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Submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy to the School of Agriculture, Biomedicine and Environment, La Trobe University, Victoria, Australia.

A thesis submitted in total fulfilment of the degree of Master of Science to the School of Agriculture, Biomedicine and Environment (SABE), La Trobe University, Victoria, Australia.

Submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, La Trobe University, Victoria, Australia.

Breast cancer has the highest incidence rate and the second highest mortality rate among diagnosed malignant tumors worldwide. In terms of the molecular mechanisms involved in breast cancer, there are many splicing factors that contribute to the aggressiveness of the cancer. One of them is hnRNPA2B1. Previous research shows that hnRNPA2B1 has a positive correlation with breast cancer cell proliferation but with respect to breast cancer metastasis, research studies have reported both negative and positive correlations. While hnRNPA2B1 is a splicing factor, it is itself alternatively spliced as well; out of the isoforms produced consequently, one that includes exon2 produces nuclear protein while the one excluding exon2 produces cytoplasmic protein. Since the previous research has focused on hnRNPA2B1 as a whole, here, we investigate the effect of each isoform of hnRNPA2B1 on both breast cancer cell proliferation and migration. Our results show that transfection with hnRNPA2B1 AMO meaning the cytoplasmic hnRNPA2B1 caused lesser cell proliferation and cell migration of breast cancer cells.

Funder: Wellcome Trust, Bioinformatic research relies on large-scale computational infrastructures which have a nonzero carbon footprint but so far, no study has quantified the environmental costs of bioinformatic tools and commonly run analyses. In this work, we estimate the carbon footprint of bioinformatics (in kilograms of CO2 equivalent units, kgCO2e) using the freely available Green Algorithms calculator (www.green-algorithms.org, last accessed 2022). We assessed 1) bioinformatic approaches in genome-wide association studies (GWAS), RNA sequencing, genome assembly, metagenomics, phylogenetics, and molecular simulations, as well as 2) computation strategies, such as parallelization, CPU (central processing unit) versus GPU (graphics processing unit), cloud versus local computing infrastructure, and geography. In particular, we found that biobank-scale GWAS emitted substantial kgCO2e and simple software upgrades could make it greener, for example, upgrading from BOLT-LMM v1 to v2.3 reduced carbon footprint by 73%. Moreover, switching from the average data center to a more efficient one can reduce carbon footprint by approximately 34%. Memory over-allocation can also be a substantial contributor to an algorithm's greenhouse gas emissions. The use of faster processors or greater parallelization reduces running time but can lead to greater carbon footprint. Finally, we provide guidance on how researchers can reduce power consumption and minimize kgCO2e. Overall, this work elucidates the carbon footprint of common analyses in bioinformatics and provides solutions which empower a move toward greener research.

Epilepsy is a neurological condition affecting roughly 60 million people worldwide and40,000 people in Ireland. Epilepsy has a range of aetiologies, dependant on subtype, ranging from complex and multifactorial to monogenic. The research in this thesis is an exploration of the role of genetic variation in various types of epilepsy. First, I present the largest genome-wide association study (GWAS) of epilepsy to date derived from data on over 29,000 cases of epilepsy obtained from clinical settings and52,538 controls. This GWAS identified 22 significant loci associated with ‘genetic generalised epilepsy’, and none with ‘focal epilepsy’. This GWAS was meta-analysed with data on over21,000 epilepsy cases and 1 million population controls obtained from biobanks. Meta-analysing the primary and biobank GWAS revealed an additional 8 loci associated with epilepsy. Heritability analysis revealed that genetic generalised epilepsy has the highest SNP-based heritability estimate of any disease, and the third highest SNP-based heritability estimate compared to the traits body mass index (BMI) and height. I then present research into the genetic basis of the developmental and epileptic encephalopathies (DEEs). The DEEs are severe, childhood-onset epilepsies which occur in instances with no family history of epilepsy and co-present with intellectual disability. Given their severe, early-onset phenotype the DEEs have traditionally been presumed to be monogenic disorders. Using a method known as polygenic risk scoring (PRS) I show that the DEEs have an increased common genetic burden for epilepsy, relative to controls. I also show that this increased PRS burden is present in DEE cases both with and without an identifiable, likely deleterious rare genetic variant. This increased polygenic burden directly challenges the assumption that DEEs are purely monogenic disorders. People with epilepsy are at an increased risk for a variety of psychiatric disorders, relative to the general population, with one in three people with epilepsy experiencing some sort of psychiatric illness over the course of their lifetime. I explored the role of PRS for psychiatric illnesses in people with epilepsy. Using data from the UK Biobank on over 7,000 epilepsy samples, I showed that people with epilepsy have an increased polygenic burden for schizophrenia, depression, and attention deficit hyperactivity disorder (ADHD), relative to population controls. Meta-analyses of these data with epilepsy samples from the EpiPGX consortium found a further enrichment of PRS for schizophrenia and depression in people with drug-resistant epilepsy, who are most at risk of psychiatric illness. Levetiracetam (LEV) is a commonly prescribed, effective, anti-seizure medicine. Psychiatric side-effects effect roughly 20% of epilepsy patients taking LEV, with roughly 1% experiencing acute psychosis. We found that people with epilepsy who experience LEV-induced psychosis had an enrichment of PRS for schizophrenia, relative to epilepsy patients who experience no psychiatric side-effects to LEV-treatment. Rare-variant analysis found no evidence of an increased burden of rare genetic variants in people who experience LEV-induced psychosis. Finally, I present research into the genetic basis of drug-resistant epilepsy. Roughly 30% of people with epilepsy will continue to have seizures after treatment with an appropriate anti-seizure medicine (ASM). I investigated whether the response to individual ASMs was associated with common genetic variation. GWAS were conducted on responders to specific ASMs or groups of functionally related ASMs, using non-responders as controls did not find any significant loci associated with ASM response, nor did PRS analyses based on risk variants for epilepsy and neuropsychiatric disorders, and ASM resistance itself. The research presented in this thesis mark various important contributions to our understand of the genetics of epilepsy.

