This thesis presents short DNA alignment tools optimization. These short DNA reads are products of next\nobreakdash-generation sequencing technologies. The results produced by existing align\-ment tools can be influenced by various parameters. For this purpose, an optimization framework to find the optimal values of selected parameters was developed. This framework is based on differencial evolution algorithm and its main goal is to maximize the alignment accuracy. The functionality of the framework was tested on both real and generated data sets of short DNA reads. An accurate alignment is crucial for correct prediction of various genetic characteristics.

Dans cette thèse, nous nous intéressons au problème de recherche incrémentale de motifs dans des séquences fortement similaires (On-line Pattern Matching on Highly Similar Sequences), issues de technologies de séquençage à haut débit (SHD). Ces séquences ne diffèrent que par de très petites quantités de variations et présentent un niveau de similarité très élevé. Il y a donc un fort besoin d'algorithmes efficaces pour effectuer la recherche rapide de motifs dans de tels ensembles de séquences spécifiques. Nous développons de nouveaux algorithmes pour traiter ce problème. Cette thèse est répartie en cinq parties. Dans la première partie, nous présentons un état de l'art sur les algorithmes les plus connus du problème de recherche de motifs et les index associés. Puis, dans les trois parties suivantes, nous développons trois algorithmes directement dédiés à la recherche incrémentale de motifs dans un ensemble de séquences fortement similaires. Enfin, dans la cinquième partie, nous effectuons une étude expérimentale sur ces algorithmes. Cette étude a montré que nos algorithmes sont efficaces en pratique en terme de temps de calcul / In this thesis, we are interested in the problem of on-line pattern matching in highly similar sequences, On-line Pattern Matching on Highly Similar Sequences, outcoming from Next Generation Sequencing technologies (NGS). These sequences only differ by a very small amount. There is thus a strong need for efficient algorithms for performing fast pattern matching in such specific sets of sequences. We develop new algorithms to process this problem. This thesis is partitioned into five parts. In the first part, we present a state of the art on the most popular algorithms of finding problem and the related indexes. Then, in the three following parts, we develop three algorithms directly dedicated to the on-line search for patterns in a set of highly similar sequences. Finally, in the fifth part, we conduct an experimental study on these algorithms. This study shows that our algorithms are efficient in practice in terms of computation time.

MSc (Zoology) / Department of Zoology / See the attached abstract below

Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

TITLE: Mutation and substitution rates in sexual and asexual forms: a clue to the persistence of sex in a model group of Cobitis? AUTOR: Jan Röslein DEPARTMENT: Ústav živočišné fyziologie a genetiky AVČR, v.v.i. SUPERVISOR: Mgr. Karel Janko, Ph.D. ABSTRACT: Subject of this thesis is to test several hypotheses about the evolution of asexual reproduction in model group of fish family Cobitis and its mutual competition among sexual and asexual forms, which touches one of the oldest unresolved issues of biology. Specifically, the work deals with the accumulation of non-synonymous mutations, which accelerated accumulation in the genome of clonal lineages theoretically leads to increased extinction compared with sexually reproducing populations (so-called. The theory of Muller's ratchet and Kondrashov's hatchet). This thesis is based on a normalized cDNA sequencing data from oocytes and liver tissue, which has served as a base matrix (generated based on non-normalized cDNA data) for transcriptome sequencing (RNAseq). Consequently, the RNAseq data have served as validation for acquired polymorphisms, detection of differential expression of allele- specific expression (ASE) hybrid biotypes. This diploma thesis balances among the edges of vast spectrum of hypotheses regarding the evolution of the genus hybrid...

