Statistical methods for the integrative analysis of single-cell multi-omics data

Single-cell profiling techniques have provided an unprecedented opportunity to study cellular heterogeneity at the molecular level. This represents a remarkable advance over traditional bulk sequencing methods, particularly to study lineage diversification and cell fate commitment events in heterogeneous biological processes. While the large majority of single-cell studies are focused on quantifying RNA expression, transcriptomic readouts provide only a single dimension of cellular heterogeneity. Recently, technological advances have enabled multiple biological layers to be probed in parallel one cell at a time, unveiling a powerful approach for investigating multiple dimensions of cellular heterogeneity. However, the increasing availability of multi-modal data sets needs to be accompanied by the development of suitable integrative strategies to fully exploit the data generated. In this thesis I worked in collaboration with different research groups to introduce innovative experimental and computational strategies for the integrative study of multi-omics at single-cell resolution. The first contribution is the development of scNMT-seq, a protocol for the simultaneous profiling of RNA expression, DNA methylation and chromatin accessibility in single cells. I demonstrate how this assay provides a powerful approach for investigating regulatory relationships between the epigenome and the transcriptome within individual cells. The second contribution is Multi-Omics Factor Analysis (MOFA), a statistical framework for the unsupervised integration of multi-omics data sets. MOFA is a Bayesian latent variable model that can be viewed as a statistically rigorous generalization of Principal Component Analysis to multi-omics data. The method provides a principled approach to retrieve, in an unsupervised manner, the underlying sources of sample heterogeneity while at the same time disentangling which axes of heterogeneity are shared across multiple modalities and which are specific to individual data modalities. The third contribution is the generation of a comprehensive molecular roadmap of mouse gastrulation at single-cell resolution. We employed scNMT-seq to simultaneously profile RNA expression, DNA methylation and chromatin accessibility for hundreds of cells, spanning multiple time points from the exit from pluripotency to primary germ layer specification. Using MOFA, and other tools, I performed an integrative analysis of the multi-modal measurements, revealing novel insights into the role of the epigenome in regulating this key developmental process. The fourth contribution is an extended formulation of the MOFA model tailored to the analysis of large-scale single-cell data with complex experimental designs. I extended the model to incorporate a flexible regularisation that enables the joint analysis of multiple omics as well as multiple sample groups (batches and/or experimental conditions). In addition, I implemented a GPU-accelerated stochastic variational inference framework, thus enabling the scalable analysis of potentially millions of samples

A Biophysical Model for Analysis of Transcription Factor Interaction and Binding Site Arrangement from Genome-Wide Binding Data

BACKGROUND:How transcription factors (TFs) interact with cis-regulatory sequences and interact with each other is a fundamental, but not well understood, aspect of gene regulation. METHODOLOGY/PRINCIPAL FINDINGS:We present a computational method to address this question, relying on the established biophysical principles. This method, STAP (sequence to affinity prediction), takes into account all combinations and configurations of strong and weak binding sites to analyze large scale transcription factor (TF)-DNA binding data to discover cooperative interactions among TFs, infer sequence rules of interaction and predict TF target genes in new conditions with no TF-DNA binding data. The distinctions between STAP and other statistical approaches for analyzing cis-regulatory sequences include the utility of physical principles and the treatment of the DNA binding data as quantitative representation of binding strengths. Applying this method to the ChIP-seq data of 12 TFs in mouse embryonic stem (ES) cells, we found that the strength of TF-DNA binding could be significantly modulated by cooperative interactions among TFs with adjacent binding sites. However, further analysis on five putatively interacting TF pairs suggests that such interactions may be relatively insensitive to the distance and orientation of binding sites. Testing a set of putative Nanog motifs, STAP showed that a novel Nanog motif could better explain the ChIP-seq data than previously published ones. We then experimentally tested and verified the new Nanog motif. A series of comparisons showed that STAP has more predictive power than several state-of-the-art methods for cis-regulatory sequence analysis. We took advantage of this power to study the evolution of TF-target relationship in Drosophila. By learning the TF-DNA interaction models from the ChIP-chip data of D. melanogaster (Mel) and applying them to the genome of D. pseudoobscura (Pse), we found that only about half of the sequences strongly bound by TFs in Mel have high binding affinities in Pse. We show that prediction of functional TF targets from ChIP-chip data can be improved by using the conservation of STAP predicted affinities as an additional filter. CONCLUSIONS/SIGNIFICANCE:STAP is an effective method to analyze binding site arrangements, TF cooperativity, and TF target genes from genome-wide TF-DNA binding data