FastQC quality reports for each multiplexed fastq file (two lanes of sequencing corresponding to two raw fastq files)

Specialized metabolites are chemical tools produced by organisms to aid in their interaction with the surrounding environment. These diverse compounds can often function as metabolic weapons (e.g. antibiotics), structural components (e.g. lignins), or even attractants (e.g. flavonoids). Because of their frequent utilization in niche environments, specialized metabolite production is often lineage- or even species-specific. Therefore, knowledge between specialized metabolic systems is often nontransferable, which poses a major obstacle in the characterization of these bioactive and commercially relevant compounds. Beyond resolving the chemical composition of a specialized metabolite, the identification of responsible pathway genes and the evolutionary processes responsible for their formation is an arduous task. These gaps in knowledge are further widened by the lack of genomic resources available for specialized metabolite producing species. In this work, we present the genome assemblies of two organisms, each with unique specialized metabolic pathways: the Chinese medicinal plant Lithospermum erythrorhizon and the toxic golden alga Prymnesium parvum. Leveraging the predicted proteome of L. erythrorhizon, we investigated the evolutionary history of specialized metabolic genes responsible for the production of shikonin, a 1,4-naphthoquinone specialized metabolite. We identified a retrotransposition-mediated duplication event responsible for the creation of the core shikonin biosynthesis gene, PGT. In addition, we performed a global coexpression network analysis to identify regulatory and enzymatic gene candidates involved in the shikonin biosynthesis pathway. We also built phylogenetic trees of known and candidate shikonin genes to reveal patterns of lineage-specific gene duplication and retroduplication. Like plants, unicellular algae are known for their production of diverse, often toxic, specialized metabolites. However, these species are often enigmatic. For example, previous studies have documented large phenotypic variation in both toxin chemotypes and levels among different strains of P. parvum. To investigate the genetic basis of this variation, we generated near chromosome level assemblies of two P. parvum strains and performed a broad genome survey of thirteen additional strains. As a result, we identified a commonly studied reference strain, UTEX 2797, as a hybrid with two distinct subgenomes. We also provide evidence of significant variation in haploid genome size across the species. Collectively, these studies supply genetic resources for the future study of these organisms, as well as provide insight into the evolution of their specialized metabolic pathways.

Figure S1. Cha��ne op��ratoire typology entailing sets of increasingly elaborate steps used to remove and sculpt medieval walrus rostra found in Greenland and Europe. The Kyiv specimens have been classified using this scheme. Type 1 rostra were only modified by cut marks made during removal from the skull. In type 2, the tusk alveoli were thinned by rough parallel cuts that faceted the surface of each socket. Type 3 rostra also exhibit decorative carving of the nasal aperture. In type 4 the tusk sockets are smoothly rather than roughly facetted and the ventral margin between the tusks is always carved. Fragmentary rostra lacking some information can be attributed to broader categories (e.g. type 2/3). Image reproduced from Figure 2 of Barrett et al. (2020), where it was published open access under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Figure S2. Distribution of medieval European finds of walrus rostra that have been genetically analysed. Potential medieval trade routes are also shown. Mitogenomic clade assignment is indicated for each specimen. Stars mark the locations of control samples from Greenland, Iceland, Finnmark and Svalbard. The modern distributions of the eastern and western genetic clades of Atlantic walrus are shaded. Base map after Barrett et al. (2020) and references therein.

Most recent version of the Supplementary Tables for the article entitled "Integrative analysis and prediction of human R-loop binding proteins". Supplementary figures for the manuscript titled "Integrative analysis and prediction of human R-loop binding proteins" by Kumar*, Fournier* and Stirling currently under review.

A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Computer Science and Information Technology, School of Engineering and Mathematical Sciences, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Domestication is the process by which a previously wild population is managed by humans, thereby being subjected to a different set of selective pressures than experienced in its natural setting. Its opposite, feralization, is therefore when a domesticate escapes or is released from a captive setting, reasserting natural selective pressures. The genomics underpinning both domesti- cation and feralization have not been studied in insects; the Western honey bee (Apis mellifera L.) is a good model for this system, as honey bees exist in both a managed and feral state, and have extensive historic and genomic resources to document population changes. My goal in this thesis was to 1) improve upon our understanding of honey bee importation and genetics to the United States to support demographic assertions, and 2) to sequence managed and feral stocks of honey bees to identify the population structure and 3) genetic differences underpinning domestication. Ultimately, I reconstructed 400 years of honey bee importation and management history, creating the most comprehensive understanding to date of importation dates and locations, historical man- agement practices, and genetic bottlenecks. Additionally, I summarized thirty years of honey bee genome sequencing to provide a road map for future studies. Then, I conducted whole genome pooled sequencing on six managed and three feral stocks of honey bees from the United States. The mitochondrial and whole genome ancestry of feral colonies holds relics from their importation history, while managed colonies show evidence of more recent importation events. The managed stocks in my sample set have higher overall genetic diversity, but exhibit little differentiation, but feral stocks exhibit varying levels of differentiation, indicating different levels of ferality likely dictated by the level of reproductive isolation from managed colonies.

Contains the script used for analyses in three different formats (Jupyter Notebook, R Markdown and PDF).- all_analyses_JupyterNotebook.ipynb: Jupyter Notebook containing the script used for analyses.- all_analyses_RMarkdown.md: Same script as in the Jupyter Notebook, but in R Markdown format.- all_analyses_codeInPDF.pdf: Same script as in the Jupyter Notebook, but in PDF format.

