Genome-wide association of multiple complex traits in outbred mice by ultra low-coverage sequencing

We thank the High-Throughput Genomics Group at the Wellcome Trust Centre for Human Genetics and the Wellcome Trust Sanger Institute for the generation of the sequencing data. This work was funded by Wellcome Trust grant 090532/Z/09/Z (J.F.). Primary phenotyping of the mice was supported by the Mary Lyon Centre and Mammalian Genetics Unit (Medical Research Council, UK Hub grant G0900747 91070 and Medical Research Council, UK grant MC U142684172). D.A.B acknowledges support from NIH R01AR056280. The sleep work was supported by the state of Vaud (Switzerland) and the Swiss National Science Foundation (SNF 14694 and 136201 to P.F.). The ECG work was supported by the Netherlands CardioVascular Research Initiative (Dutch Heart Foundation, Dutch Federation of University Medical Centres, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences) PREDICT project, InterUniversity Cardiology Institute of the Netherlands (ICIN; 061.02; C.A.R., C.R.B). Na Cai is supported by the Agency of Science, Technology and Research (A*STAR) Graduate Academy. The authors wish to acknowledge excellent technical assistance from: Ayako Kurioka, Leo Swadling, Catherine de Lara, James Ussher, Rachel Townsend, Sima Lionikaite, Ausra S. Lionikiene, Rianne Wolswinkel and Inge van der Made. We would like to thank Thomas M Keane and Anthony G Doran for their help in annotating variants and adding the FVB/NJ strain to the Mouse Genomes Project.

Acoustic and Magnetic Techniques for the Isolation and Analysis of Cells in Microfluidic Platforms

Cancer comprises a collection of diseases, all of which begin with abnormal tissue growth from various stimuli, including (but not limited to): heredity, genetic mutation, exposure to harmful substances, radiation as well as poor dieting and lack of exercise. The early detection of cancer is vital to providing life-saving, therapeutic intervention. However, current methods for detection (e.g., tissue biopsy, endoscopy and medical imaging) often suffer from low patient compliance and an elevated risk of complications in elderly patients. As such, many are looking to “liquid biopsies” for clues into presence and status of cancer due to its minimal invasiveness and ability to provide rich information about the native tumor. In such liquid biopsies, peripheral blood is drawn from patients and is screened for key biomarkers, chiefly circulating tumor cells (CTCs). Capturing, enumerating and analyzing the genetic and metabolomic characteristics of these CTCs may hold the key for guiding doctors to better understand the source of cancer at an earlier stage for more efficacious disease management.

The isolation of CTCs from whole blood, however, remains a significant challenge due to their (i) low abundance, (ii) lack of a universal surface marker and (iii) epithelial-mesenchymal transition that down-regulates common surface markers (e.g., EpCAM), reducing their likelihood of detection via positive selection assays. These factors potentiate the need for an improved cell isolation strategy that can collect CTCs via both positive and negative selection modalities as to avoid the reliance on a single marker, or set of markers, for more accurate enumeration and diagnosis.

The technologies proposed herein offer a unique set of strategies to focus, sort and template cells in three independent microfluidic modules. The first module exploits ultrasonic standing waves and a class of elastomeric particles for the rapid and discriminate sequestration of cells. This type of cell handling holds promise not only in sorting, but also in the isolation of soluble markers from biofluids. The second module contains components to focus (i.e., arrange) cells via forces from acoustic standing waves and separate cells in a high throughput fashion via free-flow magnetophoresis. The third module uses a printed array of micromagnets to capture magnetically labeled cells into well-defined compartments, enabling on-chip staining and single cell analysis. These technologies can operate in standalone formats, or can be adapted to operate with established analytical technologies, such as flow cytometry. A key advantage of these innovations is their ability to process erythrocyte-lysed blood in a rapid (and thus high throughput) fashion. They can process fluids at a variety of concentrations and flow rates, target cells with various immunophenotypes and sort cells via positive (and potentially negative) selection. These technologies are chip-based, fabricated using standard clean room equipment, towards a disposable clinical tool. With further optimization in design and performance, these technologies might aid in the early detection, and potentially treatment, of cancer and various other physical ailments.

The Roles of Phenotypic Plasticity and Plant-Microbe Interactions in the Evolution of Complex Traits in Boechera stricta

All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.

