 to 6 nucleotide motifs. Because of their genomic abundance and high level of polymorphism, SSRs is designed as molecular markers to apply in a variety of researches. In recent year, the rapidly-developing next generation sequencing technology (NGST) has impacted the ways of mining SSRs. NGST not only has the advantage of higher speed and lower cost but also offers the opportunities to discover novel SSRs. However, in a pilot study, the budget may be limited and one can only afford a low-coverage sequencing project regarding to the genome of interest. The situation may be more severe when the genome size is large. In this study, we aimed to investigate the relation between the mined SSR counts and the sequencing depth for a genome whose sequence which is not yet available by simulations at low coverage sequencing. The simulation was two-fold. First, we separate whole rice genome to establish three databases. Second, we simulated a genome with approximate complexity by recombining known rice genome subsequences. Moreover, we mimicked 454 sequencing results under different coverage using 454sim and mined SSRs accordingly. The results showed that the number of mined SSRs increased as the sequencing depth increased. More importantly, this procedure provided a mean to estimate the number of mined SSRs without whole genome sequence and hence to assist to set budget in advance.

碩士
大葉大學
資訊管理學系碩士班
93
With the development of information technology, the trend of e-learning has been improved. E-learning has some problems about the uncorrected guideline of learning path. The e-learning content standard SCORM has also considered the sequencing and navigation of learning object and made clear defined in relation standard. But how to plan the suitable arranges the sequencing strategies, accord with the learner's degree, cognitive style and demand, it is the key point of influencing the quality of the teaching material. So this research will use the data mining to find the rule of e-learning path and analyze its type of learning path to provide the material editor reference basis in the compiling the tactics of the preface. Therefore, this research establishes a learning environment, recodes the learning trace of learner, and stores it to portfolio database. And we mine the learning path rule of learner, analyze its type, provide the reference of sequencing strategies for editor. Providing teacher sequencing strategies achieve the teaching material recombined, reused, and adaptive learning. The research objective is listed in the following: 1. Establish the learning management system fit in with SCORM. 2. Establish the learning material and provide the learner to learn. 3. Store the portfolio of learner, and set up the portfolio database. 4. Mine and analyze learning path rule and type. Key Words : Portfolio, learning path, SCORM

Pedobacter lusitanus NL19 is a Gram -negative bacterium from the famil y Sphingobacteriaceae, which was isolated from a deactivated uranium mine in Portugal. This strain produces non ribosomal peptides (NRPs), calle d pedopeptins. The production of these peptides is repressed by high concentrations of peptone from casein (PC). In addition to biosynthetic gene clusters (BGCs) encoding NRPs, the NL19 strain genome also has BGCs of other secondary metabolites (SMs), incl uding lanthipeptides (4 BGCs: ped8, ped 14, ped15 and ped17). Lanthipeptides are ribosomally synthesized and post -translationally modified peptides (RiPPs), which exhibit a wide variety of biological activities, including antimicrobial and antiallodyni c. Lanthipeptides are characterized by the presence of lanthionine (Lan) and meth yllanthionine (MeLan) residues and are divided into four classes, defined by the enzymes that catalyze the reactions needed for the installation of these residues. T his s tudy focused on the NL19 strain and the main objectives were to determine the effect of high concentrations of PC: i) on the transcription of lanthipeptides and ii) on the proteome of the strain, with special interest in proteins involved in the biosy nthesis of SMs. Due to the COVID -19 pandemics and confinement, a third objective was defined , w hich aimed to identify and analy z e, in silico, lanthipeptide BGCs from the genomes of other genera of th e family Sphingobacteriaceae. Objective i) involved the transcriptional analysis of BGC ped 1 5 that encodes two precursor peptides and other biosynthetic proteins. It was necessary to s e quence the upstream region of this cluster, through primer walking, whi ch allowed the identification of six other structural peptide genes. The transcriptional analysis was performed by RT -qPCR and revealed that high concen trations of PC do not affect the expression of the BGC ped15, contrary to what was found fo