A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, La Trobe University, Victoria, Australia.

This thesis is submitted for the total fulfilment of the requirements for a PhD to the Department of Mathematics and Statistics, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, La Trobe University, Victoria.

Aging is characterized by changes in transcriptional outputs that correlate with physiological changes observed as we age, including decreased function, and increased cell death. Importantly, many of these changes are conserved across tissues and organisms . Because one of the molecular hallmarks of aging is epigenetic dysregulation, we are interested in understanding how age-associated changes in chromatin contribute to the aging transcriptome. To accomplish this, we use the Drosophila visual system as a model for aging, with a particular focus on photoreceptor neurons. To perform cell-type specific genomic studies in Drosophila, we previously developed a nuclei immuno-enrichment method that was compatible with RNA-seq. However, due to low nuclei yields, this protocol was not amenable to chromatin-based studies, such as ChIP-seq and ATAC-seq. In Chapter 1, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein that increased yields without compromising efficiency. We further demonstrate that this protocol is compatible with several chromatin profiling techniques, such as Assay of Transposable-Accessible Chromatin (ATAC)-seq, Chromatin Immunoprecipitation (ChIP-seq), and CUT&Tag. Chromatin accessibility is enriched for transcription factors. Thus, in Chapter 2, we profiled accessible chromatin in aging photoreceptors and integrated this data with RNA-seq to identify transcription factors that showed differential activity in aging Drosophila photoreceptors. Surprisingly, we found that 57 transcription factors had differential binding activity during aging, including two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When we disrupted the Clock:Cycle complex by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, we observed changes in expression of 15–20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, we showed that expression of ClkDN in photoreceptors leads to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, our data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress. Previous work in the Weake lab found that long, highly expressed genes were more susceptible to be downregulated with age. DNA:RNA hybrids are co-transcriptional structures that form when the nascent RNA hybridizes with the template strand, resulting in a displaced non-template ssDNA. Importantly, accumulation of R-loops is associated with transcriptional inhibition and genomic instability, both hallmarks of aging. In Chapter 3, I characterized R-loop in maintaining proper transcriptional outputs and regulating visual function during aging. Bulk assays to measure R-loop levels revealed a significant increase in nuclear R-loops with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, over-expression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. Together, these studies underscore the importance of understanding how age-related changes in genomic processes, such as circadian transcription and maintenance of R-loops, contribute to physiological changes observed during aging.

Sorghum [Sorghum bicolor (L.) Moench] is the fifth most widely grown cereal crop in the world that serves as a staple food for millions of people. Grain mold of sorghum, caused by a consortium of fungal pathogens, is a leading constraint to sorghum production. A second sorghum disease with significant economic impact is anthracnose caused by the ascomycete fungus Colletotrichum sublineolum (Cs). Grain mold causes yield reduction and is highly detrimental to food quality due to contamination by toxigenic fungi and mycotoxins while anthracnose results in significant yield reduction in susceptible cultivars. Genetic resistance is considered the only effective and sustainable way to control both diseases, but the genetic control of these diseases are not well understood. In this project, we implemented genetic, genomic and molecular approaches to identify loci and/or genes underlying resistance to the two diseases. The results presented in Chapters 2 to 5 provide new insights to the genetic and genomic architecture of resistance to grain mold and anthracnose. Chapter 1 provides background information and review of the literature on the pathology of the two diseases, the contrasting and shared mechanisms of genetic resistance and approaches to QTL and gene identification. Chapter 2 and Chapter 3 describe genome wide association studies (GWAS) conducted on sorghum landrace accessions from Ethiopia. Results of both sets of GWAS were recently published (Nida et al., 2019, Journal of Cereal Science 85, 295-304, https://doi.org/10.1016/j.jcs.2018.12.016; Nida et al., 2021, Theoretical Applied Genetics, https://doi.org/10.1007/s00122-020-03762-2). Chapter 4 describes global transcriptome profiles of early stage of the developing grain from resistant and susceptible sorghum genotypes which uncovered process that correlate with resistance or susceptibility to grain mold. Results of this study was also recently published (Nida et al., 2021, BMC Genomics 22, 295, https://doi.org/10.1186/s12864-021-07609-y. Finally, Chapter 5 summarizes two anthracnose resistance genes identified through whole genome resequencing and genetic mapping.In Chapter 2, genomic regions associated with grain mold resistance were identified through GWAS conducted using sorghum landraces. A major grain mold resistance locus containing tightly linked and sequence related MYB transcription factor genes were identified based on association between SNPs and grain mold resistance scores of 1425 accessions. The locus contains YELLOW SEED1 (Y1, Sobic.001G398100), a likely non-functional pseudo gene (Y2, Sobic.001G398200), and YELLOW SEED3 (Y3, Sobic.001G397900). SNPs and other sequence polymorphisms that alter the Y1 and Y3 genes correlated with susceptibility to grain mold and provided a strong genetic evidence. Although Y1 has long been known as a regulator of kernel color and the biosynthesis of 3-deoxyanthocynidin phytoalexins, it was not annotated in the sorghum genome. The data suggest that the MYB genes and their grain and glume specific expressions determine responses to molding fungi.Chapter 3 focuses on GWAS conducted on a subset of early to medium flowering accessions to identify grain mold resistance loci. In addition, because of the caveats associated with grain flavonoid mediated mold resistance, we specifically aimed to identify resistance loci independent of grain flavonoids. A multi-environment grain mold phenotypic data and 173,666 SNPs were used to conduct GWAS using 635 accessions and a subset of non-pigmented accessions, potentially producing no tannins and/or phenols. A novel sorghum KAFIRIN gene encoding a seed storage protein, and LATE EMBRYOGENESIS ABUNDANT 3 (LEA3) gene encoding a protein with differential accumulation in seeds were identified. The KAFIRIN and LEA3 loci were also grain mold resistance factors in accessions with non-pigmented grains. Moreover, the known SNP (S4_62316425) in TAN1 gene, a regulator of tannin accumulation in sorghum grain was significantly associated with grain mold resistance. These data suggest the critical role of loci harboring seed protein genes for resistance to sorghum grain mold.In Chapter 4, global transcriptome profiles of developing grain of resistant and susceptible sorghum genotypes were studied. The developing kernels of grain mold resistant RTx2911 and susceptible RTx430 sorghum genotypes were inoculated with a mixture of fungal pathogens mimicking the species complexity of the diseases under natural infestation. Global transcriptome changes corresponding to multiple molecular and cellular processes, and biological functions including defense, secondary metabolism, and flavonoid biosynthesis were observed with differential regulation in the two genotypes. Genes encoding pattern recognition receptors (PRRs), regulators of growth and defense homeostasis, antimicrobial peptides, pathogenesis-related proteins, zein seed storage proteins, and phytoalexins showed increased expression correlating with resistance. The data suggest a pathogen inducible defense system in the developing grain of sorghum that involves the chitin PRR, MAPKs, key transcription factors, downstream components regulating immune gene expression and accumulation of defense molecules.Finally, Chapter 5 deals with anthracnose resistance loci and subsequent genetic mapping and identification of two resistance genes. The sorghum line SAP135 was previously described for its broad-spectrum resistance to anthracnose. To identify the specific resistance gene, a mapping population was generated by crossing SAP135 with the susceptible line TAM428. Bulked-segregant analysis (BSA) combined with whole genome re-sequencing of resistant and susceptible pools (BSA-seq) of the mapping population defined a single major peak on chromosome 8 for resistance to the Cs strain Csgrg which was designated as ANTHRACNOSE RESISTANCE GENE 4 (ARG4). ARG4 was co-localized with a locus identified in a parallel but an independent mapping study conducted using the sorghum line P9830 against another Cs strain Csgl1. Fine mapping revealed that the resistance loci from the two populations delineated two tightly linked loci, the latter locus designated as ANTHRACNOSE RESISTANCE GENE 5 (ARG5). ARG4 (Sobic.008G166400) and ARG5 (Sobic.008G177900) encode canonical NBS-LRR proteins widely known intracellular immune receptors. Interestingly, SAP135 carries a functional ARG4 but lacks ARG5 whereas P9830 harbors a functional ARG5 and lacks ARG4 and both show sequence homology to wheat rust resistance genes. Csgrg and Csgl1 are both virulent on sorghum lines TAM428 and BTx623, thus both lines carry susceptible alleles of ARG4 and ARG5. Supplemental information for the unpublished chapters are presented in the appendix section of this thesis.