Cystic fibrosis (CF) is genetically conditioned, autosomal recessive disease that occurs in the European population with a prevalence of about 1:2500 - 1:1800. In this disease we observe a mutation of the CTFR gene with subsequent fault in chloride channels. Such afflicted individuals usually suffer from chronic respiratory problems, pancreatic insufficiency, high concentration of chloride ions in sweat and obstructive azoospermia. Genetic testing of CFTR gene is indicated in individuals who meet the CF clinical picture and a positive sweat test (increased concentration of chlorides in the sweat). Genetic testing of the CFTR gene is usually done by using commercial kits detecting the most common mutations of the CFTR gene in the Czech Republic. If the testing results are negative, it is further performed an MLPA method that captures the larger deletions and duplications of gene, eventually a sequencing of all exons is. Despite the well-established algorithm of the testing, some patients suffering from symptoms of CF are left without genetic findings. Thanks to development of next generation sequencing, it is possible to make the diagnosis of CF more effective and uncover the variants that were not captured by previous methods.

Next generation sequencing technologies are changing the way scientific experiments and diseases diagnostics are performed and thus will allow what is called personalized medicine. The sense of presented thesis is to make survey of new approaches to DNA sequencing and demonstrate usage and constraints of bioinformatic analytical tools available to day. Discussed techniques are then applied to the case study of finding molecular basis for rare hereditary disease. Introductory part deals with overview of commercially available sequencing techniques (454 Life Science, Applied Biosystems, Illumina, Helicos). Fundamentals of each method are described and possible further development is outlined. Post sequencing data analysis is than discussed in details. In practical section we demonstrate genome analysis techniques successfully used to reveal causal mutation in the gene responsible for adult form of autozomal neuronal ceroid lipofuscinosis (ANCL). Combination of linkage analysis (Merlin), copy number variant analysis (Genome-Wide Human SNP Array 6.0), analysis of expression profiles (HumanRef-8 v2 Expression BeadChips) and exome sequencing (SOLiD™ 4 System) has been applied to members of one ANCL family. We also paid attention to comparison, evaluation and selection of available mapping algorithms used in...

Internet Protocol multimedia subsystem (IMS) is an architecture for services such as voice over Internet Protocol (VoIP) in IP based communication systems. IMS is standardized by the 3GPP standardization forum, and was first released in 2002. Since then IMS has not had the wide adoption by operators as first anticipated. As 3G already supported voice and video, the operators could not justify the expense of IMS. The current emergence of the fourth generation mobile communication system named Long Term Evolution (LTE) has, however, increased the need for knowledge of IMS and of creating services for it. LTE networks are IP only networks that provide low latency. In order to use LTE for making phone calls, VoIP technologies are needed. IMS is the architecture intended to be used for Voice over LTE (VoLTE). The need for tools for education within IMS was seen in 2006 by Enea Experts in Linköping, Sweden. The author of this thesis designed an IMS for educational purposes, but the project was never fully completed. This thesis will reexamine the design decisions previously made by the author. The requirements stated by the customer remain: that an IMS with basic signaling and logging should be easy to install, maintain, and evolve at a low cost. A literature study of IMS and VoLTE is presented to contribute with knowledge in these areas. The previous design and implementation made by the author is presented and analyzed. The third-party software that the previous implementation was based on is reexamined. Existing open source components are analyzed in order to identify how they can be used to solve the problem and to identify what remains to be developed in order to fulfill the requirements. New design suggestions, presented in today´s context, are proposed and verified using analytical reasoning and experiments. The outcome of the final work is new verified design decisions for the customer to use when implementing a new IMS for educational purposes. The thesis should also provide useful insights which instructors and students can use to teach and learn more about IMS. / Internet Protocol multimedia subsystem (IMS) är en arkitektur för tjänster, som IP-telefoni (Voice over Internet Protocol, VoIP), i IP baserade kommunikationssystem. IMS standardi¬seras av standardiseringsforumet 3GPP och första utgåvan släpptes år 2002. IMS fick dock inte det breda genomslag bland operatörer som förväntats. Eftersom 3G redan hade stöd för tal och video kunde operatörerna inte se skäl till ytterligare utgifter för IMS. Den fjärde generationens mobila kommunikationssystem, Long Term Evolution (LTE) är helt IP-baserat och ger lägre fördröjningar i nätet. För att kunna ringa telefonsamtal via LTE krävs VoIP-teknik. IMS är en arkitektur avsedd för att användas för Voice over LTE (VoLTE). Den nuvarande utvecklingen av LTE har därför ökat behovet av kunskap om IMS och av utveckling av IMS-tjänster. Enea Experts i Linköping insåg behovet av verktyg för utbildning inom IMS år 2006. Författaren av det här examensarbetet designade därför ett IMS för utbildningssyfte. Projektet slutfördes dock aldrig. Syftet med examensarbetet är att ompröva de tidigare designbesluten. Kundens krav kvarstår: att ett IMS med grundläggande signalering och loggning bör vara enkelt att installera, enkelt att underhålla och möjligt att utveckla till en låg kostnad. Arbetet innehåller en litteraturstudie av IMS och VoLTE för att ge en inblick i dessa områden. Den tidigare designen och implementationen presenteras och analyseras. Tredjeparts mjukvara, som den tidigare implementationen baserades på, omprövas. Befintliga programvaror med öppen källkod analyseras i syfte att kartlägga hur de kan användas för att lösa uppgiften, samt att identifiera vad som återstår att utveckla för att uppfylla kraven. Nya beslut kring design presenteras och besluten verifieras med experiment och analytiskt resonemang. Resultatet av detta examensarbete innefattar nya verifierade beslut kring design som kunden kan använda vid utveckling av ett nytt IMS för utbildningssyfte. Arbetet erbjuder också värdefulla insikter som instruktörer och elever kan använda för att undervisa samt för att lära sig mer om IMS.