r the pedope ptin s BGC . In general, the RT -qPCR results validated the available RNA -seq results and showed that the transcriptional repression caused by high concentrations of P C is not transversal to t he production of other SMs. Objective ii) involved t he analysis of the proteome of the NL19 strain grow n in high concentrations of PC (and its control) by nano LC -ESI -MS/MS and allowed the detection of the differential expression of some proteins relat ed to the biosynthesis of SMs, including the pedopeptins nonribo somal peptide synth etases and a lanthipeptide precursor encoded in the ped8 BGC. Objective iii) involved the analysis of 446 BGCs of the family Sphingobacteriaceae with the antiSMASH and Bi G -SCAPE tools. Class I and class III lanthipeptide BGCs were ide ntified in the gene ra Mucilaginibacter and Sphingobacterium. Class III BGCs encode La nKC enzymes with slightly different lyase domains, which may indicate that these enzymes use a different me chanism for the installation of Lan /MeLan. This stu dy contributes to the bo dy of knowledge of the bacterial response to the manipulation of culture media, in particular in the production of SMs with biotechnological potential as lanthipeptide s. In addition, this study identified the potential of bacterial genera already known, but still unde rex plored, for the production of new lanthipeptides, whose biosynthetic, structural and functional characterization is unknown.
Pedobacter lusitanus NL19 é uma bactéria de Gram-negativo, da família Sphingobacteriaceae, que foi isolada de uma mina de urânio desativada em Portugal. Esta estirpe produz péptidos não ribossomais (NRPs), designados por pedopeptinas. A produção destes péptidos é reprimida em meio com elevadas concentrações de peptona de caseína (PC). Para além de clusters biosintéticos (BGCs) que codificam NRPs, o genoma da estirpe NL19 também possui BGCs de outros metabolitos secundários (SMs), incluindo lantipéptidos (4 BGCs: ped8, ped14, ped15 e ped17). Lantipéptidos são péptidos de síntese ribossomal com modificações pós-traducionais (RiPPs), que exibem uma ampla variedade de atividades biológicas, incluindo antimicrobiana e antialodínica. Estes péptidos são caracterizados pela presença de resíduos de lantionina (Lan) e metillantionina (MeLan) e dividem-se em quatro classes, definidas pelas enzimas que catalisam as reações que originam esses resíduos. Este estudo focou-se na estirpe NL19 e os principais objetivos foram determinar o efeito de elevadas concentrações de PC: i) na transcrição de lantipéptidos e ii) no proteoma da estirpe, em particular proteínas envolvidas na síntese de SMs. Devido à pandemia COVID-19 e consequente confinamento, foi definido um terceiro objetivo, que consistiu na identificação e análise in silico de BGCs de lantipéptidos presentes nos genomas de outros géneros da família Sphingobacteriaceae. O objetivo i) envolveu a análise transcricional do BGC ped15, que codifica dois péptidos percursores e outras proteínas biosintéticas. Para tal, procedeu-se à sequenciação da região a montante deste cluster, por primer walking, o que permitiu identificar outros seis genes de péptidos percursores. A análise transcricional foi realizada por RT-qPCR e revelou que elevadas concentrações de PC não alteram a expressão do BGC ped15, ao contrário do que acontece com o BGC das pedopeptinas. De uma forma geral, os resultados de RT-qPCR validaram os resultados de RNA-seq disponíveis, e mostram que o efeito repressor da PC não é transversal à produção de todos os SMs. O objetivo ii) incluiu a análise do proteoma da estirpe NL19 cultivada com elevadas concentrações de PC (e respetivo controlo) por nano LC-ESI-MS/MS e permitiu detetar a expressão diferencial de várias proteínas relacionadas com a biosíntese de SMs, incluindo as péptido sintetases não ribossomais das pedopeptinas e de um precursor de lantipéptido codificado no BGC ped8. O objetivo iii) envolveu a análise de 446 BGCs da família Sphingobacteriaceae com as ferramentas bioinformáticas antiSMASH e BiG-SCAPE. Foram identificados BGCs de lantipéptidos de classe I e classe III nos géneros Mucilaginibacter e Sphingobacterium. Os BGCs de classe III codificam enzimas LanKC com domínios liase um pouco distintos, o que pode indicar que estas enzimas utilizam um mecanismo de formação de Lan/MeLan relativamente diferente daquele já conhecido. Este estudo contribui para o conhecimento da resposta bacteriana à manipulação de meios de cultura, em particular na produção de SMs com potencial biotecnológico como os lantipéptidos. Para além disso, permitiu identificar o potencial de géneros bacterianos já conhecidos, mas até agora inexplorados, para produzir novos lantipétidos, cuja caracterização biosintética, estrutural e funcional é ainda desconhecida.
