Insertion and deletion (indel), a common form of genetic variation, has been shown to cause or contribute to human genetic diseases and cancer. Despite this importance and being the second most abundant variant type in the human genome, indels have not been studied as much as the single nucleotide polymorphism (SNP). With the advance of next-generation sequencing technology, many indel calling tools have been developed. However, performance comparison of commonly used tools has shown that (1) the tools have limited power in identifying indels and there are significant number of indels undetected, and (2) there is significant disagreement among the indel sets produced by the tools. These findings indicate the necessity of improving the existing tools or developing new algorithms to achieve reliable and consistent indel calling results. Two indels are biologically equivalent if the resulting sequences are the same. Storing biologically equivalent indels as distinct entries in databases causes data redundancy and misleads downstream analysis. It is thus desirable to have a unified system for identifying and representing equivalent indels. This dissertation describes UPS-indel, a utility tool that creates a universal positioning system for indels so that equivalent indels can be uniquely determined by their coordinates in the new system. Results show that UPS-indel identifies more redundant indels than existing algorithms. While mapping short reads to the reference genome, a significant number of short reads are unmapped and excluded from downstream analyses, thereby causing information loss in the subsequent variant calling. This dissertation describes Genesis-indel, a computational pipeline that explores the unmapped reads to identify missing novel indels. Results analyzing sequence alignment of 30 breast cancer patients show that Genesis-indel identifies many novel indels that also show significant enrichment in oncogenes and tumor suppressor genes, demonstrating the importance of rescuing indels hidden in the unmapped reads in cancer and disease studies. Somatic mutations play a vital role in transforming healthy cells into cancer cells. Therefore, accurate identification of somatic mutations is essential. Many somatic mutations callers are available with different strengths and weaknesses. An ensemble approach integrating the power of the callers is warranted. This dissertation describes SomaticHunter, an ensemble of two callers, namely Platypus and VarDict. Results on synthetic tumor data show that for both SNPs and indels, SomaticHunter achieves recall comparable to the state-of-the-art somatic mutation callers and the highest precision, resulting in the highest F1 score. / Doctor of Philosophy / Insertion and deletion (indel), a common form of genetic variation in the human genome, is associated with genetic diseases and cancer. However, indels are heavily understudied due to experimental and computational challenges. This dissertation addresses the computational challenges in three aspects. First, the current approach of representing indels is ambiguous and causes significant database redundancy. A universal positioning system, UPS-indel, is proposed to represent equivalent indels unambiguously and the UPS-indel algorithm is theoretically proven to find all equivalent indels and is thus exhaustive. Second, a significant number of indels are hidden in DNA reads not mapped to the reference genome. Genesis-indel, a computational pipeline that explores the unmapped reads to identify novel indels that are initially missed, is developed. Genesis-indel has been shown to uncover indels that can be important genetic markers for breast cancer. Finally, mutations occurring in somatic cells play a vital role in transforming healthy cells into cancer cells. Therefore, accurate identification of somatic mutation is essential for a better understanding of cancer genomes. SomaticHunter, an ensemble of two sensitive variant callers, is developed. Simulated studies using whole genome and whole exome sequences have shown that SomaticHunter achieves recall comparable to state-of-the-art somatic mutation callers while delivering the highest precision and therefore resulting in the highest F1 score among all the callers compared.

Next-generation sequencing (NGS) technologies have made it possible to rapidly sequence the human genome, heralding a new era of health-care innovations based on personalized genetic information. However, these NGS technologies generate data at a rate that far outstrips Moore\'s Law. As a consequence, analyzing this exponentially increasing data deluge requires enormous computational and storage resources, resources that many life science institutions do not have access to. As such, cloud computing has emerged as an obvious, but still nascent, solution. This thesis intends to investigate and design an efficient framework for running and managing large-scale data-intensive scientific applications in the cloud. Based on the learning from our parallel implementation of a genome analysis pipeline in the cloud, we aim to provide a framework for users to run such data-intensive scientific workflows using a hybrid setup of client and cloud resources. We first present SeqInCloud, our highly scalable parallel implementation of a popular genetic variant pipeline called genome analysis toolkit (GATK), on the Windows Azure HDInsight cloud platform. Together with a parallel implementation of GATK on Hadoop, we evaluate the potential of using cloud computing for large-scale DNA analysis and present a detailed study on efficiently utilizing cloud resources for running data-intensive, life-science applications. Based on our experience from running SeqInCloud on Azure, we present CloudFlow, a feature rich workflow manager for running MapReduce-based bioinformatic pipelines utilizing both client and cloud resources. CloudFlow, built on the top of an existing MapReduce-based workflow manager called Cloudgene, provides unique features that are not offered by existing MapReduce-based workflow managers, such as enabling simultaneous use of client and cloud resources, automatic data-dependency handling between client and cloud resources, and the flexibility of implementing user-defined plugins for data transformations. In-general, we believe that our work attempts to increase the adoption of cloud resources for running data-intensive scientific workloads. / Master of Science