Today the county councils in Sweden prescribe rollators, but in the near future, the retail market for rollators will increase simultaneously with increased pressure within customer requirements. Among the users there is an increased awareness and urge to have a rollator that is up to date according to today’s development of the society. Which the current rollators on the market does not do. The demands from the customer have therefore started to change and are increasing within the coming generations. This is a result of the availability and possibility of all types of information regarding the subject. This means that there is a gap on the market that is growing, which leaves room for developing of a new product, to be able to keep up with technological progress. The goal was to deliver a reasonable concept proposal for a new rollator that with further development could be taken on to the next phase of development and production. The work also aims to clarify the next generation’s customer demands. Identify the issues surrounding today's prescriptions of rollators, as well as clarify the values that are imbedded in the product. The work has been based on a method called Pretzel, which is a product developing process developed by the company Scalae. Mapping of the society's value and norms connected to the rollators shows that the biggest problem among people is the early acceptance of the aid, this results in late prescribing of rollators. Which means that unnecessary injury often occurs, that is in not only unfortunately for the injured person but also a big cost for the society. Especially in comparison with the prescription cost of a rollator. These problems are thereby deep-rooted in values that closely connected to the slow development of rollators. The technological development today is extremely fast and new products are launched on the market at increasingly speed. For example, it would be very unusually to have the same model of mobile phone as your mother had when she was young. But when it comes to rollators, it is most possible that you may get the same model as your grandmother. To decrease the resistance against rollators the final concept is inspired by existing products on the market that already are accepted by the society. Although in other contexts, such as strollers and shopping carts. By using design and functions that the user is already are familiar with reach acceptance faster and reduce the risk of injuries. The final concept is called the Dramator and is a hybrid between a shopping cart and a rollator. / Idag förskrivs rollator av Landstingen samt kommuner, men inom en snar framtid kan marknaden för retail komma att öka radikalt i samband med att kraven från kunden ökar. Det råder en allt mer ökad medvetenhet och en vilja hos kunden att få en produkt som hänger med i samhällets utveckling, vilket dagens rollatorer inte gör. Då möjligheten och tillgängligheten till all sorts information ökar bidrar detta till att framtida generationer kommer att ställa högre krav på produkten än vad som ställs idag. Detta innebär att det just nu håller på att bildas ett glapp på marknaden och att en ny produkt som hänger med teknikens framfart måste utvecklas. Målet var att komma fram till ett konceptförslag, som sedan skulle kunna tas vidare till nästa fas för vidareutveckling och produktion. Arbetet syftar även till att bringa klarhet i kundkraven hos nästa generation och problem kring dagens förskrivningar, samt klarlägga värderingar som råder kring produkten. Arbetet har utgått från en metod som heter Pretzel, som är en process för degenerering och produktutveckling framtagen av företaget Scalae. Kartläggningen av värderingarna och samhällets syn på rollatorn, visar på problem som resulterar i att många rollatorer utskrivs försent. Detta innebär att onödiga personskador hinner uppkomma som i sin tur kostar samhället mycket pengar i jämförelse med förskrivningskostnaden av en rollator. Problemen ligger djup rotade i värderingar som går hand i hand men den långsamma utvecklingen av rollatorer. Den tekniska utvecklingen går idag oerhört fort och nya produkter lanseras på marknaden med rasande fart. Att till exempel ha samma mobiltelefonmodell som sin mamma är i dagens samhälle högst orimligt, men när det gäller rollatorer kan du till och med få samma modell som din farmor hade.Konceptet är inspirerat av existerande produkter på marknaden som är socialt accepterande, fast i andra sammanhang, så som barnvagnar och klassiska Dramaten-väskan. Genom att använda formspråk och funktioner som användaren redan är bekant med är målet acceptansprocessen för användaren kan kortas ner så att hjälpmedlet kan börja användas i tid och på så sätt minska risken för fallskador. Det slutliga konceptet kallas för Dramatorn och är en hybrid mellan en dramatenväska och en rollator.

