I propose statistical methods and software for the analysis of single-cell transcriptome sequencing (scRNA-seq) and metagenomics data. Specifically, I present a general and flexible zero-inflated negative binomial-based wanted variation extraction (ZINB-WaVE) method, which extracts low-dimensional signal from scRNA-seq read counts, accounting for zero inflation (dropouts), over-dispersion, and the discrete nature of the data. Additionally, I introduce an application of the ZINB-WaVE method that identifies excess zero counts and generates gene and cell-specific weights to unlock bulk RNA-seq differential expression pipelines for zero-inflated data, boosting performance for scRNA-seq analysis. Finally, I present a method to estimate bacterial abundances in human metagenomes using full-length 16S sequencing reads.
The recent development of single-cell RNA sequencing (scRNA-seq) technologies has revolutionized transcriptomic studies by revealing the genome-wide gene expression levels within individual cells. In contrast to bulk RNA sequencing, scRNA-seq technology captures cell-specific transcriptome landscapes, which can reveal crucial information about cell-to-cell heterogeneity across different tissues, organs, and systems and enable the discovery of novel cell types and new transient cell states. According to search results from PubMed, from 2009-2023, over 5,000 published studies have generated datasets using this technology. Such large volumes of data call for high-quality statistical methods for their analysis. In the three projects of this dissertation, I have explored and developed statistical methods to model the marginal and joint gene expression distributions and determine the latent structure type for scRNA-seq data. In all three projects, synthetic data simulation plays a crucial role. My first project focuses on the exploration of the Beta-Poisson hierarchical model for the marginal gene expression distribution of scRNA-seq data. This model is a simplified mechanistic model with biological interpretations. Through data simulation, I demonstrate three typical behaviors of this model under different parameter combinations, one of which can be interpreted as one source of the sparsity and zero inflation that is often observed in scRNA-seq datasets. Further, I discuss parameter estimation methods of this model and its other applications in the analysis of scRNA-seq data. My second project focuses on the development of a statistical simulator, scDesign2, to generate realistic synthetic scRNA-seq data. Although dozens of simulators have been developed before, they lack the capacity to simultaneously achieve the following three goals: preserving genes, capturing gene correlations, and generating any number of cells with varying sequencing depths. To fill in this gap, scDesign2 is developed as a transparent simulator that achieves all three goals and generates high-fidelity synthetic data for multiple scRNA-seq protocols and other single-cell gene expression count-based technologies. Compared with existing simulators, scDesign2 is advantageous in its transparent use of probabilistic models and is unique in its ability to capture gene correlations via copula. We verify that scDesign2 generates more realistic synthetic data for four scRNA-seq protocols (10x Genomics, CEL-Seq2, Fluidigm C1, and Smart-Seq2) and two single-cell spatial transcriptomics protocols (MERFISH and pciSeq) than existing simulators do. Under two typical computational tasks, cell clustering and rare cell type detection, we demonstrate that scDesign2 provides informative guidance on deciding the optimal sequencing depth and cell number in single-cell RNA-seq experimental design, and that scDesign2 can effectively benchmark computational methods under varying sequencing depths and cell numbers. With these advantages, scDesign2 is a powerful tool for single-cell researchers to design experiments, develop computational methods, and choose appropriate methods for specific data analysis needs. My third project focuses on deciding latent structure types for scRNA-seq datasets. Clustering and trajectory inference are two important data analysis tasks that can be performed for scRNA-seq datasets and will lead to different interpretations. However, as of now, there is no principled way to tell which one of these two types of analysis results is more suitable to describe a given dataset. In this project, we propose two computational approaches that aim to distinguish cluster-type vs. trajectory-type scRNA-seq datasets. The first approach is based on building a classifier using eigenvalue features of the gene expression covariance matrix, drawing inspiration from random matrix theory (RMT). The second approach is based on comparing the similarity of real data and simulated data generated by assuming the cell latent structure as clusters or a trajectory. While both approaches have limitations, we show that the second approach gives more promising results and has room for further improvements.
Spatial transcriptomic technologies measure gene expression at increasing spatial resolution, approaching individual cells. One limitation of current technologies is that spatial measurements may contain contributions from multiple cells, hindering the discovery of cell type-specific spatial patterns of localization and expression. In this thesis, I will explore the development of Robust Cell Type Decomposition (RCTD), a computational method that leverages cell type profiles learned from single-cell RNA sequencing data to decompose mixtures, such as those observed in spatial transcriptomic technologies. Our RCTD approach accounts for platform effects introduced by systematic technical variability inherent to different sequencing modalities. We demonstrate RCTD provides substantial improvement in cell type assignment in Slide-seq data by accurately reproducing known cell type and subtype localization patterns in the cerebellum and hippocampus. We further show the advantages of RCTD by its ability to detect mixtures and identify cell types on an assessment dataset. Finally, we show how RCTD’s recovery of cell type localization uniquely enables the discovery of genes within a cell type whose expression depends on spatial environment. Spatial mapping of cell types with RCTD has the potential to enable the definition of spatial components of cellular identity, uncovering new principles of cellular organization in biological tissue.