[ES] Las distrofias hereditarias de la retina (DHR) son un conjunto de trastornos caracterizados por la muerte, generalmente, progresiva de fotorreceptores y células del epitelio pigmentario retiniano. Las DHR afectan a más de 2 millones de personas en el mundo, produciendo una pérdida parcial y, en muchos casos, total de la visión, para la cual no existe ningún tratamiento que revierta o frene la progresión de la enfermedad. Se caracterizan por una elevada heterogeneidad clínica y genética, identificándose hasta la fecha mutaciones en más de 250 genes responsable de algún tipo de DHR. Esta tesis doctoral se ha centrado en el diagnóstico genético de 208 pacientes afectados de DHR no sindrómica (DHR-NS), mediante secuenciación masiva dirigida a un panel de 117 genes y cinco regiones intrónicas profundas en las que previamente se habían descrito mutaciones. Las variantes identificadas en los pacientes se priorizaron de acuerdo a la mínima frecuencia alélica y se clasificaron según los criterios de la guía del American College of Medical Genetics and Genomics. Las variantes de número de copias identificadas mediante la secuenciación del panel se validaron utilizando multiplex ligation-dependent probe amplification o array genómico. A todos los pacientes se les realizó un estudio oftalmológico, más o menos exhaustivo, incluyendo pruebas electrofisiológicas. Además, se realizaron ensayos in vitro con minigenes para comprobar la repercusión de ciertas variantes sobre el procesamiento del ARNm. Para ello, se empleó el plásmido pSPL3 en el que se introdujo la mutación a estudio, la transformación se realizó en bacterias E. coli electrocompetentes y la transfección en células HEK-293. Mediante la secuenciación del panel de genes, se obtuvo una tasa diagnóstica del 60%. Las mutaciones en los pacientes resueltos se distribuyeron en 30 genes diferentes, de las cuales 38 se describen por primera vez en este trabajo. Los genes mutados con mayor frecuencia en estos pacientes fueron ABCA4, USH2A, RPGR y PRPH2. Adicionalmente, se identificaron ocho variantes de número de copias en los genes EYS, ABCA4, PRPF31, MERTK, CRB1 y ROM1, demostrando el enorme potencial de estas tecnologías. En total, se reclasificaron el 10% de las familias estudiadas y se observó variabilidad fenotípica intra e inter familiar en pacientes con mutaciones en C1QTNF5, CERKL y PROM1. Además, destacamos que el 50% de las mujeres heterocigotas para RPGR y sintomáticas presentaron retinosis pigmentaria (RP) con asimetría interocular. Asimismo, se identificó un caso con posible digenismo entre los genes CNGA1 y CNGB1, y la duplicación completa de ROM1 en dos familias no relacionadas diagnosticadas de RP. Por otra parte, se han identificado seis familias con mutaciones en dos genes que potencialmente ambos podrían ser la causa de la DHR. En el ensayo funcional mediante minigenes, se observó que seis de las variantes analizadas presentaron un splicing aberrante. Los resultados de esta tesis destacan la utilidad clínica de la secuenciación masiva dirigida para las DHR-NS y la importancia de un examen oftalmológico completo que nos permita establecer nuevas asociaciones genotipo-fenotipo y ampliar el conocimiento de este grupo de enfermedades. Identificar la causa de la enfermedad es esencial para mejorar el manejo del paciente, realizar un asesoramiento genético preciso y beneficiarse de los futuros tratamientos y ensayosclínicos basados en terapia génica. / [CA] Les distròfies hereditàries de la retina (DHR) són un conjunt de trastorns caracteritzats per la mort, generalment, progressiva de fotoreceptors i cèl¿lules de l'epiteli pigmentari retiniano. Les DHR afecten més de 2 milions de persones al món, produint una pèrdua parcial i, en molts casos, total de la visió, per la qual no hi ha cap tractament que revertisca o frene la progressió de la malaltia. Es caracteritzen per una elevada heterogeneïtat clínica i genètica, identificant-se fins a la data mutacions en més de 250 gens responsable d'algun tipus de DHR. Esta tesi doctoral s'ha centrat en el diagnòstic genètic de 208 pacients afectats de DHR no sindròmica (DHR-NS), per mitjà de seqüenciació massiva dirigida a un panell de 117 gens i cinc regions intròniques profundes en les que prèviament s'havien descrit mutacions. Les variants identificades en els pacients es van prioritzar d'acord a la mínima freqüència allèlica i es van classificar segons els criteris de la guia de l'American College of Medical Genetics and Genomics. Les variants de nombre de còpies identificades per mitjà de la seqüenciació del panell es van validar utilitzant multiplex ligation-dependent probe amplification o array genòmic. A tots els pacients se'ls va realitzar un estudi oftalmològic, més o menys exhaustiu, incloent proves electrofisiològiques. A més, es van realitzar assajos in vitro amb minigens per comprovar la repercussió de certes variants sobre el processament de l'ARNm. Per a això, es va emprar el plasmidi pSPL3 en què es va introduir la mutació a estudi, la transformació es va realitzar en bacteris E. coli electrocompetentes i la transfecció en cèl-lules HEK-293. Per mitjà de la seqüenciació del panell de gens, es va obtindre una taxa diagnòstica del 60%. Les mutacions en els pacients resolts es van distribuir en 30 gens diferents, de les quals 38 es descriuen per primera vegada en este treball. Els gens mutats amb major freqüència en estos pacients van ser ABCA4, USH2A, RPGR i PRPH2. Addicionalment, es van identificar huit variants de nombre de còpies en els gens EYS, ABCA4, PRPF31, MERTK, CRB1 i ROM1, demostrant l'enorme potencial d'estes tecnologies. En total, es van reclassificar el 10% de les famílies estudiades i es va observar variabilitat fenotípica intra i inter familiar en pacients amb mutacions en C1QTNF5, CERKL i PROM1. A més, destaquem que el 50% de les dones heterozigotes per RPGR i simptomàtiques van presentar retinosi pigmentària (RP) amb asimetria interocular. Així mateix, es va identificar un cas amb possible digenismo entre els gens CNGA1 i CNGB1, i la duplicació completa de ROM1 en dues famílies no relacionades diagnosticades de RP. D'altra banda, s'han identificat sis famílies amb mutacions en dos gens que potencialment ambdós podrien ser la causa de la DHR. En l'assaig funcional per mitjà de minigens, es va observar que sis de