Massive parallel sequencing (NGS) is useful in detecting and later classifying somatic driver mutations in cancer tumours. False-positive variants occur in the NGS workflow and they may be mistaken for low frequency somatic cancer mutations in a patient sample. This pushes the need for decreasing the noise rate in the NGS workflow since it may improve the detection of rare allele frequency variants, in particular cancer mutations. In this project, the aim was to reduce the level of false-positive variants in an NGS workflow. The scope was limited to looking at substitution errors and their neighbouring nucleotides. Alongside this, it was also a way to understand how different types of substitution errors are distributed in the data, if their frequencies are affected by neighbouring nucleotides and how data processing may affect these substitution rates. A bioinformatic pipeline was set up where a commercially available genomic DNA sample with known variants was subjected to different trimming and filtering settings. The goal was to reduce the substitution error rate as much as possible, without removing any true variants from the data. The optimised settings were trimming the sequencing reads with 5 bp from the tail and filtering sequencing reads that contained 5 or more substitutions. Three additional samples, whereof two were clinical and the third commercial, were tested with these settings. The results showed that in all samples, C:G>T:A substitutions were of a higher frequency compared to the rest of the substitution types. For all samples, A:T>C:G substitutions, where the neighbouring nucleotide was a C or a G on each side, had a higher frequency compared to A:T>C:G substitutions with other neighbouring nucleotides on both sides. Those substitution types were especially targeted by the trimming. For the two commercial samples, substitutions that resulted in the nucleotide combinations >XAA or >XTT were of a higher frequency compared to the same substitution types that did not result in those nucleotide combinations. Filtering reads with 5 or more substitutions particularly targeted these substitution types. Consequently, filtering had a greater effect on the commercial samples, compared to the clinical samples. Overall, trimming and filtering helped reduce transversions more than the transitions, increasing the transition/transversion ratio after processing the data. The results suggest that trimming and filtering can be a useful method to computationally reduce the transversion errors introduced in an NGS workflow, but transition errors to a lesser extent, in particular A:T>G:C transitions. To confirm these findings, more samples should be tested using this methodology. To better understand the effect of trimming and filtering on variant calling, the scope could in the future be expanded to also look at small insertions and deletions.