Mestrado em Biotecnologia

Pattern discovery is an important area of data mining referring to a class of techniques designed for the extraction of interesting patterns from the data. A pattern is some kind of a local structure that captures correlations and dependencies present in the elements of the data. In general, pattern discovery is about finding all patterns of `interest' in the data and a popular measure of interestingness for a pattern is its frequency of occurrence in the data. Thus the problem of frequent pattern discovery is to find all patterns in the data whose frequency of occurrence exceeds some user defined threshold. However, frequency of a pattern is not the only measure for finding patterns of interest and there also exist other measures and techniques for finding interesting patterns. This thesis is concerned with efficient discovery of inherent patterns from long sequence (or temporally ordered) data. Mining of such sequentially ordered data is called temporal data mining and the temporal patterns that are discovered from large sequential data are called episodes. More specifically, this thesis explores efficient methods for finding small and relevant subsets of episodes from sequence data that best characterize the data. The thesis also discusses methods for comparing datasets, based on comparing the sets of patterns representing the datasets. The data in a frequent episode discovery framework is abstractly viewed as a single long sequence of events. Here, the event is a tuple, (Ei; ti), where Ei is referred to as an event-type (taking values from a finite alphabet set) and ti is the time of occurrence. The events are ordered in the non-decreasing order of the time of occurrence. The pattern of interest in such a sequence is called an episode, which is a collection of event-types with a partial order defined over it. In this thesis, the focus is on a special type of episode called serial episode, where there is a total order defined among the collection of event-types representing the episode. The occurrence of an episode is essentially a subset of events from the data whose event-types match the set of eventtypes associated with the episode and the order in which they occur conforms to the underlying partial order of the episode. The frequency of an episode is some measure of how often it occurs in the event stream. Many different notions of frequency have been defined in literature. Given a frequency definition, the goal of frequent episode discovery is to unearth all episodes which have a frequency greater than a user-defined threshold. The size of an episode is the number of event-types in the episode. An episode β is called a subepisode of another episode β, if the collection of event-types of β is a subset of the corresponding collection of α and the event-types of β satisfy the same partial order relationships present among the corresponding event-types of α. The set of all episodes can be arranged in a partial order lattice, where each level i contains episodes of size i and the partial order is the subepisode relationship. In general, there are two approaches for mining frequent episodes, based on the way one traverses this lattice. The first approach is to traverse this lattice in a breadth-first manner, and is called the Apriori approach. The other approach is the Pattern growth approach, where the lattice is traversed in a depth-first manner. There exist different frequency notions for episodes, and many Apriori based algorithms have been proposed for mining frequent episodes under the different frequencies. However there do not exist Pattern-growth based methods for many of the frequency notions. The first part of the thesis proposes new Pattern-growth methods for discovering frequent serial episodes under two frequency notions called the non-overlapped frequency and the total frequency. Special cases, where certain additional conditions, called the span and gap constraints, are imposed on the occurrences of the episodes are also considered. The proposed methods, in general, consist of two steps: the candidate generation step and the counting step. The candidate generation step involves finding potential frequent episodes. This is done by following the general Pattern growth approach for finding the candidates, which is the depth-first traversal of the lattice of all episodes. The second step, which is the counting step, involves counting the