les variants analitzades van presentar un splicing aberrant. Els resultats d'esta tesi destaquen la utilitat clínica de la seqüenciació massiva dirigida per a les DHR-NS i la importància d'un examen oftalmològic complet que ens permeta establir noves associacions genotip-fenotip i ampliar el coneixement d'este grup de malalties. Identificar la causa de la malaltia és essencial per millorar el maneig del pacient, realitzar un assessorament genètic precís i beneficiar-se dels tractaments basats en teràpia gènica. / [EN] Inherited retinal dystrophies (IRD) are a group of disorders generally characterized by the progressive death of photoreceptors and retinal pigment epithelial cells. IRD affect more than 2 million people in the world, producing a partial and, in many cases, total loss of vision, for which there is no treatment that reverses or slows the progression of the disease. They are characterized by a high clinical and genetic heterogeneity, identifying mutations in more than 250 genes responsible for some type of IRD to date. This doctoral thesis has focused on the genetic diagnosis of 208 patients affected by non-syndromic IRD (NS-IRD), through targeted genomic sequencing focuses on a panel of 117 genes and five deep intronic regions in which mutations have previously been identified. The identified variants in patients are prioritized according to the minimum allelic frequency and are classified according to the criteria of the American College of Medical Genetics and Genomics guide. The copy number variants identified by panel sequencing were validated using multiplex ligation-dependent probe amplification or genomic array. All patients underwent an ophthalmological study, more or less comprehensive, including electrophysiological tests. In addition, in vitro assays with minigenes were performed to verify the impact of certain variants on mRNA processing. To do this, pSPL3 plasmid in which the mutation was introduced was used, the transformation was performed in electrocompetent E. coli bacteria and transfection in HEK-293 cells. By the panel sequencing, a diagnostic rate of 60% was obtained. The mutations in the resolved patients were distributed in 30 different genes, of which 38 were described for the first time in this work. The most frequently mutated genes in these patients were ABCA4, USH2A, RPGR and PRPH2. In addition, eight copy number variants were identified in the genes EYS, ABCA4, PRPF31, MERTK, CRB1 and ROM1, that demonstrated the enormous potential of these technologies. In total, 10% of the families studied were reclassified and intra and inter-family phenotypic variability was detected in patients with mutations in C1QTNF5, CERKL and PROM1. In addition, we highlight that 50% of the symptomatic and heterozygous RPGR women showed retinitis pigmentosa (RP) with interocular asymmetry. Likewise, we identified a case with possible digenism between the CNGA1 and CNGB1 genes, and the complete duplication of ROM1 in two unrelated families diagnosed with RP. On the other hand, six families have been identified with mutations in two genes that could potentially be the cause of IRD. Functional assays by minigenes showed that six of the studied variants affected splicing. The results of this thesis highlight the clinical utility of targeted genomic sequencing in IRD and the importance of a complete ophthalmological examination that allows us to establish new genotype-phenotype associations and expand the knowledge of this group of diseases. Identifying the cause of the disease is essential to improve patient management, to perform an accurate genetic counseling and to take advantage of treatments based on gene therapy. / Rodríguez Muñoz, A. (2020). High-throughput strategies for molecular diagnosis of nonsyndromic inherited retinal dystrophies [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/139095

Forest trees represent a unique group of organisms combined with ecological and economic importance. Owing to their random mating system and widespread geographical distribution, they harbor abundance genetic variation both within and among populations. Despite their importance, research in forest trees has been underrepresented majorly due to their large and complex genome and scarce funding. However, recent climate change and other associated problems such as insect outbreaks, diseases and stress related damages have urged scientists to focus more on trees. Furthermore, the advent in high-throughput sequencing technologies have allowed trees to be sequenced and used as reference genome, which provided deeper understanding between genotype and environment. Whole genome sequencing is still not possible for organisms having large genomes including most tree species, and it is still not feasible economically for population genomic studies which require sequencing hundreds of samples. To get around this problem, genomic reduction is required. Sequence capture has been one of the genomic reduction techniques enabled studying the subset of the DNA of interest. In this paper, our primary goal is to outline challenges, provide guidance about the utility of sequence capture in trees, and to leverage such data in genome-wide association analyses to find the genetic variants that underlie complex, adaptive traits in spruce and pine, as well as poplar. Results of this research will facilitate bridging the genomic information gap between trees and other organisms. Moreover, it will provide better understanding how genetic variation governs phenotype in trees, which will facilitate both marker assisted selection for improved traits as well as provide guidance to determine forest management strategies for reforestation to mitigate the effects of climate change. / Ph. D. / Forests are under extensive threat including increased demand in wood consumption, climate changes and associated diseases and stress related damages. Up until very recently, researches in trees have been relatively slower owing to their large and complex genomes. However, this has dramatically changed mainly due to the advancement in sequencing technology. There have been more and more studies performed identifying novel genes that are responsible for improved characteristics. In this study, we provided guidance about how to better utilize sequencing technology and identified genes that are potentially related with adaptation in trees.