Sequence and Structural Determinants of Specificity Differences between Paralogous Transcription Factors

Transcription factors (TFs) control the temporal and spatial expression of target genes by interacting with DNA in a sequence-specific manner. Recent advances in high throughput experiments that measure TF-DNA interactions in vitro and in vivo have facilitated the identification of DNA binding sites for thousands of TFs. However, it remains unclear how each individual TF achieves its specificity, especially in the case of paralogous TFs that recognize distinct target genomic sites despite sharing very similar DNA binding motifs. In my work, I used a combination of high throughput in vitro protein-DNA binding assays and machine-learning algorithms to characterize and model the binding specificity of 11 paralogous TFs from 4 distinct structural families. My work proves that even very closely related paralogous TFs, with indistinguishable DNA binding motifs, oftentimes exhibit differential binding specificity for their genomic target sites, especially for sites with moderate binding affinity. Importantly, the differences I identify in vitro and through computational modeling help explain, at least in part, the differential in vivo genomic targeting by paralogous TFs. Future work will focus on in vivo factors that might also be important for specificity differences between paralogous TFs, such as DNA methylation, interactions with protein cofactors, or the chromatin environment. In this larger context, my work emphasizes the importance of intrinsic DNA binding specificity in targeting of paralogous TFs to the genome.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

Interspecific competition between the potato cyst nematode species Globodera pallida and G. rostochiensis

The two potato cyst nematode species, Globodera pallida and G. rostochiensis, are among the most important pests of potato. PCN are difficult to manage, while the two species respond differently to the main control methods. An increase in the incidence of G. pallida had been reported and is generally attributed to greater effectiveness of control measures against G. rostochiensis. The status of PCN in Ireland was studied using PCR. The results demonstrated qPCR to be an efficient means of high-throughput PCN sampling, being able to accurately identify both species in mixed-species populations. Species discrimination using qPCR revealed an increase in the incidence of G. pallida in Ireland in the absence of G. pallida-selective control measures. The population dynamics of G. pallida and G. rostochiensis in Ireland were studied in mixed- and single-species competition assays in vivo. G. pallida proved to be the more successful species, with greater multiplication in mixed- than single-species populations, with G. rostochiensis showing the opposite. This effect was similarly observed in staggered inoculation trials and population proportion trials. It was hypothesised that the greater G. pallida competitiveness could be attributed to its later hatch. G. pallida exhibited a later peak in hatching activity and more prolonged hatch, relative to G. rostochiensis. G. rostochiensis hatch was significantly reduced in mixedspecies hatching assays. G. pallida hatch was significantly higher when hatch was induced in potato root leachates containing G. rostochiensis-specific compounds, indicating that G. pallida hatch is stimulated upon perception of G. rostochiensis–derived compounds. Rhizotron studies revealed that root damage, caused by feeding of the early-hatching G. rostochiensis, resulted in increased lateral root proliferation and significantly increased G. pallida multiplication. Split-root trials indicated a significant G. pallida-induced ISR effect. G. rostochiensis multiplication was significantly reduced in split-root rhizotrons when G. pallida colonised roots before or after G. rostochiensis infection.

Metabarcoding approach for non-indigenous species surveillance in marine coastal waters

In this study, high-throughput sequencing (HTS) metabarcoding was applied for the surveillance of plankton communities within the southeastern (SE) Baltic Sea coastal zone. These results were compared with those from routine monitoring survey and morphological analyses. Four of five nonindigenous species found in the samples were identified exclusively by metabarcoding. All of them are considered as invasive in the Baltic Sea with reported impact on the ecosystem and biodiversity. This study indicates that, despite some current limitations, HTS metabarcoding can provide information on the presence of exotic species and advantageously complement conventional approaches, only requiring the same monitoring effort as before. Even in the currently immature status of HTS, this combination of HTS metabarcoding and observational records is recommended in the early detection of marine pests and delivery of the environmental status metrics of nonindigenous species.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

Point-and-shoot: rapid quantitative detection methods for on-site food fraud analysis – moving out of the laboratory and into the food supply chain