During the transition to multicellular life, our cells acquired new ways to communicate with each other to transition from cell-level fitness to organism-level fitness. Communication between these genes is often disrupted in cancer allowing tumours to lose features of multicellularity and exhibit behaviors more commonly associated with unicellular life such as rapid cell division, selfish accumulation of nutrients and immortality. We are using a computational approach to investigate how features of multicellularity are broken down during multiple stages of cancer progression to build a comprehensive molecular landscape of cancer progression, which is immensely valuable for the development of more robust therapeutic strategies.

This thesis is under the specialisation 'Genobioinf'. Evolutionary diversification has enabled humans to adapt to climatic and dietary changes and respond to pathogens. My research focuses on Southeast Asian populations, which have been underrepresented in genomic studies. I employed a statistical method called the Fine-Mapping of Adaptation Variation (FineMAV), that pinpoints parts of the human genome that have undergone changes in response to their environment and resulted in evolutionary adaptation. I developed a software that other researchers can use to generate genome-wide FineMAV scores for human genomic sequencing datasets of their interest and help identify variants that can be subsequently studied using cellular assays or animal models.

The development of numerous high-throughput technologies has enabled us to interrogate human epidermal growth factor 2 (HER2) positive breast cancer on a “panomic” level. These technologies allow us to examine the exome, transcriptome, proteome, and epigenome simultaneously. To demonstrate the effectiveness of research projects across multiple “omes” three studies were performed, each encompassing at least two different “omes”.The first project looked at single nucleotide polymorphism (SNPs) in the homologous recombination repair (HRR) and HER2 pathways, compared them to proteomic data, and linked the findings with clinical outcome. I found 6 SNPs that correlate with recurrence free survival (RFS) when comparing patients who received TCH-based (docetaxel (T), carboplatin (C), and trastuzumab (H)) treatment versus those who received other treatment regimens, and an ERBB3 SNP that has an impact on protein phosphorylation in the PI3K/AKT and MAPK/ERK signaling pathways.For the second and third projects, I focused on the phenomenon known as bivalency, which encompasses two histone 3 trimethylations, located at lysine 4 (H3K4me3) and lysine 27 (H3K27me3). In the second project, I looked at how they differ between estrogen receptor (ER) positive and negative HER2+ breast cancer cell lines, and what impact they had on gene expression. I confirmed that there was a strong correlation between H3K4me3 with higher gene expression and H3K27me3 with low to no gene expression, and that there are several differences in genes within the HER and ER pathways. Additionally, I compared the bivalency profiles of several downstream genes of these two pathways with gene expression in patients, and found genes differentially expressed, and significantly correlated with RFS.The third project continued the bivalency studies by looking and how bivalency status in ER+/- HER2+ cell lines change in the presence of a positive and a negative environmental stimulus, and how that changes gene expression. E2, the ligand for the ER, was chosen as the positive stimulus, and trastuzumab, an anti-HER2 drug, was chosen as the negative stimulus. For this project, a new method was adapted by taking the framework of both the sequential chromatin immunoprecipitation followed by sequencing (reChIP-seq) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to form what we call reCUT&RUN. After the reCUT&RUN protocol was developed, several workflows were tested to optimize the analysis of the sequencing data generated. KEGG pathway analysis revealed that treatment had little impact on the distribution of the H3K4me3 and H3K27me3 marks. However, those found to be truly bivalent using reCUT&RUN differed between the control, E2, and trastuzumab-treated groups. Finally, the inclusion of RNA-seq and differential expression analysis revealed that over 70% of genes that were significantly differentially expressed had the H3K4me3 mark and less than 1% were H3K27me3. This indicates that the bivalent marks may play a role in which genes could have their expression impacted by treatment, including several genes involved in epithelial-mesenchymal transition and the regulation of H3K4me3 and H3K27me3 enrichment. This chapter not only demonstrated the usefulness of the reCUT&RUN protocol over other conventional methods, but it also gives new insight to the role that bivalent markers may play in treatment response.Overall, this thesis demonstrates the power of interrogating HER2+ breast cancer on a panomic level. By doing so, we can better characterize the complex interactions that take place across the exome, transcriptome, proteome and epigenome, allowing us to develop a better model to understand the complexities of oncogenesis and how patients respond to therapy.