Yes / Background. Increased availability of Next Generation Sequencing (NGS) techniques allows, for the first time, to distinguish relapses from reinfections in patients with multiple Buruli ulcer (BU) episodes. Methodology. We compared the number and location of single nucleotide polymorphisms (SNPs) identified by genomic screening between four pairs of Mycobacterium ulcerans isolates collected at the time of first diagnosis and at recurrence, derived from a collection of almost 5000 well characterized clinical samples from one BU treatment center in Benin. Principal Findings. The findings suggest that after surgical treatment—without antibiotics—the second episodes were due to relapse rather than reinfection. Since specific antibiotics were introduced for the treatment of BU, the one patient with a culture available from both disease episodes had M. ulcerans isolates with a genomic distance of 20 SNPs, suggesting the patient was most likely reinfected rather than having a relapse. Conclusions. To our knowledge, this study is the first to study recurrences in M. ulcerans using NGS, and to identify exogenous reinfection as causing a recurrence of BU. The occurrence of reinfection highlights the contribution of ongoing exposure to M. ulcerans to disease recurrence, and has implications for vaccine development. / This work was supported by the UBS Optimus Foundation (Zurich, Switzerland) and the Department of Economy, Science and Innovation of the Flemish Government (Belgium). KV was supported by a VLADOC PhD scholarship of VLIRUOS (Belgium).

Acredita-se que o Brasil é o centro de diversidade de vírus transmitidos por ácaros do gênero Brevipalpus (VTB). Alguns desses VTB infectam culturas fundamentais para o agronegócio brasileiro como citros e café, além de maracujá e de várias plantas ornamentais. Na última década os genomas de dois deles, Citrus leprosis virus C (CiLV-C) e Coffee ringspot virus (CoRSV) foram sequenciados, mas ainda é escasso o conhecimento sobre a diversidade genética e processos evolutivos envolvidos na população dessas espécies. Neste contexto, o objetivo deste trabalho foi caracterizar molecularmente novas estirpes de CiLV-C e CoRSV que infectam citros e café, respectivamente. E revelar as relações filogenéticas com espécies de VTB conhecidas, assim como avaliar a variabilidade genética da população de CiLVC no Brasil. Para o estudo de CiLV-C, 47 amostras de Citrus sinensis apresentando sintomas típicos da leprose dos citros foram coletadas em diferentes regiões do Brasil no período de 2011-2015. A presença de CiLV-C foi detectada por RT-PCR em todas as amostras coletadas e, posteriormente, foi realizado o sequenciamento de quatro regiões do genoma viral (p29, p15, RI e MP) de cada isolado. As sequências obtidas foram utilizadas no estudo de filogenia e variabilidade da população de CiLV-C no Brasil. Foi demonstrado que a população de CiLV-C apresenta uma variabilidade relativamente baixa; entretanto, foi identificada a existência de duas linhagens dentro da espécie, nomeadas Cor e SJRP. Os genomas completos de CiLV-C SJRP e também do dicorhavirus tentativo CoRSV identificado em Limeira, SP, foram obtidos mediante o sequenciamento de RNA de pequeno tamanho (siRNA). Cada sequência foi validada mediante o sequenciamento de fragmentos gerados por RT-PCR ao longo do genoma. CiLV-C SJRP apresenta cerca de 85% de identidade de nucleotídeo com o membro-tipo do gênero Cilevirus e exibe evidências de recombinação com isolados da linhagem Cor, a prevalente no território brasileiro. Globalmente, o genoma de CoRSV Limeira apresenta mais de 90% de identidade de nucleotídeo com isolado CoRSV Lavras, o que indica que ambos os isolados são membros da mesma espécie tentativa de dichorhavirus. / South America is most likely the center of diversity of Brevipalpus transmitted viruses (BTV). Some of these BTV infect major crops of the Brazilian agribusiness such as citrus and coffee. Passion fruit and several other ornamental plants are affected as well. The genome of two of these viruses, Citrus leprosis virus C (CiLV-C) and Coffee ringspot virus (CoRSV) were sequenced, but the knowledge about several molecular characteristics and processes involved in the evolution of their populations are still scarce. Thus, the objective of this study was to molecularly characterize new isolates of BTV infecting citrus and coffee, reveal the phylogenetic relationships with known species of BTV, and assess the genetic variability of the population of CiLV-C in Brazil. For CiLV-C studies, 47 samples of Citrus sinensis showing typical symptoms of leprosis were collected in different Brazilian regions during 2011-2015. The presence of CiLV-C was detected by RT-PCR in all the collected samples and four regions of the viral genome (p29, p15, IR and MP) of each isample were sequenced. It has been shown that the CiLV-C population has relatively