Statistical approaches to harness high throughput sequencing data in diverse biological systems

The development of novel statistical approaches to questions specific to biological systems of interest is becoming more valuable as we tackle increasingly complex problems. This thesis explores three distinct biological systems in which high throughput sequencing data is utilised, varying in research area, organism, number of sequencing platforms and datasets integrated, and structure such as matched samples; showcasing the variety of study designs and thus the need for tailored statistical approaches. First, we characterise allelic imbalance from RNA-Seq data including stringent filtering criteria and a count based likelihood ratio test. This work identified genes of particular importance in livestock genomics such as those related to energy use. Second, we outline a novel methodology to identify highly expressed genes and cells for single cell RNA-Seq data. We derive a gamma-normal mixture model to identify lowly and highly expressed components, and use this to identify novel markers for olfactory sensory neuron (OSN) maturity across publicly available mouse neuron datasets. In addition we estimate single cell networks and find that mature OSN single cell networks are more centralised than immature OSN single cell networks. Third, we develop two novel frameworks for relating information from Whole Exome DNA-Seq and RNA-Seq data when i) samples are matched and when ii) samples are not necessary matched between platforms. In the latter case, we relate functional somatic mutation driver gene scores to transcriptional network correlation disturbance using a permutation testing framework, identifying potential candidate genes for targeted therapies. In the former case, we estimate directed mutation-expression networks for each cancer using linear models, providing a useful exploratory tool for identifying novel relationships among genes. This thesis demonstrates the importance of tailored statistical approaches to further understanding across many biological systems

Diverse Routes toward Early Somites in the Mouse Embryo.

Somite formation is foundational to creating the vertebrate segmental body plan. Here, we describe three transcriptional trajectories toward somite formation in the early mouse embryo. Precursors of the anterior-most somites ingress through the primitive streak before E7 and migrate anteriorly by E7.5, while a second wave of more posterior somites develops in the vicinity of the streak. Finally, neuromesodermal progenitors (NMPs) are set aside for subsequent trunk somitogenesis. Single-cell profiling of T-/- chimeric embryos shows that the anterior somites develop in the absence of T and suggests a cell-autonomous function of T as a gatekeeper between paraxial mesoderm production and the building of the NMP pool. Moreover, we identify putative regulators of early T-independent somites and challenge the T-Sox2 cross-antagonism model in early NMPs. Our study highlights the concept of molecular flexibility during early cell-type specification, with broad relevance for pluripotent stem cell differentiation and disease modeling.Research in the authors’ laboratories is supported by the Wellcome Trust, MRC, CRUK, Blood Cancer UK, NIH-NIDDK, the Sanger-EBI Single Cell Centre; by core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute; and by core funding from Cancer Research UK and the European Molecular Biology Laboratory. J.A.G. was funded by Wellcome Trust award [109081/Z/15/A]. C.G. was funded by the Swedish Research Council (2017-06278). This work was funded as part of a Wellcome Strategic Award to study cell fate decisions during gastrulation (105031/D/14/Z) awarded to Wolf Reik, Berthold Göttgens, John Marioni, Jennifer Nichols, Ludovic Vallier, Shankar Srinivas, Benjamin Simons, Sarah Teichmann, and Thierry Voet

Synthesis of nano lead oxide for the application of lead-acid energy storage devices

Over the past decades, efforts have been emphasized on electric vehicles (EV) as a major solution to reduce carbon emission for greener environment. While one of the core design components of EV is the energy storage device which is to ensure sustainable power supply in the drive train system. However, existing energy storage applied in EV is relatively expensive, so, affordable and feasible energy storage needs to be explored to achieve the objective of fuel economy. As a matter of fact, technology of lead-acid battery has been around for more than a century, the established manufacturing and recycling processes, as well as overall simplistic product designs with relatively high power and energy densities, are attracting global researchers to harness their ultimate innovation. This is done via the essence of nanotechnology in transforming existing product design of lead-acid battery so that it is applicable in EV power train. Despite the challenges are inevitable, global researchers accept that electrode material with novel microstructural is the key solution to the problem. Recently, nanodendritic PbO2 has been the attractive material with unique morphology and enhanced electrochemical performance for lead-acid electrochemical storage devices. Thus far, many studies have only been investigated this unique material via electrodeposition technique. In this study, flower-like PbO consisting of three dimensional nanoflakes was synthesized and used as a starting precursor to form nanodendritic PbO2 via an electrochemical oxidation at constant voltage in the presence of electrolyte. According to the XRD results, the as-synthesized PbO was perfectly indexed to the diffraction peaks of pure PbO with a mixture of orthorhombic and tetragonal structures. The same is true for PbO2 which was electrochemically oxidized from the as-synthesized PbO. Meanwhile, both SEM results show that the as-synthesized PbO was characterized with flower-like structures providing high active surface area to form nanodendritic PbO2 via electrochemical oxidation at constant voltage based on periodic bond chain theory. The formed nanodendritic PbO2 delivered a first discharge capacity of 170 mAhg-1 at 200 mAg-1 and displayed improved cyclic voltammetry curve. This suggested that the formation of nanodendrites on the primary surface of agglomerated PbO2 provides larger crystallite network structures for better material utilization at high discharge rate. Upon the completion of current experimental work, a few general conclusions of this work have unfolded other research which focus on fabricating a prototype of lead-acid hybrid supercapacitor for evaluation

