Genetic screens have laid much of the groundwork for our current understanding of biology, and mutagenesis screens in Caenorhabditis elegans have proven to be a particularly useful tool in determining the molecular components of biological processes. The Million Mutation Project (MMP) is a collection of mutagenized C. elegans strains that have been clonally propagated and whole-genome sequenced. Utilizing the MMP, I have performed a screen for touch insensitive mutants to assess the phenotypic coverage of the set, to obtain new alleles of known genes, and to potentially identify touch phenotype-causing mutations in genes that have not yet been linked to the touch response. In this thesis I first present my rationale for screening the MMP set for touch phenotypes, then review what has already been learned about genes required for the function of the neurons that sense gentle touch in C. elegans. I describe my approach to phenotyping the MMP set and present statistics on response distributions. As expected, most of the MMP strains that I determined to have strong touch insensitive phenotypes had mutations in genes identified in previous touch phenotype mutageneses. However, some of the phenotype-causing MMP alleles cause protein-coding changes in regions that were not known to be affected by previously characterized alleles. The genomic data from the MMP also allowed me to consider protein-altering mutations in known touch genes that did not result in a detectable phenotype. Finally, I address the set of strains for which we have not identified candidate causative mutations. Although I have not discovered any previously unknown touch genes through my screen of the MMP, it is still quite possible that the touch insensitive mutants I have identified will lead us to identify additional genes needed for gentle touch sensation. Ultimately, my screen has successfully demonstrated the utility of the MMP set and provided new insights as to the structure and function of the genetic determinants of gentle touch sensation in C. elegans.

Genomics has become an important tool in agriculture. Many modern crop breeding approaches such as genomic selection and genome editing require detailed information of the genomic composition of a crop species. However, the assembly of high-quality genome sequences is prone to technical artifacts that arise from inaccuracies in the sequencing technology and assembly algorithms. This is particularly true for the genomes of cereal crops, which are often very large, repeat-rich, and polyploid. Until recently, the highly continuous assembly of such cereal crop genomes from short-read data was mainly possible with proprietary assembly tools. In this work, we combined data generated with several short-read sequencing protocols and genomics technologies, including paired-end and mate-pair reads with multiple insert sizes, 10X linked reads, Hi-C contacts, and optical maps to assemble a chromosome level reference genome of Digitaria exilis (fonio millet) with open-source tools. Fonio millet is a semi-domesticated cereal orphan crop native to West Africa that has a high potential for desert agriculture. We implemented the TRITEX pipeline - a recently developed open-source pipeline for the assembly of large Triticeae genomes. We modified the pipeline to include 10X and Hi-C reads into the assembly process independently. We then compared the TRITEX assembly to the fonio reference genome, which had previously been assembled from the same input data but using proprietary algorithms. We found the two assemblies highly similar in content with high concordance in the local order (0.91 Pearson coefficient for alignments). However, we detected many small putative discrepancies between the two assemblies. While the TRITEX assembly was able to produce a highly continuous genome assembly, further work is needed to characterize the putative discrepancies in more detail.

Pyrenophora teres f. teres is the causal agent of net form net blotch of barley. P. teres f. teres is prevalent globally across all barley growing regions and globally is the most devastating foliar disease of barley. Though economically important, the molecular mechanism whereby P. teres f. teres causes disease is poorly understood and investigations into these mechanisms have been hindered by a lack of genomic resources. To set a genomic foundation for P. teres f. teres the reference isolate 0-1 was sequenced and assembled using PacBio single molecule real-time (SMRT) sequencing and scaffolded into 12 chromosomes to provide the first finished genome of P. teres f. teres. High confidence gene models were generated for the reference genome of isolate 0-1 using a combination of pure culture and in planta RNA sequencing. An additional four P. teres f. teres isolates were sequenced and assembled to the same quality as the reference isolate 0-1 and used in a comparative genomic study. Comparisons of the five P. teres f. teres isolates showed a two-speed genome architecture with the genome being partitioned into core and accessory genomic compartments. Accessory genomic compartments clustered in sub-telomeric regions of the P. teres f. teres genome with a majority of previously identified quantitative trait loci (QTL) associated with avirulence/virulence being spanned by these accessory regions. Using these genomic resources, with a bi-parental mapping population and a natural population for QTL analysis and genome wide association study (GWAS), respectively, we identified a candidate gene for the previously mapped AvrHar. QTL analysis identified a locus extending off the end of P. teres f. teres chromosome 5 and GWAS analysis identified significant associations with a gene encoding a small secreted protein. The candidate AvrHar gene was validated using CRISPR-Cas9-RNP gene disruption in parental isolates 15A and 0-1. Disruption of AvrHar in isolate 15A did not result in a phenotypic change while disruption of the 0-1 allele resulted in a complete loss of pathogenicity. This is the first identification of an effector from P. teres f. teres validated using CRISPR-Cas9-RNP gene editing. / North Dakota Barley Council