Major food adulteration and contamination events occur with alarming regularity and are known to be episodic, with the question being not if but when another large-scale food safety/integrity incident will occur. Indeed, the challenges of maintaining food security are now internationally recognised. The ever increasing scale and complexity of food supply networks can lead to them becoming significantly more vulnerable to fraud and contamination, and potentially dysfunctional. This can make the task of deciding which analytical methods are more suitable to collect and analyse (bio)chemical data within complex food supply chains, at targeted points of vulnerability, that much more challenging. It is evident that those working within and associated with the food industry are seeking rapid, user-friendly methods to detect food fraud and contamination, and rapid/high-throughput screening methods for the analysis of food in general. In addition to being robust and reproducible, these methods should be portable and ideally handheld and/or remote sensor devices, that can be taken to or be positioned on/at-line at points of vulnerability along complex food supply networks and require a minimum amount of background training to acquire information rich data rapidly (ergo point-and-shoot). Here we briefly discuss a range of spectrometry and spectroscopy based approaches, many of which are commercially available, as well as other methods currently under development. We discuss a future perspective of how this range of detection methods in the growing sensor portfolio, along with developments in computational and information sciences such as predictive computing and the Internet of Things, will together form systems- and technology-based approaches that significantly reduce the areas of vulnerability to food crime within food supply chains. As food fraud is a problem of systems and therefore requires systems level solutions and thinking.

The Tao survivorship of schistosomes: implications for schistosomiasis control

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is a major public health problem which contributes substantially to the economic and financial burdens of many nations in the developing world. An array of survival strategies, such as the unique structure of the tegument which acts as a major host-parasite interface, immune modulation mechanisms, gene regulation, and apoptosis and self-renewal have been adopted by schistosome parasites over the course of long-term evolution with their mammalian definitive hosts. Recent generation of complete schistosome genomes together with numerous biological, immunological, high-throughput "-omics" and gene function studies have revealed the Tao or strategies that schistosomes employ not only to promote long-term survival, but also to ensure effective life cycle transmission. New scenarios for the future control of this important neglected tropical disease will present themselves as our understanding of these Tao increases.

Clinical implications of recent findings in schistosome proteomics

Schistosomiasis is a neglected tropical disease of clinical significance that, despite years of research, still requires an effective vaccine and improved diagnostics for surveillance, control and potential elimination. Furthermore, the causes of host pathology during schistosomiasis are still not completely understood. The recent sequencing of the genomes of the three key schistosome species has enabled the discovery of many new possible vaccine and drug targets, as well as diagnostic biomarkers, using high-throughput and sensitive proteomics methods. This review focuses on the literature of the last 5 years that has reported on the use of proteomics to both better understand the biology of the schistosome parasites and the disease they cause in definitive mammalian hosts.

Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G₁ -S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1Amutant OCCC. Mol Cancer Ther; 15(7); 1472-84. Ó2016 AACR.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Frequency of mutations and polymorphisms in borderline ovarian tumors of known cancer genes.

Borderline ovarian tumors represent an understudied subset of ovarian tumors. Most studies investigating aberrations in borderline tumors have focused on KRAS/BRAF mutations. In this study, we conducted an extensive analysis of mutations and single-nucleotide polymorphisms (SNPs) in borderline ovarian tumors. Using the Sequenom MassArray platform, we investigated 160 mutations/polymorphisms in 33 genes involved in cell signaling, apoptosis, angiogenesis, cell cycle regulation and cellular senescence. Of 52 tumors analyzed, 33 were serous, 18 mucinous and 1 endometrioid. KRAS c.35G>A p.Gly12Asp mutations were detected in eight tumors (six serous and two mucinous), BRAF V600E mutations in two serous tumors, and PIK3CA H1047Y and PIK3CA E542K mutations in a serous and an endometrioid BOT, respectively. CTNNB1 mutation was detected in a serous tumor. Potentially functional polymorphisms were found in vascular endothelial growth factor (VEGF), ABCB1, FGFR2 and PHLPP2. VEGF polymorphisms were the most common and detected at four loci. PHLPP2 polymorphisms were more frequent in mucinous as compared with serous tumors (P=0.04), with allelic imbalance in one case. This study represents the largest and most comprehensive analysis of mutations and functional SNPs in borderline ovarian tumors to date. At least 25% of borderline ovarian tumors harbor somatic mutations associated with potential response to targeted therapeutics.