low variability; although the existence of two lineages named Cor and SJRP were identified in this work. The complete genomes of one isolate of the lineage SJRP (CiLV-C SJRP) and that of the tentative dicorhavirus CoRSV found in Limeira, SP, were obtained by small RNA (siRNA) Sequencing. Validation was performed by sequencing fragments generated by RT-PCR using specific primers throughout the genome. CiLV-C SJRP has about 85% nucleotide identity with the genome of the type-member of the Cilevirus genus and shows evidence of recombination with isolates of the lineage Cor, which are prevalent in Brazil. CoRSV isolate Limeira has more than 90% of nucleotide identity with CoRSV Lavras, indicating that both isolates are members of the same tentative species of dichorhavirus.

Acredita-se que o Brasil é o centro de diversidade de vírus transmitidos por ácaros do gênero Brevipalpus (VTB). Alguns desses VTB infectam culturas fundamentais para o agronegócio brasileiro como citros e café, além de maracujá e de várias plantas ornamentais. Na última década os genomas de dois deles, Citrus leprosis virus C (CiLV-C) e Coffee ringspot virus (CoRSV) foram sequenciados, mas ainda é escasso o conhecimento sobre a diversidade genética e processos evolutivos envolvidos na população dessas espécies. Neste contexto, o objetivo deste trabalho foi caracterizar molecularmente novas estirpes de CiLV-C e CoRSV que infectam citros e café, respectivamente. E revelar as relações filogenéticas com espécies de VTB conhecidas, assim como avaliar a variabilidade genética da população de CiLVC no Brasil. Para o estudo de CiLV-C, 47 amostras de Citrus sinensis apresentando sintomas típicos da leprose dos citros foram coletadas em diferentes regiões do Brasil no período de 2011-2015. A presença de CiLV-C foi detectada por RT-PCR em todas as amostras coletadas e, posteriormente, foi realizado o sequenciamento de quatro regiões do genoma viral (p29, p15, RI e MP) de cada isolado. As sequências obtidas foram utilizadas no estudo de filogenia e variabilidade da população de CiLV-C no Brasil. Foi demonstrado que a população de CiLV-C apresenta uma variabilidade relativamente baixa; entretanto, foi identificada a existência de duas linhagens dentro da espécie, nomeadas Cor e SJRP. Os genomas completos de CiLV-C SJRP e também do dicorhavirus tentativo CoRSV identificado em Limeira, SP, foram obtidos mediante o sequenciamento de RNA de pequeno tamanho (siRNA). Cada sequência foi validada mediante o sequenciamento de fragmentos gerados por RT-PCR ao longo do genoma. CiLV-C SJRP apresenta cerca de 85% de identidade de nucleotídeo com o membro-tipo do gênero Cilevirus e exibe evidências de recombinação com isolados da linhagem Cor, a prevalente no território brasileiro. Globalmente, o genoma de CoRSV Limeira apresenta mais de 90% de identidade de nucleotídeo com isolado CoRSV Lavras, o que indica que ambos os isolados são membros da mesma espécie tentativa de dichorhavirus. / South America is most likely the center of diversity of Brevipalpus transmitted viruses (BTV). Some of these BTV infect major crops of the Brazilian agribusiness such as citrus and coffee. Passion fruit and several other ornamental plants are affected as well. The genome of two of these viruses, Citrus leprosis virus C (CiLV-C) and Coffee ringspot virus (CoRSV) were sequenced, but the knowledge about several molecular characteristics and processes involved in the evolution of their populations are still scarce. Thus, the objective of this study was to molecularly characterize new isolates of BTV infecting citrus and coffee, reveal the phylogenetic relationships with known species of BTV, and assess the genetic variability of the population of CiLV-C in Brazil. For CiLV-C studies, 47 samples of Citrus sinensis showing typical symptoms of leprosis were collected in different Brazilian regions during 2011-2015. The presence of CiLV-C was detected by RT-PCR in all the collected samples and four regions of the viral genome (p29, p15, IR and MP) of each isample were sequenced. It has been shown that the CiLV-C population has relatively low variability; although the existence of two lineages named Cor and SJRP were identified in this work. The complete genomes of one isolate of the lineage SJRP (CiLV-C SJRP) and that of the tentative dicorhavirus CoRSV found in Limeira, SP, were obtained by small RNA (siRNA) Sequencing. Validation was performed by sequencing fragments generated by RT-PCR using specific primers throughout the genome. CiLV-C SJRP has about 85% nucleotide identity with the genome of the type-member of the Cilevirus genus and shows evidence of recombination with isolates of the lineage Cor, which are prevalent in Brazil. CoRSV isolate Limeira has more than 90% of nucleotide identity with CoRSV Lavras, indicating that both isolates are members of the same tentative species of dichorhavirus.