The three-dimensional (3D) structure of the genome plays a crucial role in gene expression regulation. Chromatin conformation capture technologies (Hi-C) have revealed that the genome is organized in a hierarchy of topologically associated domains (TADs), sub-TADs, and chromatin loops which is relatively stable across cell-lines and even across species. These TADs dynamically reorganize during development of disease, and exhibit cell- and conditionspecific differences. Identifying such hierarchical structures and how they change between conditions is a critical step in understanding genome regulation and disease development. Despite their importance, there are relatively few tools for identification of TADs and even fewer for identification of hierarchies. Additionally, there are no publicly available tools for comparison of TADs across datasets. These tools are necessary to conduct large-scale genome-wide analysis and comparison of 3D structure. To address the challenge of TAD identification, we developed a novel sliding window-based spectral clustering framework that uses gaps between consecutive eigenvectors for TAD boundary identification. Our method, implemented in an R package, SpectralTAD, has automatic parameter selection, is robust to sequencing depth, resolution and sparsity of Hi-C data, and detects hierarchical, biologically relevant TADs. SpectralTAD outperforms four state-of-the-art TAD callers in simulated and experimental settings. We demonstrate that TAD boundaries shared among multiple levels of the TAD hierarchy were more enriched in classical boundary marks and more conserved across cell lines and tissues. SpectralTAD is available at http://bioconductor.org/packages/SpectralTAD/. To address the problem of TAD comparison, we developed TADCompare. TADCompare is based on a spectral clustering-derived measure called the eigenvector gap, which enables a loci-by-loci comparison of TAD boundary differences between datasets. Using this measure, we introduce methods for identifying differential and consensus TAD boundaries and tracking TAD boundary changes over time. We further propose a novel framework for the systematic classification of TAD boundary changes. Colocalization- and gene enrichment analysis of different types of TAD boundary changes revealed distinct biological functionality associated with them. TADCompare is available on https://github.com/dozmorovlab/TADCompare.

The group of Mycobacteria is one of the most intensively studied bacterial taxa, as they cause the two historical and worldwide known diseases: leprosy and tuberculosis. Mycobacteria not identified as tuberculosis or leprosy complex, have been referred to by ‘environmental mycobacteria’ or ‘Nontuberculous mycobacteria (NTM). Mycobacterium kansasii (M. kansasii) is one of the most frequent NTM pathogens, as it causes pulmonary disease in immuno-competent patients and pulmonary, and disseminated disease in patients with various immuno-deficiencies. There have been five documented subtypes of this bacterium, by different molecular typing methods, showing that type I causes tuberculosis-like disease in healthy individuals, and type II in immune-compromised individuals. The remaining types are said to be environmental, thereby, not causing any diseases. The aim of this project was to conduct a comparative genomic study of M. kansasii types I-V and investigating the gene expression level of those types. From various comparative genomics analysis, provided genomics evidence on why M. kansasii type I is considered pathogenic, by focusing on three key elements that are involved in virulence of Mycobacteria: ESX secretion system, Phospholipase c (plcb) and Mammalian cell entry (Mce) operons. The results showed the lack of the espA operon in types II-V, which renders the ESX- 1 operon dysfunctional, as espA is one of the key factors that control this secretion system. However, gene expression analysis showed this operon to be deleted in types II, III and IV. Furthermore, plcB was found to be truncated in types III and IV. Analysis of Mce operons (1-4) show that mce-1 operon is duplicated, mce-2 is absent and mce-3 and mce-4 is present in one copy in M. kansasii types I-V. Gene expression profiles of type I-IV, showed that the secreted proteins of ESX-1 were slightly upregulated in types II-IV when compared to type I and the secreted forms of ESX-5 were highly down regulated in the same types. Differentially expressed genes in types II-IV were also evaluated and validated by qPCR for selected genes. This study gave a general view of the genome of this bacterium and its types, highlighted some different aspects of its subtypes and supplemented by gene expression data.

