>Magister Scientiae - MSc / Next generation sequencing (NGS) technology platforms have accelerated ability to produce completed genome assemblies. Recently, collaborators at Tygerberg Medical School outsourced the sequencing of Oryx bacillus, a member of the Mycobacterium tuberculosis complex (MTC). A total of 31,271,059 short reads were generated and required filtering, assembly and annotation using bioinformatics algorithms. In this project, an NGS assembly pipeline was implemented, tailored specifically for SOLiD sequence data. The raw reads were aligned to seven fully sequenced and annotated MTC members, namely, Mycobacterium tuberculosis H37Rv, H37Ra, CDC1551, F11, KZN 1435, Mycobacterium bovis AF2122/97 and Mycobacterium bovis BCG str. Pasteur 1173P2 using NovoalignCS. Depth and breadth of sequence coverage across each base of the reference genome was calculated using BEDTools, and structural variation. Structural variation at the nucleotide level including deletions, insertions and single nucleotidepolymorphisms (SNPs) were called using three tools, GATK, SAMtools and Nesoni. These variations were further filtered using in-house PERL scripts. Putative functional roles for the alterations at the DNA level were extrapolated from the overlap with essential genes present in annotated MTC members. Approximately 20,730,631 short reads (59.78%) out of a total of 31,271,059 reads aligned to the seven reference genomes. The per base sequence coverage calculations revealed an average of 1,243 unaligned regions. These unaligned regions overlapped with mycobacterial regions of difference (RD) and genetic phage elements acquired by the MTC through horizontal gene transfer and are genes prevalent in the clinical isolates of M. tuberculosis. A total of 2,680 genetic variations were identified and categorised into 845 synonymous and 1,724 non-synonymous SNPs together with 44 insertions and 67 deletions. Some of the variant alleles overlapped known genes to be involved in TB drug resistance. While the biological significance of our findings remain to be elucidated, it nonetheless deserves further attention, because SNPs have the potential to impact on strain phenotype by gene disruption. Therefore, any hypotheses generated from these large-scale analyses will be tested by our collaborators at Tygerberg medical school.

The ability of Next-Generation Sequencing (NGS) to produce massive quantities of genomic data inexpensively has allowed to study the structure of viral populations from an infected host at an unprecedented resolution. As a result of a high rate of mutation and recombination events, an RNA virus exists as a heterogeneous "swarm". Virologists and computational epidemiologists are widely using NGS data to study viral populations. However, discerning rare variants is muddled by the presence of errors introduced by the sequencing technology. We develop and implement time- and cost-efficient strategy for NGS of multiple viral samples, and computational methods to analyze large quantities of NGS data and to handle sequencing errors. In particular, we present: (i) combinatorial pooling strategy for massive NGS of viral samples; (ii) kGEM and 2SNV — methods for viral population haplotyping; (iii) ShotMCF — a Multicommodity Flow (MCF) based method for frequency estimation of viral haplotypes; (iv) QUASIM — an agent-based simulator of viral evolution taking in account viral variants and immune response.