Noroviruses are the major cause of acute viral gastroenteritis worldwide. Thus, rapid and reliable pathogen detection and control are crucial to avoid epidemic outbreaks. Peptides which bind to these viruses with high specificity and affinity could serve as small and stable recognition elements in biosensing applications for a point-of-care diagnostic of noroviruses. They can be identified by screening large phage display libraries using the biopanning technique. In the present study, this method was applied to identify norovirus-binding peptide motifs. For this purpose, a biopanning based on column chromatography was established, and three rounds of selections were performed. After the second round, the cosmix-plexing recombination technique was implemented to enhance the chance of obtaining peptides with very high affinity. Biopanning data evaluation was based on next-generation sequencing (NGS), to show that this innovative method can enable a detailed analysis of the complete sequence spectrum obtained during and after biopanning. Highly enriched motifs could be characterized by their large proportion of the amino acids W, K, R, N, and F. Neighbourhood analysis was exemplarily performed for selected motifs, showing that the motifs FAT, RWN, and KWF possessed the fingerprints with the largest differences relative to the original library. This thesis thus presents next-generation sequencing-based analysis tools, which could now be transferred to any other biopanning project. The identified peptide motifs represent promising candidates for a future examination of their norovirus-specific binding. A new option for testing such phage-target interactions in the context of biopanning selections was studied in the second part of the thesis. For this purpose, a phage-based microarray was developed as a miniaturized binding assay. As a prerequisite, the different immobilization behaviour of phages on positively and negatively charged surfaces was studied, and a non-contact printing technique for bacteriophages was developed. Subsequently, the interaction of phages and antibodies directed against phage coat proteins was characterized in enzyme-linked immunosorbent assays, and the protocol was successfully transferred to the non-contact printed phage spots. At the proof-of-concept level, the phage array could finally be integrated into a microfluidic setup, showing a higher signal-to-background ratio relative to the static phage array. These results point the way towards a microfluidic phage array, allowing online monitoring, automation, and parallelisation of the phage array analysis. / Noroviren gelten als Hauptursache akuter viraler Magen-Darm-Erkrankungen. Nur eine zeitnahe und verlässliche Detektion und Kontrolle dieser Pathogene kann epidemische Ausbrüche vermeiden. Um dies zu ermöglichen, könnten Peptide, die an diese Viren mit hoher Spezifität und Affinität binden, als kleine und stabile Erkennungselemente in biosensorischen Anwendungen eingesetzt werden. Solche Peptide können mithilfe der Biopanning-Technik identifiziert werden, die auf dem Screening großer Phagen-Display-Bibliotheken beruht. In der vorliegenden Arbeit wurde diese Methode genutzt, um Norovirus-bindende Peptidmotive zu identifizieren. Dazu wurde ein auf Säulenchromatographie basierendes Biopanning entwickelt und drei Selektionsrunden durchgeführt. Die Cosmix-Plexing-Rekombinationstechnik wurde nach der zweiten Runde eingesetzt, um die Wahrscheinlichkeit der Gewinnung hochaffiner Binder zu erhöhen. Die Auswertung der Biopanningdaten erfolgte mittels Hochdurchsatzsequenzierung (Next-Generation Sequencing). Es konnte gezeigt werden, dass diese innovative Methode die detailierte Analyse des kompletten Sequenzspektrums während und nach dem Biopanning ermöglicht. Stark angereicherte Motive konnten durch ihren hohen Anteil an den Aminosäuren W, K, R, N und F charakterisiert werden. Eine Nachbarschaftsanalyse wurde exemplarisch für ausgewählte Motive durchgeführt. Dabei wurden die stärksten Unterschiede im Fingerprint im Vergleich zur Ausgangsbibliothek bei den Motiven FAT, RWN und KWF gefunden. Diese Dissertation stellt damit auf Next-Generation Sequencing basierende Analysetechniken vor, die für weitere Biopanningprojekte übernommen werden können. Die identifizierten Peptidmotive könnten als vielversprechende Kandidaten zukünftig auf ihre Norovirus-spezifische Bindung hin getestet werden. Eine neue Möglichkeit, solche Phagen-Analyt-Interaktionen zu untersuchen, wurde im zweiten Teil der Dissertation untersucht. Dafür wurde als miniaturisierter Bindungsassay ein Phagen-basiertes Mikroarray entwickelt. Als Voraussetzung wurde zunächst das unterschiedliche Immobilisierungsverhalten von Bakteriophagen auf positiv und negativ geladenen Oberflächen untersucht und eine kontaktfreie Drucktechnik für Bakteriophagen etabliert. Anschließend wurde die Interaktion von Phagen und gegen sie gerichteten Antikörpern in Enzym-gekoppelten Immunadsorptionstests charakterisiert und das Protokoll erfolgreich auf die kontaktfrei gedruckten Phagenspots übertragen. Schließlich wurde erstmals die grundsätzliche Möglichkeit gezeigt, das Array in ein mikrofluidisches Setup zu integrieren, was zu einem höheren Signal-zu-Hintergrund-Verhältnis im Vergleich zum statischen Array führte. Diese Ergebnisse zeigen damit den Weg zu einem mikrofluidischen Phagen-Array auf, das sowohl die Möglichkeit des Online-Monitorings als auch der Automatisierung und Parallelisierung der Phagen-Array-Analyse bietet.