Since 2004 sequencing has undergone a revolutionary change with the advent of first the 454 sequencer, followed by the introduction of the Solexa/Illumina chemistries. This has led to the ability to sequence the whole genomes of a large number of microorganisms in a short space of time. Microbiology’s last revolution was in the introduction of PCR, which allowed for faster detection of pathogens, particularly viruses. With whole genome sequencing (WGS) many of the time-consuming steps carried out by a reference laboratory can be skipped. These include organism detection, speciation, antimicrobial susceptibility testing, typing and molecular epidemiology – all carried out in a single sequencing run followed by bioinformatics analysis. So far the merits of WGS in microbiology have only been demonstrated in highly specialised scientific laboratories in high-income countries. However, with continuingly decreasing costs and increasing throughput, many public health laboratories are now acquiring sequencers and their use will inevitably spread to middle-income countries. In this thesis I explore the use of WGS in three specific areas and include details on how to develop and assess bioinformatics pipelines. First, I shall demonstrate that WGS can be used to assess highly divergent regions within microorganisms by using the example of the weaknesses in current approaches to the molecular detection of methicillin-resistant Staphylococcus aureus isolates. In particular, I shall highlight how current molecular tests have limitations in detecting drug resistance when the regions of the genome conferring resistance have significant mutations. Second, I will examine how WGS can provide insights into the biology of the current vaccine against tuberculosis, Bacillus Calmette Guerin (BCG), particularly how the continued passage of the seedlot for this vaccine has led to very different versions being used around the globe. Finally, I will demonstrate how WGS can be used to develop a more targeted public health approach to tuberculosis, in particular how antimycobacterial drug resistance evolves within a population and how not all clades of Mycobacterium tuberculosis should be treated the same. Whole genome sequencing clearly has a lot to offer the fields of microbiology and communicable disease control. The next step is in translating its use into public health policy and ensuring that global approaches are unified to best benefit the populations served.

Aquatic primary productivity mainly depends on pelagic phytoplankton. The globally abundant marine picocyanobacteria Prochlorococcus comprises a significant fraction of the photosynthetic biomass in most tropical, oligotrophic oceans. The Red Sea is an enclosed narrow body of water characterized by continuous solar irradiance, and negligible annual rainfall, in addition to elevated temperatures and salinity levels, which mimics a global warming scenario. Analysis of 16S rRNA sequences of bacterioplankton communities indicated the predominance of a high-light adapted ecotype (HL II) of Prochlorococcus at the surface of the Northern and Central Red Sea. To this end, we analyzed the distribution of Prochlorococcus at multiple depths within and below the euphotic zone in different regions of the Red Sea, using clone libraries of the 16S–23S rRNA internal transcribed spacer (ITS) region. Results indicated a high diversity of Prochlorococcus ecotypes at the 100 m depth in the water column and an unusual dominance of HL II-related sequences in deeper waters of the Red Sea. To further investigate the microdiversity of Prochlorococcus over a wider biogeographical scope, we used a 454-pyrosequencing approach to analyze rpoC1 gene pyrotags. Samples were collected from the surface of the water column to up to 500 m at 45 stations that span the Red Sea’s main basin from 4 north to south. Phylogenetic analysis of abundant rpoC1 OTUs revealed genotypes of recently discovered strains that belong to the high-light and lowlight clades. In addition, we used a rapid community-profiling tool (GraftM) and quantitatively analyzed rpoC1 gene abundance from 45 metagenomes to assess the Prochlorococcus community structure across vertical and horizontal physicochemical gradients. Results revealed the clustering of samples according to their depth and a strong influence on ecotypic distribution by temperature and oxygen levels. In efforts to better understand how the cells survive the unusual features of the Red Sea, a Prochlorococcus strain of the HL II adapted clade from the euphotic zone was cultured, enabling morphological analyses and growth rates measurements for the strain. In addition, we successfully sequenced and annotated the genome of the strain, which was then used for genomic comparison with other ecotypes. Interestingly, the set of unique genes identified in the draft genome included genes encoding proteins involved in salt tolerance mechanisms. The expression level and pattern of these genes in the Red Sea water column was explored through metatranscriptomic mapping and revealed their occurrence throughout, independent of the diel cycle. This led to the hypothesis that Prochlorococcus populations in the highly saline Red Sea are able to biosynthesize additional compatible solutes via several pathways to counterbalance the effects of salt stress. The results presented in this dissertation provide the first glimpse on how the environmental parameters of the Red Sea can affect the evolution, diversity and distribution patterns of Prochlorococcus ecotypes.