La spermatogenèse est un processus complexe qui dépend de la coopération de nombreux gènes. Son produit final le spermatozoïde, est un sujet d’étude idéal car il renferme à la fois des indices d’événements passés ainsi que des informations qui seront transmises à l'ovocyte lors de la fécondation. L'identification de nouveaux acteurs de la spermatogenèse, des modifications spécifiques de l'ADN du sperme ou la présence de transcrits spécifiques pourraient servir comme biomarqueurs dans le diagnostic de l’infertilité. Cette thèse avait pour but d’analyser le génome, le transcriptome et l’épigénome de spermatozoïdes dans le contexte de l'infertilité masculine. Nous avons identifié de nouvelles causes génétiques et confirmé la présence d'anomalies de méthylation dans le sperme d'hommes infertiles. Nous avons découvert 20 mutations dans le gène SOX8, chez des patients atteints de trouble du développement sexuel ou d'infertilité masculine ou féminine, qui apparaît comme un régulateur du développement et de la fonction gonadique. Par séquençage d’exome, une mutation dans le gène ATAD2 modeleur de la chromatine spécifique de la lignée germinale mâle fut également identifiée. Par RNA-seq et MeDIP-chIP du sperme d’hommes fertiles et infertiles, nous avons caractérisé la signature transcriptionnelle du sperme. La majorité des ARNs spermatiques humain est remarquablement conservée chez les mammifères placentaires suggérant des fonctions ancestrales importantes. Enfin, nos données transcriptomiques et épigénétiques tendent à indiquer qu’une expression et une régulation adéquates des gènes impliqués dans le remodelage de la chromatine constituent un facteur clé pour la fertilité masculine. / Spermatogenesis is a complex process which depends on the cooperation of many genes. The end-product, the spermatozoon, is an ideal subject for study since it carries both clues of the past events and information which will be transmitted to the oocyte at fertilization. The identification of main actors of spermatogenesis, specific modifications of sperm DNAs or sperm specific isoforms could improve our understanding of a such complex mechanism and could serve as a determination of biomarkers or diagnostic tools for fertility. The aim of the project was to go further three omes: genome, epigenome and transcriptome of mature human sperm in the context of male infertility. We identified new genetic causes of male infertility and confirmed the presence of methylation abnormalities in sperm cells of infertile men. Firstly, SOX8 gene was found mutated in a cohort of 20 patients with disorder of sex development and male or female infertility. Similarly, to NR5A1, SOX8 appears to be a novel regulator of gonadal development and function. Then by exome-sequencing, we identified a homozygous nonsense mutation in the male germline-specific chromatin modeler ATAD2. Furthermore, RNA-seq and MeDIP-chIP of sperm from fertile and infertile men along with bioinformatics analyzes of the generated data, enabled us to characterize more deeply the normal sperm transcriptional signature. We also found that the majority of human sperm RNAs are remarkably preserved in placental mammals suggesting crucial ancestral functions. Finally, proper expression and regulation of chromatin remodelers seem to be critical for male fertility, as revealed by both the transcriptomic and the epigenetic data.

Bio-systems are inherently complex information processing systems. Furthermore, physiological complexities of biological systems limit the formation of a hypothesis in terms of behavior and the ability to test hypothesis. More importantly the identification and classification of mutation in patients are centric topics in today’s cancer research. Next generation sequencing (NGS) technologies can provide genome-wide coverage at a single nucleotide resolution and at reasonable speed and cost. The unprecedented molecular characterization provided by NGS offers the potential for an individualized approach to treatment. These advances in cancer genomics have enabled scientists to interrogate cancer-specific genomic variants and compare them with the normal variants in the same patient. Analysis of this data provides a catalog of somatic variants, present in tumor genome but not in the normal tissue DNA. In this dissertation, we present a new computational framework to the problem of predicting the number of mutations on a chromosome for a certain patient, which is a fundamental problem in clinical and research fields. We begin this dissertation with the development of a framework system that is capable of utilizing published data from a longitudinal study of patients with acute myeloid leukemia (AML), who’s DNA from both normal as well as malignant tissues was subjected to NGS analysis at various points in time. By processing the sequencing data at the time of cancer diagnosis using the components of our framework, we tested it by predicting the genomic regions to be mutated at the time of relapse and, later, by comparing our results with the actual regions that showed mutations (discovered at relapse time). We demonstrate that this coupling of the algorithm pipeline can drastically improve the predictive abilities of searching a reliable molecular signature. Arguably, the most important result of our research is its superior performance to other methods like Radial Basis Function Network, Sequential Minimal Optimization, and Gaussian Process. In the final part of this dissertation, we present a detailed significance, stability and statistical analysis of our model. A performance comparison of the results are presented. This work clearly lays a good foundation for future research for other types of cancer.