Introduces readers to core algorithmic techniques for next-generation sequencing (NGS) data analysis and discusses a wide range of computational techniques and applications This book provides an in-depth survey of some of the recent developments in NGS and discusses mathematical and computational challenges in various application areas of NGS technologies. The 18 chapters featured in this book have been authored by bioinformatics experts and represent the latest work in leading labs actively contributing to the fast-growing field of NGS. The book is divided into four parts: Part I focuses on computing and experimental infrastructure for NGS analysis, including chapters on cloud computing, modular pipelines for metabolic pathway reconstruction, pooling strategies for massive viral sequencing, and high-fidelity sequencing protocols. Part II concentrates on analysis of DNA sequencing data, covering the classic scaffolding problem, detection of genomic variants, including insertions and deletions, and analysis of DNA methylation sequencing data. Part III is devoted to analysis of RNA-seq data. This part discusses algorithms and compares software tools for transcriptome assembly along with methods for detection of alternative splicing and tools for transcriptome quantification and differential expression analysis. Part IV explores computational tools for NGS applications in microbiomics, including a discussion on error correction of NGS reads from viral populations, methods for viral quasispecies reconstruction, and a survey of state-of-the-art methods and future trends in microbiome analysis. Computational Methods for Next Generation Sequencing Data Analysis: Reviews computational techniques such as new combinatorial optimization methods, data structures, high performance computing, machine learning, and inference algorithms Discusses the mathematical and computational challenges in NGS technologies Covers NGS error correction, de novo genome transcriptome assembly, variant detection from NGS reads, and more This text is a reference for biomedical professionals interested in expanding their knowledge of computational techniques for NGS data analysis. The book is also useful for graduate and post-graduate students in bioinformatics.
The large-scale, high-dimensional, and sparse single-cell RNA sequencing (scRNA-seq) data have raised great challenges in the pipeline of data analysis. A large number of statistical and machine learning methods have been developed to analyze scRNA-seq data and answer related scientific questions. Although different methods claim advantages in certain circumstances, it is difficult for users to select appropriate methods for their analysis tasks. Benchmark studies aim to provide recommendations for method selection based on an objective, accurate, and comprehensive comparison among cutting-edge methods. They can also offer suggestions for further methodological development through massive evaluations conducted on real data. In Chapter 2, we conduct the first, systematic benchmark study of nine cutting-edge computational doublet-detection methods. In scRNA-seq, doublets form when two cells are encapsulated into one reaction volume by chance. The existence of doublets, which appear as but are not real cells, is a key confounder in scRNA-seq data analysis. Computational methods have been developed to detect doublets in scRNA-seq data; however, the scRNA-seq field lacks a comprehensive benchmarking of these methods, making it difficult for researchers to choose an appropriate method for their specific analysis needs. Our benchmark study compares doublet-detection methods in terms of their detection accuracy under various experimental settings, impacts on downstream analyses, and computational efficiency. Our results show that existing methods exhibited diverse performance and distinct advantages in different aspects. In Chapter 3, we develop an R package DoubletCollection to integrate the installation and execution of different doublet-detection methods. Traditional benchmark studies can be quickly out-of-date due to their static design and the rapid growth of available methods. DoubletCollection addresses this issue in benchmarking doublet-detection methods for scRNA-seq data. DoubletCollection provides a unified interface to perform and visualize downstream analysis after doublet-detection. Additionally, we created a protocol using DoubletCollection to execute and benchmark doublet-detection methods. This protocol can automatically accommodate new doublet-detection methods in the fast-growing scRNA-seq field. In Chapter 4, we conduct the first comprehensive empirical study to explore the best modeling strategy for autoencoder-based imputation methods specific to scRNA-seq data. The autoencoder-based imputation method is a family of promising methods to denoise sparse scRNA-seq data; however, the design of autoencoders has not been formally discussed in the literature. Current autoencoder-based imputation methods either borrow the practice from other fields or design the model on an ad hoc basis. We find that the method performance is sensitive to the key hyperparameter of autoencoders, including architecture, activation function, and regularization. Their optimal settings on scRNA-seq are largely different from those on other data types. Our results emphasize the importance of exploring hyperparameter space in such complex and flexible methods. Our work also points out the future direction of improving current methods.
Major methodological and technological advances in sequencing have inspired ambitious biological questions that were previously elusive. Addressing such questions with novel and complex data requires statistically rigorous tools. In this dissertation, I develop, evaluate, and apply statistical and computational methods for analysis of high-throughput sequencing data. A unifying theme of this work is that all these methods are aimed at RNA-seq data. The first method focuses on characterizing gene expression in RNA-seq experiments with ordered conditions. The second focuses on single-cell RNA-seq data, where we develop a method for normalization to account for a previously unknown technical artifact in the data. Finally, we develop a simulation in order to recapitulate the source of the artifact [in silico].