frequencies of the episodes. The thesis presents efficient methods for counting the occurrences of serial episodes using occurrence windows of subepisodes for both the non-overlapped and total frequency. The relative advantages of Pattern-growth approaches over Apriori approaches are also discussed. Through detailed simulation results, the effectiveness of this approach on a host of synthetic and real data sets is shown. It is shown that the proposed methods are highly scalable and efficient in runtime as compared to the existing Apriori approaches. One of the main issues in frequent pattern mining is the huge number of frequent patterns, returned by the discovery methods, irrespective of the approach taken to solve the problems. The second part of this thesis, addresses this issue and discusses methods of selecting a small subset of relevant episodes from event sequences. There have been a few approaches, discussed in the literature, for finding a small subset of patterns. One set of methods are information theory based methods, where patterns that provide maximum information are searched for. Another approach is the Minimum Description Length (MDL) principle based summarization schemes. Here the data is encoded using a subset of patterns (which forms the model for the data) and its occurrences. The subset of patterns that has the maximum efficiency in encoding the data is the best representative model for the data. The MDL principle takes into account both the encoding efficiency of the model as well as model complexity. A method, called Constrained Serial episode Coding(CSC), is proposed based on the MDL principle, which returns a highly relevant, non-redundant and small subset of serial episodes. This also includes an encoding scheme, where the model representation and the encoding of the data are efficient. An interesting feature of this algorithm for isolating a small set of relevant episodes is that it does not need a user-specified threshold on frequency. The effectiveness of this method is shown on two types of data. The first is data obtained from a detailed simulator for a reconfigurable coupled conveyor system. The conveyor system consists of different intersecting paths and packages flow through such a network. Mining of such data can allow one to unearth the main paths of package ows which can be useful in remote monitoring and visualization of the system. On this data, it is shown that the proposed method is able to return highly consistent sub paths, in the form of serial episodes, with great encoding efficiency as compared to other known related sequence summarization schemes, like SQS and GoKrimp. The second type of data consists of a collection of multi-class sequence datasets. It is shown that the selected episodes from the proposed method form good features in classi cation. The proposed method is compared with SQS and GoKrimp, and it is shown that the episodes selected by this method help in achieving better classification results as compared to other methods. The third and nal part of the thesis discusses methods for comparing sets of patterns representing different datasets. There are many instances when one is interested in comparing datasets. For example, in streaming data, one is interested in knowing whether the characteristics of the data are the same or have changed significantly. In other cases, one may simply like to compare two datasets and quantify the degree of similarity between them. Often, data are characterized by a set of patterns as described above. Comparing sets of patterns representing datasets gives information about the similarity/dissimilarity between the datasets. However not many measures exist for comparing sets of patterns. This thesis proposes a similarity measure for comparing sets of patterns which in turn aids in comparison of di erent datasets. First, a kernel for comparing two patterns, called the Pattern Kernel, is proposed. This kernel is proposed for three types of patterns: serial episodes, sequential patterns and itemsets. Using this kernel, a Pattern Set Kernel is proposed for comparing different sets of patterns. The effectiveness of this kernel is shown in classification and change detection. The thesis concludes with a summary of the main contributions and some suggestions for extending the work presented here.

Dissertação de Mestrado, Estudos Integrados dos Oceanos, 19 de Outubro de 2016, Universidade dos Açores.