No description available.

Tourette Syndrome (TS) is a neurodevelopmental disorder that starts in childhood. It is marked by multiple motor and vocal tics and a fluctuating course with recurrent time periods of symptom exacerbation followed by symptom remission. TS is considered a disorder of complex etiology that involves the interaction of multiple genetic and environmental factors. Previous studies have implicated regulation of the immune system in TS patients, although this remains a matter of debate. <br>Here we present a genome-wide gene expression study comparing data from TS patients at time points of symptom exacerbation and remission as well as upon symptom onset for newly diagnosed patients, with differentially gene expression analysis, co-expression analysis and pathway analysis. Although the fold change of each regulated gene was very mild, it is the first time that changes in the regulation of the immune system have been implicated at different time points in TS patients.<br><br>

Single nucleotide polymorphism (SNP) methodology is being used as a means to determine genetic merit in beef cattle by interrogating animal genomes and associating the findings with performance traits. The ability to predict future trait performance is highly attractive to beef cattle producers as they can make important management and financial decisions earlier and with more certainty. To fully realize the potential of SNP testing technology the methodology must be vetted to assure producer confidence. The purpose of this project is to assess three sources of information for beef cattle trait assessment. These information sources are: SNP testing, Expected Progeny Differences (EPDs) and direct animal measures. To conduct this study, young beef bulls (n=181) consigned to the SIU Beef Evaluation Station were utilized in an 84-day period to obtain direct measures. The SIU Beef Evaluation Station uses the Calan-Broadbent confinement feeding system which allows researchers to monitor individual animal feed intake and weight gain. Feed efficiency traits are important to the cattle industry since feed is generally among the largest input cost to producers. The evaluation of bulls also assesses reproductive and carcass traits which are also important to the producer’s financial success.Individual animal performance information was sent to the bull’s respective breed association for determination of EPD’s. Blood samples were submitted to a commercial company for SNP testing (Igentiy Gold and Igenity Beef Profile, Neogen, Lincoln, NE). Data was analyzed using pairwise comparisons by source of information. Pearson correlations were used to determine the direction and the strength for sources of information to vary together. Data was determined to be correlated when the correlation coefficient was 0.3 < r < - 0.3. No correlation was observed between RFISIU :RFINEO (r = 0.042), RFINEO:F/GSIU (r = - 0.09), RFISIU:ADGNEO (r = 0.091), RFISIU:ADGSIU (r = - 0.039), RFINEO:ADGNEO (r = 0.236), BWNEO:BWSIU (r = 0.115), FRAMESIU:BWSIU (r = 0.111), FRAMESIU:BWEPD (r = 0.159), FRAMESIU:ADGSIU (r = 0.148), FRAMESIU:ADGNEO (r = -0.005), BWSIU:BWEPD (r = 0.256), and BWNEO:BWEPD (r = 0.226). Correlations were observed between RFISIU:F/GSIU (r = 0.455), ADGSIU :ADGNEO (r = 0.353), and FRAMESIU:BWNEO (r = 0.326).This study determined that beef bulls should continue to be performance tested due to discrepancies between sources of information for key animal performance traits. Assessment of SNPs used in the commercial test should continue.