Bioconversion of hemicelluloses into simpler sugars leads to production of a significant amount of pentose sugars, such as D-xylose. However, efficient utilization of pentoses by conventional yeast production strains remains challenging, especially due to inhibition by hexose co-fermentation. Wild yeast strains can provide new industrially relevant characteristics to bypass these inhibitions and efficiently utilize pentose sugars. To explore this strategy, we isolated gut-residing yeasts from the termite Macrotermes bellicosus collected in Comoé National Park, Côte d´Ivoire. The yeasts were classified through their ITS and LSU sequence and genomes of three new strains and species were sequenced and annotated. We identified a novel yeast species, which we name Barnettozyma botsteinii sp. nov. 1118T (MycoBank: 833563, CBS 16679T and IBT 710) and two new strains of Kurtzmaniella quercitrusa: var. comoensis (CBS 16678, IBT 709) and var. filamentosus (CBS 16680, IBT 711). The two K. quercitrusa strains grow 15% faster on synthetic glucose medium than Saccharomyces cerevisiae CEN.PKT in acidic conditions (pH = 3.2) and both strains grow on D-xylose as the sole carbon source at a rate of 0.35 h-1. At neutral pH, the yeast form of K. quercitrusa var. filamentosus, but not var. comoensis, switched to filamentous growth in a carbon source dependent manner, revealing phenotypic diversity among strains within the species. The utilisation of plant-derived sugars by K. quercitrusa indicates the potential for a mutualistic relationship between this yeast and the termite. Besides its metabolism, K. quercitrusa var. filamentosus also has a large potential as a production organism, because of its capacity to grow at low pH and to undergo a dimorphic shift.

Renewable plant biomass represents a rich source of fixed carbon that is poised to accelerate the growth of the bioeconomy because it is widely available, underused, and inexpensive. Similarly, because it is a ubiquitous, carbohydrate-rich feedstock that can be used in a broad range of bioprocesses, current and emerging technologies are being designed to transform these feedstocks into a variety of products including surfactants, food additives, pigments, plastics, and biofuels. However, current strategies to deconstruct recalcitrant plant materials rely on expensive enzymes with inefficient and harsh pretreatment steps. However, anaerobic fungi degrade a variety of crude, untreated biomass materials into fermentable sugars that can be converted into various products making them an appealing, low-cost solution to this problem. Although there are potential applications in industry for anaerobic fungi, it remains untapped because of the difficulties in cultivating them, sequencing their genomes, and genetically engineering them. In this work, three novel anaerobic fungi were isolated, and their genomes were sequenced to identify their genomic potential that was then leveraged to develop bioprocesses and engineering tools. Specifically, I developed methods to acquire the first gapless genomes for anaerobic fungi to provide more comprehensive insight into their capabilities. The biomass hydrolyzing abilities of one strain were characterized and leveraged as a pretreatment system for plant biomass; by partnering these anaerobic fungi with K. marxianus yeast, higher carbon conversion to fine and commodity chemicals was achieved as part of a two-stage bioproduction system. Similarly, the genomes were leveraged to identify novel genes for mevalonate production. My analysis of codon utilization due to the unusual GC composition of these genomes overcome one of the challenges with heterologous gene expression, leading to a hybrid pathway in E. coli with titers up to 2.5 g/L of mevalonate. Finally, a basic set of genetic tools was created including promoters, reporters, selection markers, and a gene-editing system that are still in development but form the fundamental toolbox for genetic engineering in anaerobic fungi. Together, this work provides a foundation for future genetic and metabolic engineering approaches that can enhance the efficiency and production of chemicals and fuels from renewable plant biomass.

File S1 contains the generalized mathematical proofs of the results in this study. The R code implementing the "P3D" approximated "Q+2K" GWAS model for epistatic effects is provided in File S2. The R code used to generate the simulated data is provided in File S3. A sample phenotypic and genotypic data which is a subset of the rice data is provided in File S4 and S5 respectively. Figures S1-S5 and Tables S1-S2 are included in a single file "Supplementary Tables and Figures.pdf".