Os campos hidrotermais são caracterizados por serem ambientes únicos, por terem características físico-químicas extremas, assim como composições únicas de espécies biológicas. Bathymodiolus azoricus é um bivalve de mar profundo predominante dos campos hidrotermais ao longo da Crista Médio-Atlântica (CMA). Ainda muito pouco é conhecido sobre a estrutura genética tanto como a existência de conectividade desta espécie. Desse modo, o objetivo deste estudo foi identificar escalas e padrões espaciais sobre conectividade entre populações, a fim de prever a suscetibilidade de esta e de outras espécies, uma vez que a área em estudo poderá ser afetada pela mineração. Uma análise à genómica populacional das amostras, que foram recolhidas em três diferentes locais (Menez Gwen, Lucky Strike, Rainbow) e foram sujeitas a genotipagem “RAD sequencing” e Polimorfismo de um Único Nucleotídeo (SNPs), destacaram dois aspetos importantes: (1) Quando todos os SNPs foram analisados, nenhuma estrutura genética foi revelada, sugerindo assim que a maioria dos marcadores não contribuem para qualquer estrutura entre amostras populacionais ou simplesmente não são significativas; (2) Quando analisados apenas os conjuntos discriminantes dos marcadores de SNPs, um padrão de estrutura genética foi claramente revelado, correspondendo assim às expectativas de distribuição espacial das amostras. Este estudo proporciona uma contribuição inovadora para a compreensão da estrutura genética nas populações hidrotermais do CMA, fornecendo assim, pela primeira vez, evidências de uma estrutura genética relativa à fauna existente nos campos hidrotermais na zona norte da CMA. Estas observações não são incompatíveis com a conectividade genética pronunciada, e alguns cuidados devem ser tomados em conta quando se sugerem conclusões. Uma possível explicação para este padrão inclui troca larvar insuficiente entre campos hidrotermais, mas também uma adaptação local. Ambos os casos irão, sem dúvida, afetar a conectividade, e assim, ter consequências na recolonização após a mineração. A recolonização em locais de possível impacto por dispersão larval a partir de fontes remotas pode ser possível. A maioria dessas larvas seriam, no entanto, localmente não adaptáveis, o que pode restringir a resposta à seleção, expansão populacional e taxas de recolonização. Mais estudos com um maior número de taxas representativas são necessários, de modo a obter uma perspetiva mais clara da conectividade populacional nos campos hidrotermais. Além disso, existe uma lacuna considerável de conhecimento sobre os mecanismos que influenciam os padrões de conectividade, dificultando o estabelecimento de conclusões. Finalmente, estudos de genética populacional deverão ser acoplados a modelos de dispersão larval, a fim de identificar possíveis barreiras à dispersão.
ABSTRACT: Hydrothermal vents are unique environments of extreme physical–chemical characteristics and biological species composition. Bathymodiolus azoricus is a deep-sea Mytilid bivalve that dominates hydrothermal vent sites along the Mid-Atlantic Ridge (MAR). Very little is known about the genetic structure and connectivity of this species. Thus, the aim of this study was to identify the spatial scales and patterns over which populations are connected, in order to predict how this and other vent species are likely to be affected by SMS mining in the area. A population genomics analysis of samples collected from three MAR vent sites (Menez Gwen, Lucky Strike, Rainbow) using RAD sequencing and Single Nucleotide Polymorphism (SNP)genotyping highlighted two important aspects: (1) When all SNPs were used no genetic structure was revealed, suggesting that most of the markers do not contribute to any structure among population samples or are simply not informative; (2) when analyzing only the most discriminant sets of SNP markers, a clear pattern of genetic structure was uncovered, corresponding to expectations of the spatial distribution of samples. This study makes a novel contribution to the understanding of genetic structure at hydrothermal vent populations on the MAR, providing, for the first time, evidence of subtle genetic structure of hydrothermal vent fauna along the northern MAR. This observation is not incompatible with pronounced genetic connectivity, and some care must be taken when drawing conclusions. Possible explanations for this pattern include limited larval exchange between vent fields, but also local adaptation. Both scenarios will impact connectivity and thus have consequences for post-mining recolonization. Recolonization of impacted sites by larvae dispersing from remote sources may be possible. Most of these larvae would, however, not be locally adapted, which might constrain the response to selection, population expansion, and ultimately recolonization rates. More studies are needed with as many representative taxa as possible in order to obtain a wider perspective of population connectivity on hydrothermal vents. Moreover, there is a considerable gap in our knowledge of the mechanisms influencing connectivity patterns, which makes it difficult to draw conclusions. Finally, population genetic studies should be coupled with models of larval dispersal in order to identify potential barriers to dispersal.
