The problem of estimating the structure of a graph from observed data is of growing interest in the context of high-throughput genomic data, and single-cell RNA sequencing in particular. These, however, are challenging applications, since the data consist of high-dimensional counts with high variance and over-abundance of zeros. Here, we present a general framework for learning the structure of a graph from single-cell RNA-seq data, based on the zero-inflated negative binomial distribution. We demonstrate with simulations that our approach is able to retrieve the structure of a graph in a variety of settings and we show the utility of the approach on real data.
Hebei University of Technology（河北工业大学）；University of Victoria（维多利亚大学）；National University of Sciences（国立科学大学）；University of Saskatchewan（萨斯喀彻温大学）
Background: Single-cell RNA sequencing (scRNA-seq) yields valuable insights about gene expression and gives critical information about complex tissue cellular composition. In the analysis of single-cell RNA sequencing, the annotations of cell subtypes are often done manually, which is time-consuming and irreproducible. Garnett is a cell-type annotation software based the on elastic net method. Besides cell-type annotation, supervised machine learning methods can also be applied to predict other cell phenotypes from genomic data. Despite the popularity of such applications, there is no existing…
Survival analysis consists of studying the elapsed time until an event of interest, such as the death or recovery of a patient in medical studies. This work explores the potential of neural networks in survival analysis from clinical and RNA-seq data. If the neural network approach is not recent in survival analysis, methods were classically considered for low-dimensional input data. But with the emergence of high-throughput sequencing data, the number of covariates of interest has become very large, with new statistical issues to consider. We present and test a few recent neural network appro…
In our Brief Communication (DOI: 10.1038/s41592-018-0033-z), we presented the method SAVER for recovering true gene expression levels in noisy single cell RNA sequencing data. We evaluated the performance of SAVER, along with comparable methods MAGIC and scImpute, in an RNA FISH validation experiment and a data downsampling experiment. In a Comment [arXiv:1908.07084v1], Li & Li were concerned with the use of the downsampled datasets, specifically focusing on clustering results obtained from the Zeisel et al. data. Here, we will address these comments and, furthermore, amend the data downsampli…
The human microbiome plays critical roles in human health and has been linked to many diseases. While advanced sequencing technologies can characterize the composition of the microbiome in unprecedented detail, it remains challenging to disentangle the complex interplay between human microbiome and disease risk factors due to the complicated nature of microbiome data. Excessive numbers of zero values, high dimensionality, the hierarchical phylogenetic tree and compositional structure are compounded and consequently make existing methods inadequate to appropriately address these issues. We prop…
The existence of doublets is a key confounder in single-cell RNA sequencing (scRNA-seq) data analysis. Computational methods have been developed for detecting doublets from scRNA-seq data. We developed an R package DoubletCollection to integrate the installation and execution of eight doublet-detection methods. DoubletCollection also provides a unified interface to perform and visualize downstream analysis after doublet detection. Here, we present a protocol of using DoubletCollection to benchmark doublet-detection methods. This protocol can automatically accommodate new doublet-detection meth…
RNA sequencing (RNA-seq) enables characterization and quantification of individual transcriptomes as well as detection of patterns of allelic expression and alternative splicing. Current RNA-seq protocols depend on high-throughput short-read sequencing of cDNA. However, as ongoing advances are rapidly yielding increasing read lengths, a technical hurdle remains in identifying the degree to which differences in read length influence various transcriptome analyses. In this study, we generated two paired-end RNA-seq datasets of differing read lengths (2x75 bp and 2x262 bp) for lymphoblastoid cell…
Single-cell RNA sequencing (scRNA-seq) provides a high throughput, quantitative and unbiased framework for scientists in many research fields to identify and characterize cell types within heterogeneous cell populations from various tissues. However, scRNA-seq based identification of discrete cell-types is still labor intensive and depends on prior molecular knowledge. Artificial intelligence has provided faster, more accurate, and user-friendly approaches for cell-type identification. In this review, we discuss recent advances in cell-type identification methods using artificial intelligence …
On June 25th, 2018, Huang et al. published a computational method SAVER on Nature Methods for imputing dropout gene expression levels in single cell RNA sequencing (scRNA-seq) data. Huang et al. performed a set of comprehensive benchmarking analyses, including comparison with the data from RNA fluorescence in situ hybridization, to demonstrate that SAVER outperformed two existing scRNA-seq imputation methods, scImpute and MAGIC. However, their computational analyses were based on semi-synthetic data that the authors had generated following the Poisson-Gamma model used in the SAVER method. We h…