Supplemental materials include 11 Suppl figures - pdf-files,6 Suppl Tables, 4 pdf and 2 excel files Suppl Results, 2 pdf files.Supplemental Results 1. Problems with the construction of haploid yeast strains with DNA polymerase alleles reducing fitness.Supplemental Results 2. Estimation of the probabilities of recurrent mutations in the same gene. Supplemental Table 1. Mutator effect of pol2rc-ΔN is reduced 60% in strain without the catalytic subunit of pol ζ, Rev3.Supplemental Table 2. Differences in mutant frequencies with or without UV-light irradiation in pol2rc-ΔN vs. wild-type strains.Supplemental Table 3. The pol2rc-ΔN elevated chromosome instability.Supplemental Table 4. (Excel file) Summary of mutations found in the course of genomic sequencing of the pol2rc-ΔN segregants.Supplementary Table 5. (Excel file) Ancestry of mutations in independent clones of the pol2rc-ΔNsegregants.Supplemental Table 6. Types of mutations found in genomes of pol2rc-ΔΝ strains. Supplemental Figure 1. Creation of haploid pol2rc-ΔN strains.Supplemental Figure 2. The absence of DPB4 does not affect the slow growth phenotype of pol2-ΔNmutants.Supplemental Figure 3. Outline of genetic analysis of diploids double heterozygous for the complete deletion of POL2 gene and pol2rc-ΔN.Supplemental Figure 4. Peculiarities in the genomic regions of pol2rc-ΔN strains relevant to the construction method.Supplemental Figure 5. High proportion of cells with abnormal morphology and nuclear material in pol2rc-ΔN cells.Supplemental Figure 6. Strains with pol2rc-ΔN grow slower than wild-type but are not temperature-sensitive or cold-sensitive.Supplemental Figure 7. Higher proportion of dead methylene blue-stained cells in cultures of pol2rc-ΔN strains in comparison to wild-type strains.Supplemental Figure 8. Many cells with pol2rc-ΔN have difficulty to start dividing. Supplemental Figure 9. Schematic of analysis to find if single cdc28 mutations can confer a growth advantage to pol2rc-ΔN strains.Supplementary Figure 10. Rescue of growth defects and sensitivity to drugs of pol2rc-ΔN by cdc28 mutations. Supplementary Figure 11. Multiple alignment of CMGC/CDK/CDC2 protein kinase orthologs.

Maintaining high genetic diversity within and among wildlife populations is an important component to the management of threatened species. Population genomics utilizes recent advancements in high-throughput next-generation sequencing to obtain genome-wide data that can yield deeper perspectives on intraspecific genetic variation and elucidate evolutionary significant units that may require conservation management or augmentation. The semi-aquatic Blanding’s Turtle (Emydoidea blandingii) has experienced drastic population declines in North America due in large part to anthropogenic activities. This species is listed as threatened or endangered across most of its range. A population genomic study can help to understand the status of this species and guide future management practices. Hence, a population genomic analysis was conducted using 3RAD to discover and analyze SNPs across the range using samples from Nebraska, Indiana, Michigan, Ohio, and Nova Scotia,. Range-wide analysis used 8,602 SNPs while analysis within the Great Lakes region used 7,893 SNPs. High amounts of missing data were found across all individuals and loci. Low levels of genetic variation relative to other turtle species were detected both across the range and within the Great Lakes region. Minimal population structure was detected range-wide via clustering and admixture analyses; however, a signal of population differentiation was detected among Nebraska, Nova Scotia, and the Great Lakes. Clustering and differentiation analyses focused on the Great Lakes region found a signal of population structure and differences between the Lake Michigan and Lake Erie watershed. These results may prove useful for conservation management of Blanding’s Turtle populations, particularly related to efforts using translocation or head-starting practices.

A thesis submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy to the School of Applied Systems Biology, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Supplementary Material describing more details of the genome assembly pipelines and assembly parameters (Sm1), the annotation of protein coding genes (SM2) and the estimation of genome size and heterozygosity by genome profiling analysis (SM3). The supplementary material also contains an overview of RNA sequencing reads from ostracods in GenBank (Table S1), the origin of the biological ostracod material of the study (Table S2), statistics of ostracod genome sequence data (Table S3), statistics of ostracod transcriptome sequence data (Table S4), statistics of ostracod resequencing data (Table S5), statistics of genome assemblies of ostracods (Table S6), statistics of protein coding gene annotations and other gene features in ostracod genomes (Table S7), and annotations and gene features of crustacean genomes of the last four years (Table S8). We also show results of GenomeScope analyses of two individuals of the ostracod Darwinula stevensoni and two of the ostracod Notodromas monacha (Figure S1A-D).

Deserts, such as the Namib, Sonoran, and Saharan, are regions that are unsuitable habitats for many organisms. However, darkling beetles (Tenebrionidae), specifically Pimeliinae, have appeared to flourish in otherwise inhospitable environments. All organisms have heat shock proteins protecting cellular components from degradation due to environmental stress such as desert heat. Modifications to heat shock proteins may provide more efficient cellular protection allowing desert-dwelling beetles to survive in regions where few other organisms are found. I performed a study which analyzed heat shock protein 40 (Hsp40) homologs across Pimeliinae by using targeted enrichment and high-throughput sequencing of seven genetic loci from 142 taxa (25 tribes) to examine protein functionality and evolution. I determined that the critical J domain of Hsp40 is conserved across pimeliine taxa. Additionally, there were a variety of cysteine shifts within different pimeliine tribes throughout six of the seven homologs, indicating possible protein structure alterations. Maximum likelihood analyses of Hsp40 homologs determined that despite the relationships between the tribes shuffling, taxa remained within their respective tribes. In an effort to examine how Hsp40s may have evolved alongside other behaviors and life histories, the tribe Adesmiini (Tenebrionidae: Pimeliinae) was examined in detail. Adesmiines thrive in the arid regions of sub-Saharan Africa, northern Africa, and the Palearctic. Despite having a few genera (i.e., Onymacris) which have been the subject of extensive life history analyses, Adesmiini has undergone few modern taxonomic studies. As a result, Adesmiini is a good candidate for phylogenetic investigation. To investigate evolutionary relationships, 510 targeted loci across 47 specimens (41 species, 10 genera) were used to produce a well-supported phylogeny. Current generic concepts were not in agreement with the resulting topology. In addition to producing a molecular phylogeny, two adesmiine traits of interest, activity time (diurnal/nocturnal) and substrate usage (psammophily), were also examined. Since Adesmiini is a predominantly diurnal tribe within a primarily nocturnal family, the activity time was mapped to the topology. From this study’s tree, it was determined that there were at least three shifts from diurnal to nocturnal throughout Adesmiini. Several charismatic adesmiines occur on the dunes of southern Africa, so the shift to inhabiting sand hills (psammophily) devoid of vegetation was also investigated. Psammophily was determined to have arisen multiple times within Adesmiini, and the topology revealed no clear indication to a single radiation of adesmiine substrate usage. Finally, a study was performed on Adesmiini using the same seven Hsp40 homologs as in the pimeliine functional genomics investigation. The resulting phylogenies indicate a correlation between Hsp40 modification and diurnal, psammophilous adesmiines.