This work was funded by EU’s FP-7 for research, technological development and demonstration under MIDAS Grant Agreement number 603418.

Les impacts environnementaux dues à l'extraction minière sont considérables. C'est l'action des microorganismes, en utilisant leur métabolisme du soufre sur les déchets miniers, qui engendre les plus grands défis. Jusqu'à présent, peu de recherches ont été effectués sur les microorganismes environnementaux pour la compréhension globale de l'action du métabolisme du soufre dans une optique de prévention et de rémédiation des impacts environnementaux de l'extraction minière. Dans cette étude, nous avons étudié une bactérie environnementale, Acidithiobacillus thiooxidans, dans le but de comprendre le métabolisme du soufre selon le milieu de culture et le niveau d'acidité du milieu. Nous avons utilisé la transcriptomique à haut débit, RNA-seq, en association avec des techniques de biogéochimie et de microscopie à électrons pour déterminer l'expression des gènes codants les enzymes du métabolisme du soufre. Nous avons trouvé que l'expression des gènes des enzymes du métabolisme du soufre chez ce microorganisme sont dépendantes du milieu, de la phase de croissance et du niveau d'acidité présent dans le milieu. De plus, les analyses biogéochimiques montrent la présence de composés de soufre réduits et d'acide sulfurique dans le milieu. Finalement, une analyse par microscopie électronique révèle que la bactérie emmagasine des réserves de soufre dans son cytoplasme. Ces résultats permettent une meilleure compréhension de son métabolisme et nous rapprochent de la possibilité de développer une technique de prédiction des réactions ayant le potentiel de causer des impacts environnementaux dus à l'extraction minière.
The environmental impact of mining extraction is important. The action of microorganisms using their sulfur metabolism to metabolise compounds in mining waste contributes to reactions that may impact water quality and the environment. Few studies have been conducted on environmental microorganisms to advance the global comprehension of their sulfur metabolism in an attempt to study their impact on the environment. In this study, we cultivate an environmental bacterium, Acidithiobacillus thiooxidans, in an attempt to understand its sulfur metabolism in different growth media and at different levels of acidity. We used high-throughput RNA sequencing in association with sulfur biogeochemistry and electron microscopy to determine the expression of the genes encoding sulfur metabolism enzymes. The expression of genes encoding sulfur metabolism enzymes was media and pH-dependent. Also, the biogeochemical analysis showed the presence of reduced sulfur intermediates and of sulfuric acid in the medium. Finally, an electron microscopic analysis revealed that the bacteria stock sulfur in the cytoplasm. These results resulted in a better comprehension of its sulfur metabolism and it opens the possibility to predict reactions in mining operations that have impact on the environment.

This thesis investigated the movement sequences of elite and junior-elite female netball athletes using a local positioning system (LPS). Study one determined the indoor validity of an LPS, specifically the Wireless ad hoc System for Positioning (WASP), for measuring distance, velocity and angular velocity whilst sprinting and walking five nonlinear courses. The criterion measure used to assess WASP validity was Vicon, a motion analysis system. During all sprinting and walking drills, WASP had an acceptable accuracy for measuring total distance covered (coefficient of variation, CV; < 5.2%). Similarly, WASP had an acceptable accuracy across all sprinting and walking drills for measuring mean velocity (CV; < 6.5%). During all sprinting drills, WASP had acceptable accuracy for measuring mean and peak angular velocity (CV; < 3%). A increased bias was observed during all walking drills, compared to sprinting, likely due to radio-frequency (RF) interference from the metal-clad indoor stadium where validation trials were conducted. Researchers and practitioners may use WASP to accurately quantify the non-linear movement of athletes during indoor court-based sports although should be aware of the increased bias during walking movement.