Noroviruses are the major cause of acute viral gastroenteritis worldwide. Thus, rapid and reliable pathogen detection and control are crucial to avoid epidemic outbreaks. Peptides which bind to these viruses with high specificity and affinity could serve as small and stable recognition elements in biosensing applications for a point-of-care diagnostic of noroviruses. They can be identified by screening large phage display libraries using the biopanning technique. In the present study, this method was applied to identify norovirus-binding peptide motifs. For this purpose, a biopanning based on column chromatography was established, and three rounds of selections were performed. After the second round, the cosmix-plexing recombination technique was implemented to enhance the chance of obtaining peptides with very high affinity. Biopanning data evaluation was based on next-generation sequencing (NGS), to show that this innovative method can enable a detailed analysis of the complete sequence spectrum obtained during and after biopanning. Highly enriched motifs could be characterized by their large proportion of the amino acids W, K, R, N, and F. Neighbourhood analysis was exemplarily performed for selected motifs, showing that the motifs FAT, RWN, and KWF possessed the fingerprints with the largest differences relative to the original library. This thesis thus presents next-generation sequencing-based analysis tools, which could now be transferred to any other biopanning project. The identified peptide motifs represent promising candidates for a future examination of their norovirus-specific binding. A new option for testing such phage-target interactions in the context of biopanning selections was studied in the second part of the thesis. For this purpose, a phage-based microarray was developed as a miniaturized binding assay. As a prerequisite, the different immobilization behaviour of phages on positively and negatively charged surfaces was studied, and a non-contact printing technique for bacteriophages was developed. Subsequently, the interaction of phages and antibodies directed against phage coat proteins was characterized in enzyme-linked immunosorbent assays, and the protocol was successfully transferred to the non-contact printed phage spots. At the proof-of-concept level, the phage array could finally be integrated into a microfluidic setup, showing a higher signal-to-background ratio relative to the static phage array. These results point the way towards a microfluidic phage array, allowing online monitoring, automation, and parallelisation of the phage array analysis. / Noroviren gelten als Hauptursache akuter viraler Magen-Darm-Erkrankungen. Nur eine zeitnahe und verlässliche Detektion und Kontrolle dieser Pathogene kann epidemische Ausbrüche vermeiden. Um dies zu ermöglichen, könnten Peptide, die an diese Viren mit hoher Spezifität und Affinität binden, als kleine und stabile Erkennungselemente in biosensorischen Anwendungen eingesetzt werden. Solche Peptide können mithilfe der Biopanning-Technik identifiziert werden, die auf dem Screening großer Phagen-Display-Bibliotheken beruht. In der vorliegenden Arbeit wurde diese Methode genutzt, um Norovirus-bindende Peptidmotive zu identifizieren. Dazu wurde ein auf Säulenchromatographie basierendes Biopanning entwickelt und drei Selektionsrunden durchgeführt. Die Cosmix-Plexing-Rekombinationstechnik wurde nach der zweiten Runde eingesetzt, um die Wahrscheinlichkeit der Gewinnung hochaffiner Binder zu erhöhen. Die Auswertung der Biopanningdaten erfolgte mittels Hochdurchsatzsequenzierung (Next-Generation Sequencing). Es konnte gezeigt werden, dass diese innovative Methode die detailierte Analyse des kompletten Sequenzspektrums während und nach dem Biopanning ermöglicht. Stark angereicherte Motive konnten durch ihren hohen Anteil an den Aminosäuren W, K, R, N und F charakterisiert werden. Eine Nachbarschaftsanalyse wurde exemplarisch für ausgewählte Motive durchgeführt. Dabei wurden die stärksten Unterschiede im Fingerprint im Vergleich zur Ausgangsbibliothek bei den Motiven FAT, RWN und KWF gefunden. Diese Dissertation stellt damit auf Next-Generation Sequencing basierende Analysetechniken vor, die für weitere Biopanningprojekte übernommen werden können. Die identifizierten Peptidmotive könnten als vielversprechende Kandidaten zukünftig auf ihre Norovirus-spezifische Bindung hin getestet werden. Eine neue Möglichkeit, solche Phagen-Analyt-Interaktionen zu untersuchen, wurde im zweiten Teil der Dissertation untersucht. Dafür wurde als miniaturisierter Bindungsassay ein Phagen-basiertes Mikroarray entwickelt. Als Voraussetzung wurde zunächst das unterschiedliche Immobilisierungsverhalten von Bakteriophagen auf positiv und negativ geladenen Oberflächen untersucht und eine kontaktfreie Drucktechnik für Bakteriophagen etabliert. Anschließend wurde die Interaktion von Phagen und gegen sie gerichteten Antikörpern in Enzym-gekoppelten Immunadsorptionstests charakterisiert und das Protokoll erfolgreich auf die kontaktfrei gedruckten Phagenspots übertragen. Schließlich wurde erstmals die grundsätzliche Möglichkeit gezeigt, das Array in ein mikrofluidisches Setup zu integrieren, was zu einem höheren Signal-zu-Hintergrund-Verhältnis im Vergleich zum statischen Array führte. Diese Ergebnisse zeigen damit den Weg zu einem mikrofluidischen Phagen-Array auf, das sowohl die Möglichkeit des Online-Monitorings als auch der Automatisierung und Parallelisierung der Phagen-Array-Analyse bietet.