Specialisation code: GENOBIONF The shrimp production industry in Malaysia can be affected by a devastating disease known as acute hepatopancreatic necrosis disease (AHPND) caused by the pathogenic bacterium, Vibrio parahaemolyticus. As one of the most deadly shrimp diseases worldwide, it has caused huge economic losses due to the high mortality rate. Here, the genetic variability of the pathogen is explored in detail and an improved detection method is developed. Gene expression studies were also undertaken to better understand the inner workings of the pathogen and disease. Altogether, this study provides new genetic insights to devise more effective disease management strategies.

Figure_S1 contains a protocol for the optimization of chromatin shearing.Figure_S2 contains a comparison of called peaks and % FRiP with overlapped-hc peaks and % FRiP-hc,respectively, for CTCF and TAL1.Figure_S3 contains a comparison of matched vs non-matched input control on the number of called peaks and FRiP scores in four retrospective TAL1 datasets. File_S1 contains detailed descriptions of supplemental File_S2 and Tables_S1-S4.File_S2 contains code used to analyze peaks and motifs.Table_S1 contains a list of datasets and metadata for all ChIP-seq samples. Table_S2 contains data on peaks and motifs statistics.Table_S3 contains a list of datasets and accompanying metadata for all input samples.Table_S4 contains isolation/sort markers, dates of collection, and cell numbers for hematopoietic progenitors. @font-face {font-family:"Cambria Math"; panose-1:2 4 5 3 5 4 6 3 2 4; mso-font-charset:0; mso-generic-font-family:roman; mso-font-pitch:variable; mso-font-signature:-536870145 1107305727 0 0 415 0;}p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-unhide:no; mso-style-qformat:yes; mso-style-parent:""; margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman",serif; mso-fareast-font-family:"Times New Roman";}.MsoChpDefault {mso-style-type:export-only; mso-default-props:yes;}div.WordSection1 {page:WordSection1;}

Supplementary Materials and Data Associated with Fan et al., "Genome and transcriptome of a pathogenic yeast, Candida nivariensis"

Supplemental Material of article entitled "Identification of the sex chromosome system in a sand fly species, Lutzomyia longipalpis s.l. "

A thesis submitted in total fulfillment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, College of Science, Health and Engineering, La Trobe University, Australia.

This thesis has been submitted in total fulfilment of the requirements for the Doctor of Philosophy to the School of Psychology and Public Health, La Trobe University, Victoria, Australia.

In this manuscript we use long-read sequencing data to generate a denovo assembly as well as fill >75% gaps in existing assembly using LRGapcloser software.

A thesis submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy to the School of Applied Systems Biology, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

This is the supplementary material that accompanies the submission Development of the Wheat Practical Haplotype Graph Database as a Resource for Genotyping Data Storage and Genotype Imputation (G3-2021-402618). It includes Figures S1 and S2 and Tables S1-S7.

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SI for the resubmission of G3-2021-402606.

Emulsion PCR (ePCR) is an important technique that permits amplification of DNA molecules in physically separated picoliter-volume water-in-oil droplets, and thus avoids formation of unproductive chimeras and other artifacts between similar DNA sequences. However, the recovery of ePCR products involves repeated extraction with hazardous organic solvents followed by purification using silica-based columns, making the overall process cumbersome. In this benchmark, we have described a quick ePCR extraction protocol for the purification of ePCR products, which directly employs silica-based DNA purification columns; products purified using this method have been found to be compatible with gene cloning and next-generation sequencing applications. The method described here makes ePCR easy, safe and within the reach of every laboratory.

Presentations summarise the results of a project analysing whole-genome sequences of archaeological emmer wheat specimens from Egypt (https://doi.org/10.1038/s41477-019-0534-5).

Supplemental figures, tables, and file

This is an introduction to the ethical issues arising from the rise in "Direct to Consumer" genetic testing. It is provided in the format of a discussion (telephone call and email exchange) between a research scientist and his clinician friend.This is a sample chapter from 'What Future Will Our Children Have? Conversations about how science is changing our lives'. [ ISBN - 13: 978-8497666923]

Figure S1 shows pairwise phenotypic profile similarity for genes annotated to the same functional pathways.Figure S2 shows pairwise phenotypic profile similarity for genes annotated to the same heteromeric protein complexes.Figure S3 shows precision for gene pairs ranked by phenotypic profile similarity, determined using |PCC|, when mutants with auxotrophic phenotypes are excluded.Figure S4 shows violin plots of the distribution of semantic similarity for subsets of gene pairs annotated with GO biological process terms.Figure S5 shows violin plots of the distribution of semantic similarity for subsets of gene pairs annotated with GO biological process terms when electronic annotations are excluded.Figure S6 shows precision-recall curves for gene pairs ranked by phenotypic profile similarity based on |PCC|.Figure S7 compares precision-recall curves for gene pairs ranked by phenotypic profile similarity based on |PCC|, |SRCC|, or mutual information.Figure S8 compares precision-recall curves for gene pairs ranked by phenotypic profile similarity determined using either quantitative fitness scores or discretized, ternary fitness scores. Figure S9 compares precision for gene pairs ranked by phenotypic profile similarity when quantitative fitness scores are partitioned into larger numbers of bins.Table S1 lists the names and EcoCyc accession IDs for the 366 pathways shown in Figure S1.Table S2 lists the names and EcoCyc accession IDs for the 271 heteromeric protein complexes shown in Figure S2.