Recently, ultra high-throughput sequencing of RNA (RNA-Seq) has been developed as an approach for analysis of gene expression. By obtaining tens or even hundreds of millions of reads of transcribed sequences, an RNA-Seq experiment can offer a comprehensive survey of the population of genes (transcripts) in any sample of interest. This paper introduces a statistical model for estimating isoform abundance from RNA-Seq data and is flexible enough to accommodate both single end and paired end RNA-Seq data and sampling bias along the length of the transcript. Based on the derivation of minimal suff…
We developed NameMyGene, a web tool and a stand alone program to easily generate putative family-based names for small RNA sequences so that laboratories can easily organize, analyze, and observe patterns from, the massive amount of data generated by next-generation sequencers. NameMyGene, also applicable to other emerging methods such as RNA-Seq, and Chip-Seq, solely uses the input small RNA sequence and does not require any additional data such as other sequence data sets. The web server and software is freely available (http://www.johnlab.org/NameMyGene) and is based on Java to ensure platf…
Deep sequencing has become one of the most popular tools for transcriptome profiling in biomedical studies. While an abundance of computational methods exists for "normalizing" sequencing data to remove unwanted between-sample variations due to experimental handling, there is no consensus on which normalization is the most suitable for a given data set. To address this problem, we developed "DANA" - an approach for assessing the performance of normalization methods for microRNA sequencing data based on biology-motivated and data-driven metrics. Our approach takes advantage of well-known biolog…
RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods ar…
RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations), while optionally adjusting for other systematic factors that affect the data collection process. There are a number of subtle yet critical aspects of these analyses, such as read counting, appropriate treatment of biological variability, quality control checks and appropriate setup of sta…
Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNA…
RNA-sequencing (RNA-seq) has become an exemplar technology in modern biology and clinical applications over the past decade. It has gained immense popularity in the recent years driven by continuous efforts of the bioinformatics community to develop accurate and scalable computational tools. RNA-seq is a method of analyzing the RNA content of a sample using the modern sequencing platforms. It generates enormous amounts of transcriptomic data in the form of nucleotide sequences, known as reads. RNA-seq analysis enables the probing of genes and corresponding transcripts which is essential for an…
Applications of single-cell RNA sequencing in various biomedical research areas have been blooming. This new technology provides unprecedented opportunities to study disease heterogeneity at the cellular level. However, unique characteristics of scRNA-seq data, including large dimensionality, high dropout rates, and possibly batch effects, bring great difficulty into the analysis of such data. Not appropriately addressing these issues obstructs true scientific discovery. Herein, we propose a unified Regularized Zero-inflated Mixture Model framework designed for scRNA-seq data (RZiMM-scRNA) to …
The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct annotation is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3-prime untr…
In this paper we introduce a novel parallel pipeline for fast and accurate mapping of RNA sequences on servers equipped with multicore processors. Our software, named HPG-Aligner, leverages the speed of the Burrows-Wheeler Transform to map a large number of RNA fragments (reads) rapidly, as well as the accuracy of the Smith-Waterman algorithm, that is employed to deal with conflictive reads. The aligner is complemented with a careful strategy to detect splice junctions based on the division of RNA reads into short segments (or seeds), which are then mapped onto a number of candidate alignment …
Single-cell RNA-Sequencing (scRNA-Seq) is a revolutionary technique for discovering and describing cell types in heterogeneous tissues, yet its measurement of expression often suffers from large systematic bias. A major source of this bias is the cell cycle, which introduces large within-cell-type heterogeneity that can obscure the differences in expression between cell types. The current method for removing the cell-cycle effect is unable to effectively identify this effect and has a high risk of removing other biological components of interest, compromising downstream analysis. We present cc…
We propose a probabilistic model for interpreting gene expression levels that are observed through single-cell RNA sequencing. In the model, each cell has a low-dimensional latent representation. Additional latent variables account for technical effects that may erroneously set some observations of gene expression levels to zero. Conditional distributions are specified by neural networks, giving the proposed model enough flexibility to fit the data well. We use variational inference and stochastic optimization to approximate the posterior distribution. The inference procedure scales to over on…
We propose a probabilistic model for interpreting gene expression levels that are observed through single-cell RNA sequencing. In the model, each cell has a low-dimensional latent representation. Additional latent variables account for technical effects that may erroneously set some observations of gene expression levels to zero. Conditional distributions are specified by neural networks, giving the proposed model enough flexibility to fit the data well. We use variational inference and stochastic optimization to approximate the posterior distribution. The inference procedure scales to over on…