Coordinated changes in gene expression kinetics underlie both mouse and human erythroid maturation

Abstract: Background: Single-cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single-cell RNA-Seq permits prediction of future expression states. Here we apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis. Results: Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression. Conclusions: By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes

geneBasis: an iterative approach for unsupervised selection of targeted gene panels from scRNA-seq.

Funder: European Molecular Biology Laboratory (EMBL) (4843)scRNA-seq datasets are increasingly used to identify gene panels that can be probed using alternative technologies, such as spatial transcriptomics, where choosing the best subset of genes is vital. Existing methods are limited by a reliance on pre-existing cell type labels or by difficulties in identifying markers of rare cells. We introduce an iterative approach, geneBasis, for selecting an optimal gene panel, where each newly added gene captures the maximum distance between the true manifold and the manifold constructed using the currently selected gene panel. Our approach outperforms existing strategies and can resolve cell types and subtle cell state differences

Genetic variation of macronutrient tolerance in Drosophila melanogaster

Carbohydrates, proteins and lipids are essential nutrients to all animals; however, closely related species, populations, and individuals can display dramatic variation in diet. Here we explore the variation in macronutrient tolerance in Drosophila melanogaster using the Drosophila genetic reference panel, a collection of similar to 200 strains derived from a single natural population. Our study demonstrates that D. melanogaster, often considered a "dietary generalist", displays marked genetic variation in survival on different diets, notably on high-sugar diet. Our genetic analysis and functional validation identify several regulators of macronutrient tolerance, including CG10960/GLUT8, Pkn and Eip75B. We also demonstrate a role for the JNK pathway in sugar tolerance and de novo lipogenesis. Finally, we report a role for tailless, a conserved orphan nuclear hormone receptor, in regulating sugar metabolism via insulin-like peptide secretion and sugar-responsive CCHamide-2 expression. Our study provides support for the use of nutrigenomics in the development of personalized nutrition.Peer reviewe

Clonal evolution in liver cancer at single-cell and single-variant resolution.

Genetic heterogeneity of tumor is closely related to its clonal evolution, phenotypic diversity and treatment resistance, and such heterogeneity has only been characterized at single-cell sub-chromosomal scale in liver cancer. Here we reconstructed the single-variant resolution clonal evolution in human liver cancer based on single-cell mutational profiles. The results indicated that key genetic events occurred early during tumorigenesis, and an early metastasis followed by independent evolution was observed in primary liver tumor and intrahepatic metastatic portal vein tumor thrombus. By parallel single-cell RNA-Seq, the transcriptomic phenotype of HCC was found to be related with genetic heterogeneity. For the first time we reconstructed the single-cell and single-variant clonal evolution in human liver cancer, and dissection of both genetic and phenotypic heterogeneity will facilitate better understanding of their relationship

The human body at cellular resolution: the NIH Human Biomolecular Atlas Program

Abstract: Transformative technologies are enabling the construction of three-dimensional maps of tissues with unprecedented spatial and molecular resolution. Over the next seven years, the NIH Common Fund Human Biomolecular Atlas Program (HuBMAP) intends to develop a widely accessible framework for comprehensively mapping the human body at single-cell resolution by supporting technology development, data acquisition, and detailed spatial mapping. HuBMAP will integrate its efforts with other funding agencies, programs, consortia, and the biomedical research community at large towards the shared vision of a comprehensive, accessible three-dimensional molecular and cellular atlas of the human body, in health and under various disease conditions

SpatialExperiment: infrastructure for spatially resolved transcriptomics data in R using Bioconductor

Summary: SpatialExperiment is a new data infrastructure for storing and accessing spatially resolved transcriptomics data, implemented within the R/Bioconductor framework, which provides advantages of modularity, interoperability, standardized operations, and comprehensive documentation. Here, we demonstrate the structure and user interface with examples from the 10x Genomics Visium and seqFISH platforms, and provide access to example datasets and visualization tools in the STexampleData, TENxVisiumData, and ggspavis packages. Availability and implementation: The SpatialExperiment, STexampleData, TENxVisiumData, and ggspavis packages are available from Bioconductor. The package versions described in this manuscript are available in Bioconductor version 3.15 onwards. Supplementary information: Supplementary tables and figures are available at Bioinformatics online