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

The 21st century marks the defining point of human history in terms of technological advancement. In 2014, we were at the edge of acquiring a complete understanding of the fundamental construct to all life forms. The capability to manipulate and recreate lives as desired will soon be at our hands and will eventually lead to the redefinition of life and humanity. This brave new world, for better or worse, will be stitched together by scientific breakthroughs in many disciplines. Nanopore fluidic system – and microfluidic in general – might be one of the key puzzles towards the future. It is seen as a likely candidate for the next generation of rapid and low-cost genetic sequencing technology, which will allow us to gain thorough insight into the genetic code of every living organism on earth. It can also have the capability to individually detect and manipulate virtually any biological molecules, possibly allowing it to be a universal diagnostic tool or a bio-molecule synthesiser. The future of nanopore fluidic system is prosperous, but the difficulties are equally challenging. Currently, both biological and solid-state nanopores are non-trivial to create. For instance, a small solid-state nanopore can only be fabricated with expansive machinery in a low-yield, low-throughput manner. To overcome this challenge, a new set of methods involving high electric field to fabricate and enlarge a solid-state nanopore has been developed. It was found that a nanopore, when subjected to a high electric field, can be enlarged in angstrom increments and cleared of unidentified obstructions that cause low-frequency ionic current fluctuations. It was also found that an intact solid-state membrane, when subjected to a high electric field for a period of time, can leave a single nanopore imprinted onto it. The process of creation is best describe as a dielectric breakdown event and can be modeled by the percolation theory for dielectric breakdown. The resulting nanopores are cylindrical in shape and are shown to be equally capable of single molecule sensing compare to pores created by other methods. To accommodate future nanopore designs and applications and to examine the scope of applicability of the new fabrication approach, more advanced nanopore devices were created on some dual-layer solid-state membranes comprising of a metallic and a dielectric layer. Experiments indicated that the method could indeed create nanopore on such advanced membranes. It was further shown that the metallic layer receded further than the dielectric layer, forming a hollow conical shape at the opening of the dielectric nanopore. Such metalized bi-layer nanopore system was found to interact strongly with short single stranded DNA molecules, resulting in prolonged DNA translocation time. A simple picture of the mechanism was proposed to explain the observation. Lastly, to extend the limit of the new fabrication approach, I attempted to fabricate nanopore on complex multi-layer membranes involving a graphene film sandwiched in several dielectric materials. It was found that the quality of the graphene film and the transfer method were vital to the success of this project. Nevertheless, preliminary results indicated that the new method could create a nanopore through this complex multi-layer membrane. The new method to fabricate and tune both simple and complex nanopores is amongst the simplest, the least costly and the most efficient one that one can imagine. The research work has already sparked a dramatic increase in scientific throughput in our laboratory and other laboratories we had collaboration with. It fueled more than a dozen projects and involved close to a thousand nanopores in total. Such projects are far from possible if they were to rely on conventional fabrication methods. However, these are insignificant if we consider the new method is simple enough that, for the very first time, general public can easily access nanofabrication and single-molecule manipulation technology. The liberation of nanotechnology to the general public symbolically marks the beginning of a brave new world.

Lower motor neuron diseases and peripheral neuropathies are two groups of diseases that include multiple rare disorders where many causes are unknown and definitive treatments are unavailable. Understanding the molecular etiology of these genetic diseases provides an opportunity for rapid diagnosis, preconception genetic counseling and, in a subset, direction for the development of future treatment options. The recent introduction of whole exome sequencing (WES) marks a new era in Mendelian genetic disease research as the majority of the coding region of the genome can be sequenced in a timely and cost-effective manner. In this study, WES was used to investigate the molecular etiology of a cohort of 37 patients presenting with lower motor neuron disease or peripheral neuropathy. A molecular diagnosis was determined for seven patients informing the diagnostic utility of WES. Novel phenotypes were found for three genes originally associated with a different disorder. Finally, the foundation has been laid, through the use of functional studies and large scale data-sharing, to identify novel disease-causing genes for lower motor neuron disease and peripheral neuropathy.

Objective: The objective of this thesis was to estimate the impact on newborn screening (NBS) results of changing screening technology from tandem mass spectrometry (MS/MS) to an approach using targeted next-generation sequencing (T-NGS) and MS/MS in parallel. Methods: We integrated results of an analysis of MS/MS screening data for phenylketonuria (PKU) and medium-chain acyl-CoA dehydrogenase (MCAD) deficiency; and a query of genetic compendia for variants of genes associated with the two disorders. Results: The introduction of T-NGS into NBS may reduce nearly 80% of false positives that are generated using the current screening approach. Based on estimated NBS results, T-NGS may be applied using a second-tier approach, which may improve specificity while maintaining sensitivity at its current level. Discussion: T-NGS may enhance the performance of NBS for PKU by improving specificity when used as a second tier test, but may be limited by feasibility and cost under current circumstances. Future studies should consider the cost-effectiveness of T-NGS for all infants undergoing NBS.