A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Applied Systems Biology, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.This thesis was a recipient of the Nancy Millis Award for theses of exceptional merit.

All code, source data files, and supplementary figures associated with the work titled "Trans-acting genetic variation affects the expression of adjacent genes" can be found here. Instructions for using the code are included in the mainScript R file.

Link to full thesis - https://hdl.handle.net/2292/54365This folder contains all additional supplementary material - datasets that were not suitable to be added to the thesis appendices.

Indiana University-Purdue University Indianapolis (IUPUI), Chromosomal mechanisms of sex determination vary greatly in phylogenetically closely related species, indicative of rapid evolutionary rates. Sex chromosome karyotypes are generally conserved within families; however, many species have derived sex chromosome configurations. Insects display a plethora of sex chromosome systems due to rapid diversification caused by changes in evolutionary processes within and between species. A good example of such a system are insects in the blow fly family Calliphoridae. While cytogenetic studies observe that the karyotype in blow flies is highly conserved (five pairs of autosomal chromosomes and one pair sex chromosome), there is variation in sex determining mechanisms and sex chromosome structure within closely related species in blow flies. The evolutionary history of sex chromosomes in blow fly species have not been fully explored. Therefore, the objective of this research was to characterize the sex chromosome structures in four species of blow flies and investigate the selective forces which have played a role in shaping the diverse sex chromosome system observed in blow flies. The blow fly species used in this study are Phormia regina, Lucilia cuprina, Chrysomya rufifacies and Chrysomya albiceps. Phormia regina,and Lucilia cuprina have a heteromorphic sex chromosome system and are amphogenic (females produce both male and female offspring in equal ratio). In contrast, Chrysomya rufifacies and Chrysomya albiceps, have a homomorphic sex chromosome system, are monogenic (females produce unisexual progeny), have two types of females (arrhenogenic females ��� male producers and thelygenic females ��� female producers), and sex of the offspring is determined by the maternal genotype. To accomplish these tasks, a total of nine male and female individual draft genomes for each of the four species (including three individual draft genomes of Chrysomya rufifacies ��� male, and the two females) were sequenced and assembled providing genomic data to explore sex chromosome evolution in blow flies. Whole genome analysis was utilized to characterize and identify putative sex chromosomal sequences of the four blow fly species. Genomic evidence confirmed the presence of genetically differentiated sex chromosomes in P. regina and L. cuprina; and genetically undifferentiated sex chromosomes in C. rufifacies and C. albiceps. Furthermore, comparative analysis of the ancestral Dipteran sex chromosome (Muller element F in Drosophila) was determined to be X-linked in P. regina and L. cuprina contributing to sex chromosome differentiation but not sex-linked in C. rufifacies and C. albiceps. Evolutionary pressures are often quantified by the ratio of substitution rates at non-synonymous (dN) and synonymous (dS) sites. Substitution rate ratio analysis (dN/dS) of homologous genes indicated a weaker purifying selection may have contributed to the loss of sex-linked genes in Muller element F genes of the undifferentiated sex chromosome as compared to the differentiated sex chromosome system. Overall, the results presented herein greatly expands our knowledge in sex chromosome evolution within blow flies and will reinforce the study of sex chromosome evolution in other species with diverse sex chromosome systems.

Humans have altered natural landscape since the agricultural revolution, but it has been most destructive since human globalization and rampant industrialization in the last two centuries. These activities deteriorate and fragments natural habitat of many wild species that creates small isolated populations that lose genetic diversity over time. Loss of genetic diversity reduces the adaptive capacity of a population to respond to future environmental change and increases their extinction risks. Implementing strategies for wildlife conservation is a challenge primarily because of our lack of understanding of the biology of many wild species, the risks they are currently facing, and their evolutionary histories. With the advent of genomic and computational techniques, it is now possible to address these concerns. In my research, I used genomics to study the evolutionary history of the Montezuma Quail (Cyrtonyx montezumae) and created monitoring tools that can be readily applied by wildlife managers for its conservation. Montezuma Quail is a small gamebird found mostly in Mexico with peripheral populations existing in Arizona, New Mexico, and Texas. Montezuma Quail are going through species wide decline in the United States and are listed as vulnerable in the state of Texas due to their small population sizes and geographic isolation from rest of the range. My results show that Texas quail are genetically distinct and significantly less diverse than Arizona quail. Analysis of whole genome sequences from multiple individuals show that due to small population sizes and isolation, Texas quail are significantly more inbred and genetic drift is the major contributor for loss of genetic diversity we see today. Inbreeding is negatively impacting Texas quail as they carry more deleterious alleles within their genome that reduce fitness of the individuals. Demographic models predict that both Arizona and Texas populations were formed via founding bottlenecks around 20,000 years ago. Texas populations have maintained small population sizes since its split from the ancestral populations and are less efficient in purging new deleterious mutations that arise post-bottleneck. The inferences from my research not only carries direct implications for Montezuma Quail conservationists, but also illustrate the power of evolutionary genomics in implementing targeted management strategies for any species that face existential threats in today’s waning world.