Analyzing the data produced by next-generation sequencing technologies relies on access to information synthesized based on previous research findings. The volume of data available in the literature is growing rapidly, and it is becoming increasingly necessary for researchers to use AI or other statistics-based approaches in the analysis of their datasets. In this project, knowledge graphs are explored as a tool for providing access to contextual gene-information available in scientific literature. The explorative method described in this thesis is based on the implementation and comparison of two approaches for knowledge graph construction, a rule-based statistical as well as a neural-network and co-occurrence based approach, -based on specific literature contexts. The results are presented both in the form of a quantitative comparison between approaches as well as in the form of a qualitative expert evaluation of the quantitative result. The quantitative comparison suggested that contrasting knowledge graphs constructed based on different approaches can provide valuable information for the interpretation and contextualization of key genes. It also demonstrated the limitations of some approaches e.g. in terms of scalability as well as the volume and type of information that can be extracted. The result further suggested that metrics based on the overlap of nodes and edges, as well as metrics that leverage the global topology of graphs are valuable for representing and comparing contextual information between knowledge graphs. The result based on the qualitative expert evaluation demonstrated that literature-derived knowledge graphs of gene-information can be valuable tools for identifying research biases related to genes and also shed light on the challenges related to biological entity normalization in the context of knowledge graph development. In light of these findings, automatic knowledge-graph construction presents as a promising approach for improving access to contextual information about genes in scientific literature. / För att analysera de stora mängder data som produceras med hjälp av next-generation sequencing krävs det att forskare har tillgång till och kan sammanställa information från tidigare forskning. I takt med att mängden data som finns tillgänglig i den vetenskapliga litteraturen ökar, så ökar även behovet av att använda AI och andra statistiska metoder för att få tillgång till denna data i analysen. I detta projekt utforskas kunskapsgrafer som verktyg för att tillgängliggöra kontextuell geninformation i vetenskapliga artiklar. Den explorativa metod som beskrivs i detta projekt är baserad på implementationen och jämförelsen av två olika tekniker för kunskapsgrafgenerering, en regelbaserad-statistisk metod samt en metod baserad på neurala-nätverk och co-occurrence, baserade på specifika kontexter inom litteraturen. Resultatet presenteras både i form av en kvantitativ jämförelse mellan metoder samt genom en kvalitativ expertutvärdering baserad på det kvantitativa resultatet. Den kvantitativa jämförelsen antydde att jämförelsen mellan kunskapsgrafer genererade med hjälp av olika metoder kan bidra med värdefull information för tolkningen och kontextualiseringen av viktiga gener. Resultatet visade även på begränsningar hos vissa metoder, till exempel gällande skalbarhet samt den mängd och typ av information som kan extraheras. Men även att metrics baserade på överlappning av hörn och kanter, samt metrics som tar hänsyn till den globala topologin i grafer kan vara användbara i jämförelsen av, samt för att representera skillnader mellan biologiska kunskapsgrafer. Resultatet från den kvalitativa expertutvärderingen visade att kunskapsgrafer baserade på geninformation extraherad från vetenskapliga artiklar kan vara värdefulla verktyg för att identifiera forskningsbias gällande gener, samt framhävde viktiga utmaningar gällande normalisering av biologiska entiteter inom området kunskapsgrafsutveckling. Baserat på dessa fynd framstår automatisk kunskapsgrafsgenerering som ett lovande tillvägagångssätt för att förbättra tillgängligheten av kontextuell geninformation i vetenskaplig litteratur.