The development of novel high-throughput sequencing (HTS) methods for RNA (RNA-Seq) has provided a very powerful mean to study splicing under multiple conditions at unprecedented depth. However, the complexity of the information to be analyzed has turned this into a challenging task. In the last few years, a plethora of tools have been developed, allowing researchers to process RNA-Seq data to study the expression of isoforms and splicing events, and their relative changes under different conditions. We provide an overview of the methods available to study splicing from short RNA-Seq data. We …
Coronavirus disease 2019 (COVID-19) has impacted almost every part of human life worldwide, posing a massive threat to human health. There is no specific drug for COVID-19, highlighting the urgent need for the development of effective therapeutics. To identify potentially repurposable drugs, we employed a systematic approach to mine candidates from U.S. FDA-approved drugs and preclinical small-molecule compounds by integrating the gene expression perturbation data for chemicals from the Library of Integrated Network-Based Cellular Signatures project with a publicly available single-cell RNA se…
The advent of DNA and RNA sequencing has revolutionized the study of genomics and molecular biology. Next generation sequencing (NGS) technologies like Illumina, Ion Torrent, SOLiD sequencing etc. have brought about a quick and cheap way to sequence genomes. Recently, third generation sequencing (TGS) technologies like PacBio and Oxford Nanopore Technology (ONT) have also been developed. Different technologies use different underlying methods for sequencing and are prone to different error rates. Though many tools exist for error correction of sequencing data from NGS and TGS methods, no stand…
High-throughput cDNA sequencing (RNA-seq) is a very powerful technique to quantify gene expression in an unbiased way. The Crustacean family is among the groups of organisms sparsely represented in current genomic databases. Here we present transcriptome data from Uca minax (red-jointed fiddler crab) as an opportunity to extend our knowledge. Next generation sequencing was performed on six tissue samples from Uca minax using the Illumina HiSeq system. Six Transcriptome libraries were created using Trinity; a free, open-source software tool for de novo transcriptome assembly of high-throughput …
scUnified: An AI-Ready Standardized Resource for Single-Cell RNA Sequencing Analysis
Computer Network Information Center, Chinese Academy of Sciences（中国科学院计算机网络信息中心）；Hangzhou Institute for Advanced Study,University of Chinese Academy of Sciences（中国科学院大学杭州先进研究所）；University of Chinese Academy of Sciences（中国科学院大学）；Department of Computer Science, Columbia University（哥伦比亚大学计算机科学系）
We introduce Sashimi plots, a quantitative multi-sample visualization of mRNA sequencing reads aligned to gene annotations. Sashimi plots are made using alignments (stored in the SAM/BAM format) and gene model annotations (in GFF format), which can be custom-made by the user or obtained from databases such as Ensembl or UCSC. We describe two implementations of Sashimi plots: (1) a stand-alone command line implementation aimed at making customizable publication quality figures, and (2) an implementation built into the Integrated Genome Viewer (IGV) browser, which enables rapid and dynamic creat…
University of California, Davis（加州大学戴维斯分校）；The Pennsylvania State University（宾夕法尼亚州立大学）；Texas A&M University（得克萨斯农工大学）；Yale University（耶鲁大学）
Spectral clustering is one of the fundamental unsupervised learning methods widely used in data analysis. Sparse spectral clustering (SSC) imposes sparsity to the spectral clustering and it improves the interpretability of the model. This paper considers a widely adopted model for SSC, which can be formulated as an optimization problem over the Stiefel manifold with nonsmooth and nonconvex objective. Such an optimization problem is very challenging to solve. Existing methods usually solve its convex relaxation or need to smooth its nonsmooth part using certain smoothing techniques. In this pap…
High-throughput sequencing of RNA transcripts (RNA-seq) has become the method of choice for detection of differential expression (DE). Concurrent with the growing popularity of this technology there has been a significant research effort devoted towards understanding the statistical properties of this data and the development of analysis methods. We report on a comprehensive evaluation of the commonly used DE methods using the SEQC benchmark data set. We evaluate a number of key features including: assessment of normalization, accuracy of DE detection, modeling of genes expressed in only one c…
We perform differential expression analysis of high-throughput sequencing count data under a Bayesian nonparametric framework, removing sophisticated ad-hoc pre-processing steps commonly required in existing algorithms. We propose to use the gamma (beta) negative binomial process, which takes into account different sequencing depths using sample-specific negative binomial probability (dispersion) parameters, to detect differentially expressed genes by comparing the posterior distributions of gene-specific negative binomial dispersion (probability) parameters. These model parameters are inferre…
Glaucoma is the leading cause of irreversible blindness in people over the age of 60, accounting for 6.6 to 8% of all blindness in 2010, but there is still much to be learned about the genetic origins of the eye disease. With the modern development of Next-Generation Sequencing (NGS) technologies, scientists are starting to learn more about the genetic origins of Glaucoma. This research uses differential expression (DE) and gene ontology (GO) analyses to study the genetic differences between mice with severe Glaucoma and multiple control groups. Optical nerve head (ONH) and retina data samples…