In recent decades, new technologies have made remarkable progress in helping to understand biological systems. Rapid advances in genomic profiling techniques such as microarrays or high-performance sequencing have brought new opportunities and challenges in the fields of computational biology and bioinformatics. Such genetic sequencing techniques allow large amounts of data to be produced, whose analysis and cross-integration could provide a complete view of organisms. As a result, it is necessary to develop new techniques and algorithms that carry out an analysis of these data with reliability and efficiency. This Special Issue collected the latest advances in the field of computational methods for the analysis of gene expression data, and, in particular, the modeling of biological processes. Here we present eleven works selected to be published in this Special Issue due to their interest, quality, and originality.
Since the invention of next-generation RNA sequencing (RNA-seq) technologies, they have become a powerful tool to study the presence and quantity of RNA molecules in biological samples and have revolutionized transcriptomic studies on bulk tissues. Recently, the emerging single-cell RNA sequencing (scRNA-seq) technologies enable the investigation of transcriptomic landscapes at a single-cell resolution, providing a chance to characterize stochastic heterogeneity within a cell population. The analysis of bulk and single-cell RNA-seq data at four different levels (samples, genes, transcripts, and exons) involves multiple statistical and computational questions, some of which remain challenging up to date. The first part of this dissertation focuses on the statistical challenges in the transcript-level analysis of bulk RNA-seq data. The next-generation RNA-seq technologies have been widely used to assess full-length RNA isoform structure and abundance in a high-throughput manner, enabling us to better understand the alternative splicing process and transcriptional regulation mechanism. However, accurate isoform identification and quantification from RNA-seq data are challenging due to the information loss in sequencing experiments. In Chapter 2, given the fast accumulation of multiple RNA-seq datasets from the same biological condition, we develop a statistical method, MSIQ, to achieve more accurate isoform quantification by integrating multiple RNA-seq samples under a Bayesian framework. The MSIQ method aims to (1) identify a consistent group of samples with homogeneous quality and (2) improve isoform quantification accuracy by jointly modeling multiple RNA-seq samples and allowing for higher weights on the consistent group. We show that MSIQ provides a consistent estimator of isoform abundance, and we demonstrate the accuracy of MSIQ compared with alternative methods through both simulation and real data studies. In Chapter 3, we introduce a novel method, AIDE, the first approach that directly controls false isoform discoveries by implementing the statistical model selection principle. Solving the isoform discovery problem in a stepwise manner, AIDE prioritizes the annotated isoforms and precisely identifies novel isoforms whose addition significantly improves the explanation of observed RNA-seq reads. Our results demonstrate that AIDE has the highest precision compared to the state-of-the-art methods, and it is able to identify isoforms with biological functions in pathological conditions. The second part of this dissertation discusses two statistical methods to improve scRNA-seq data analysis, which is complicated by the excess missing values, the so-called dropouts due to low amounts of mRNA sequenced within individual cells. In Chapter 5, we introduce scImpute, a statistical method to accurately and robustly impute the dropouts in scRNA-seq data. The scImpute method automatically identifies likely dropouts, and only performs imputation on these values by borrowing information across similar cells. Evaluation based on both simulated and real scRNA-seq data suggests that scImpute is an effective tool to recover transcriptome dynamics masked by dropouts, enhance the clustering of cell subpopulations, and improve the accuracy of differential expression analysis. In Chapter 6, we propose a flexible and robust simulator, scDesign, to optimize the choices of sequencing depth and cell number in designing scRNA-seq experiments, so as to balance the exploration of the depth and breadth of transcriptome information. It is the first statistical framework for researchers to quantitatively assess practical scRNA-seq experimental design in the context of differential gene expression analysis. In addition to experimental design, scDesign also assists computational method development by generating high-quality synthetic scRNA-seq datasets under customized experimental settings.
Single cell transcriptional profiling is critical for understanding cellular heterogeneity and identification of novel cell types and for studying growth and development of tissues and tumors. Leveraging recent advances in single cell RNA sequencing (scRNA-Seq) technology requires novel methods that are robust to high levels of technical and biological noise and scale to datasets of millions of cells. In this work, we address several challenges in the analysis work-flow of scRNA-Seq data: First, we propose novel computational approaches for unsupervised clustering of scRNA-Seq data based on Term Frequency - Inverse Document Frequency (TF-IDF) transformation that has been successfully used in text analysis. Here, we present empirical experimental results showing that TF-IDF methods consistently outperform commonly used scRNA-Seq clustering approaches. Second, we study the so called 'drop-out' effect that is considered one of the most notable challenges in scRNA-Seq analysis, where only a fraction of the transcriptome of each cell is captured. The random nature of drop-outs, however, makes it possible to consider imputation methods as means of correcting for drop-outs. In this part we study existing scRNA-Seq imputation methods and propose a novel iterative imputation approach based on efficiently computing highly similar cells. We then present results of a comprehensive assessment of existing and proposed methods on real scRNA-Seq datasets with varying per cell sequencing depth. Third, we present a computational method for assigning and/or ordering cells based on their cell-cycle stages from scRNA-Seq. And finally, we present a web-based interactive computational work-flow for analysis and visualization of scRNA-seq data.