A comprehensive molecular and phenotypic analysis of congenital and childhood cataractRachel L. Gillespie; The University of Manchester, Doctor of Philosophy, 2015Congenital and childhood cataract (CCC) is estimated to affect 3.5-6 per 10,000 children under 16 years in developed countries - a major cause of lifelong visual impairment. It is estimated that 25-50% of CCC cases are caused by genetic mutations. CCC demonstrates extreme heterogeneity with more than 100 associated genes, and may occur as an isolated anomaly of the eye (non-syndromic) or as a manifestation of a multisystem condition (syndromic). Limitations of conventional sequencing technologies have precluded precise genetic diagnosis and limited understanding of the epidemiological basis of the condition. Next generation sequencing (NGS) technologies have revolutionised the approach to the study of human disease. The aim of this research was to conduct a comprehensive molecular and phenotypic analysis of CCC using NGS.A disease-targeted NGS assay was designed to screen, in parallel, 115 genes associated with all forms of CCC. DNA from 36 patients, randomly selected from the study cohort, underwent cataract-targeted NGS. Putative cataract-causing variants were identified in 75% of individuals; 85% of non-syndromic patients and 63% of syndromic CCC patients. Cataract-targeted NGS was able to efficiently delineate disease sub-type and in some cases identified rare syndromic forms of the condition. These findings were envisaged to alter care and management of CCC patients demonstrating the potential clinical utility of the test. In a subset of cases, NGS identified CCC was a manifestation of an inborn error of metabolism. A number of these conditions were eligible for preventative treatment emphasizing the importance of early diagnosis. A strategic approach to the identification of novel recessive causes of CCC was also undertaken. Affected children from seven consanguineous families underwent pre-screening by cataract-targeted NGS to delineate those with mutations in known genes. Mutation negative patients underwent autozygosity-guided whole exome sequencing (WES) analysis. This strategic approach to disease gene discovery led to the identification three novel cataract-causing candidate genes, TRIM8, CCDC13 and GRWD1. It also led to the association of EIF2B2, known to cause adult-onset leukoencephalopathy with vanishing white matter (VWM) disease featuring pre-senile onset cataract, with childhood-onset cataract. This work demonstrated that cataract-targeted NGS offers an efficient and unbiased means of pre-screening, however, causation is difficult to assign to novel disease genes in the absence of experimental evidence. Correspondingly, in vitro analysis of a missense variant in HMX1 demonstrated the deleterious effect of the mutation on protein function. This work confirmed HMX1 as the cause of a rare oculoauricular phenotype and expanded the class of disease-causing mutations in this gene. In conclusion, this study has demonstrated that NGS is effective in the study of CCC and has provided a platform for future studies in to the genetic aetiology of the condition, as well as the molecular mechanisms underlying lens transparency and human development. The work adds to the increasing body of evidence that augurs an era of personalised genomic medicine in ophthalmology that will foresee improved patient outcomes attributable to the implementation of a stratified approach to medicine.

Knowledge of the diet of wildlife can aid wildlife biologists to better understand how a species functions within a given ecosystem. Numerous studies have identified various avenues to examine diet for species throughout the world. Wildlife biologists have used diet composition variables as a means to better understand habitat use and aid in the management and conservation of mule deer, Odocoileus hemionus. The complexity of deer diet is still unknown, and local wildlife management agencies could improve conservation strategies with more information regarding the breadth of plant selection in deer diet. Researchers have used non-invasive methods, such as microhistology via fecal analyses, to assess diet composition for mule deer. However, microhistology has several drawbacks that include accuracy in identification and differentiation of plant species, and even genus, as well as determination of accurate proportions of taxa ingested. Genetic techniques, such as next-generation sequencing (NGS), present new avenues for analyzing herbivore diets, especially through the amplification and analyses of specific regions of chloroplast DNA (cpDNA). Additionally, few studies have directly compared microhistological and NGS diet analyses results for any wildlife species. My objectives were to compare diet composition results of both microhistological and NGS diet analyses through estimating diet richness, taxonomic resolution, percent diet, and frequency of occurrence of plant taxa across samples. Mule deer fecal samples were collected on the Navajo Nation from summer and winter ranges of two distinct mule deer populations, Chuska and Carrizo. I found far greater richness and resolution from NGS of plant taxa through the identification of a greater number of species and genera among all populations, within seasons. Upon testing both methods for both populations, no significant agreement was identified for percent of families identified in the diet with both methods, across all samples. I found trends of positive correlation in the occurrence of families between both methods for Carrizo summer diet, as well as among genera and families in Carrizo winter diet. Upon further statistical analyses, I found no significant positive correlation in the occurrence of genera and families identified with both methods among all samples. Genetic techniques may present innovative methods for determining mule deer diet in various ecosystems, and may also be applied to a broad range of herbivore diet studies.