Weill Cornell Medicine（威尔康奈尔医学院）；Cornell University（康奈尔大学）；Institute for Computational Biomedicine（计算生物医学研究所）；Caryl and Israel Englander Institute for Precision Medicine（卡里尔与以色列·英格兰德精准医学研究所）；Temple University（天普大学）
Single-cell RNA-sequencing (scRNA-seq) has become a routinely used technique to quantify the gene expression profile of thousands of single cells simultaneously. Analysis of scRNA-seq data plays an important role in the study of cell states and phenotypes, and has helped elucidate biological processes, such as those occurring during development of complex organisms and improved our understanding of disease states, such as cancer, diabetes, and COVID, among others. Deep learning, a recent advance of artificial intelligence that has been used to address many problems involving large datasets, ha…
The newly developed deep-sequencing technologies make it possible to acquire both quantitative and qualitative information regarding transcript biology. By measuring messenger RNA levels for all genes in a sample, RNA-seq provides an attractive option to characterize the global changes in transcription. RNA-seq is becoming the widely used platform for gene expression profiling. However, real transcription signals in the RNA-seq data are confounded with measurement and sequencing errors, and other random biological/technical variation. How to appropriately take the variability due to errors and…
Motivation: High-throughput sequencing enables expression analysis at the level of individual transcripts. The analysis of transcriptome expression levels and differential expression estimation requires a probabilistic approach to properly account for ambiguity caused by shared exons and finite read sampling as well as the intrinsic biological variance of transcript expression. Results: We present BitSeq (Bayesian Inference of Transcripts from Sequencing data), a Bayesian approach for estimation of transcript expression level from RNA-seq experiments. Inferred relative expression is represente…
High-throughput RNA-sequencing (RNA-seq) technologies are powerful tools for understanding cellular state. Often it is of interest to quantify and summarize changes in cell state that occur between experimental or biological conditions. Differential expression is typically assessed using univariate tests to measure gene-wise shifts in expression. However, these methods largely ignore changes in transcriptional correlation. Furthermore, there is a need to identify the low-dimensional structure of the gene expression shift to identify collections of genes that change between conditions. Here, we…
Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The prot…
scGTN: Deep Siamese Graph Transformer Network for Single-cell RNA Sequencing Clustering
Sichuan University（四川大学）；University of International Business and Economics（对外经济贸易大学）；Great Bay University（大湾区大学）；The Education University of Hong Kong（香港教育大学）
Sequencing by Emergence (SEQE) is a new single-molecule nucleic acid (DNA/RNA) sequencing technology that estimates sequence as an emergent property of the binding and localization of a repertoire of short oligonucleotide probes. SEQE promises to deliver accurate, ultra-long, haplotype-phased reads at the whole genome-scale for very low cost within 10 minutes. The data SEQE generates requires entirely new inference techniques. In this paper we introduce a probabilistic model of the SEQE measurement process and an algorithm that estimates sequence by solving a convex relaxation of the correspon…
According to the National Cancer Institute, there were 9.5 million cancer-related deaths in 2018. A challenge in improving treatment is resistance in genetically unstable cells. The purpose of this study is to evaluate unsupervised machine learning on classifying treatment-resistant phenotypes in heterogeneous tumors through analysis of single cell RNA sequencing(scRNAseq) data with a pipeline and evaluation metrics. scRNAseq quantifies mRNA in cells and characterizes cell phenotypes. One scRNAseq dataset was analyzed (tumor/non-tumor cells of different molecular subtypes and patient identific…
scSiameseClu: A Siamese Clustering Framework for Interpreting single-cell RNA Sequencing Data
Computer Network Information Center, Chinese Academy of Sciences（中国科学院计算机网络信息中心）；University of Chinese Academy of Sciences（中国科学院大学）；College of Life Science, Northeast Agricultural University（东北农业大学生命科学学院）；SKL-IOTSC and Department of CIS, University of Macau（澳门大学SKL-IOTSC和CIS系）；Jilin University（吉林大学）
An important challenge in cancer systems biology is to uncover the complex network of interactions between genes (tumor suppressor genes and oncogenes) implicated in cancer. Next generation sequencing provides unparalleled ability to probe the expression levels of the entire set of cancer genes and their transcript isoforms. However, there are onerous statistical and computational issues in interpreting high-dimensional sequencing data and inferring the underlying genetic network. In this study, we analyzed RNA-Seq data from lymphoblastoid cell lines derived from a population of 69 human indiv…
Next-generation RNA sequencing (RNA-seq) technology has been widely used to assess full-length RNA isoform abundance in a high-throughput manner. RNA-seq data offer insight into gene expression levels and transcriptome structures, enabling us to better understand the regulation of gene expression and fundamental biological processes. Accurate isoform quantification from RNA-seq data is challenging due to the information loss in sequencing experiments. A recent accumulation of multiple RNA-seq data sets from the same tissue or cell type provides new opportunities to improve the accuracy of isof…