From genotype to phenotype: Through chromatin

Advances in sequencing technologies have enabled the exploration of the genetic basis for several clinical disorders by allowing identification of causal mutations in rare genetic diseases. Sequencing technology has also facilitated genome-wide association studies to gather single nucleotide polymorphisms in common diseases including cancer and diabetes. Sequencing has therefore become common in the clinic for both prognostics and diagnostics. The success in follow-up steps, i.e., mapping mutations to causal genes and therapeutic targets to further the development of novel therapies, has nevertheless been very limited. This is because most mutations associated with diseases lie in inter-genic regions including the so-called regulatory genome. Additionally, no genetic causes are apparent for many diseases including neurodegenerative disorders. A complementary approach is therefore gaining interest, namely to focus on epigenetic control of the disease to generate more complete functional genomic maps. To this end, several recent studies have generated large-scale epigenetic datasets in a disease context to form a link between genotype and phenotype. We focus DNA methylation and important histone marks, where recent advances have been made thanks to technology improvements, cost effectiveness, and large meta-scale epigenome consortia efforts. We summarize recent studies unravelling the mechanistic understanding of epigenetic processes in disease development and progression. Moreover, we show how methodology advancements enable causal relationships to be established, and we pinpoint the most important issues to be addressed by future research.publishedVersio

The Pharmacoepigenomics Informatics Pipeline and H-GREEN Hi-C Compiler: Discovering Pharmacogenomic Variants and Pathways with the Epigenome and Spatial Genome

Over the last decade, biomedical science has been transformed by the epigenome and spatial genome, but the discipline of pharmacogenomics, the study of the genetic underpinnings of pharmacological phenotypes like drug response and adverse events, has not. Scientists have begun to use omics atlases of increasing depth, and inferences relating to the bidirectional causal relationship between the spatial epigenome and gene expression, as a foundational underpinning for genetics research. The epigenome and spatial genome are increasingly used to discover causative regulatory variants in the significance regions of genome-wide association studies, for the discovery of the biological mechanisms underlying these phenotypes and the design of genetic tests to predict them. Such variants often have more predictive power than coding variants, but in the area of pharmacogenomics, such advances have been radically underapplied. The majority of pharmacogenomics tests are designed manually on the basis of mechanistic work with coding variants in candidate genes, and where genome wide approaches are used, they are typically not interpreted with the epigenome. This work describes a series of analyses of pharmacogenomics association studies with the tools and datasets of the epigenome and spatial genome, undertaken with the intent of discovering causative regulatory variants to enable new genetic tests. It describes the potent regulatory variants discovered thereby to have a putative causative and predictive role in a number of medically important phenotypes, including analgesia and the treatment of depression, bipolar disorder, and traumatic brain injury with opiates, anxiolytics, antidepressants, lithium, and valproate, and in particular the tendency for such variants to cluster into spatially interacting, conceptually unified pathways which offer mechanistic insight into these phenotypes. It describes the Pharmacoepigenomics Informatics Pipeline (PIP), an integrative multiple omics variant discovery pipeline designed to make this kind of analysis easier and cheaper to perform, more reproducible, and amenable to the addition of advanced features. It described the successes of the PIP in rediscovering manually discovered gene networks for lithium response, as well as discovering a previously unknown genetic basis for warfarin response in anticoagulation therapy. It describes the H-GREEN Hi-C compiler, which was designed to analyze spatial genome data and discover the distant target genes of such regulatory variants, and its success in discovering spatial contacts not detectable by preceding methods and using them to build spatial contact networks that unite disparate TADs with phenotypic relationships. It describes a potential featureset of a future pipeline, using the latest epigenome research and the lessons of the previous pipeline. It describes my thinking about how to use the output of a multiple omics variant pipeline to design genetic tests that also incorporate clinical data. And it concludes by describing a long term vision for a comprehensive pharmacophenomic atlas, to be constructed by applying a variant pipeline and machine learning test design system, such as is described, to thousands of phenotypes in parallel. Scientists struggled to assay genotypes for the better part of a century, and in the last twenty years, succeeded. The struggle to predict phenotypes on the basis of the genotypes we assay remains ongoing. The use of multiple omics variant pipelines and machine learning models with omics atlases, genetic association, and medical records data will be an increasingly significant part of that struggle for the foreseeable future.PHDBioinformaticsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/145835/1/ariallyn_1.pd