sccomp: Robust differential composition and variability analysis for single-cell data.

Cellular omics such as single-cell genomics, proteomics, and microbiomics allow the characterization of tissue and microbial community composition, which can be compared between conditions to identify biological drivers. This strategy has been critical to revealing markers of disease progression, such as cancer and pathogen infection. A dedicated statistical method for differential variability analysis is lacking for cellular omics data, and existing methods for differential composition analysis do not model some compositional data properties, suggesting there is room to improve model performance. Here, we introduce sccomp, a method for differential composition and variability analyses that jointly models data count distribution, compositionality, group-specific variability, and proportion mean-variability association, being aware of outliers. sccomp provides a comprehensive analysis framework that offers realistic data simulation and cross-study knowledge transfer. Here, we demonstrate that mean-variability association is ubiquitous across technologies, highlighting the inadequacy of the very popular Dirichlet-multinomial distribution. We show that sccomp accurately fits experimental data, significantly improving performance over state-of-the-art algorithms. Using sccomp, we identified differential constraints and composition in the microenvironment of primary breast cancer.

Trade-off between conservation of biological variation and batch effect removal in deep generative modeling for single-cell transcriptomics.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) technology has contributed significantly to diverse research areas in biology, from cancer to development. Since scRNA-seq data is high-dimensional, a common strategy is to learn low-dimensional latent representations better to understand overall structure in the data. In this work, we build upon scVI, a powerful deep generative model which can learn biologically meaningful latent representations, but which has limited explicit control of batch effects. Rather than prioritizing batch effect removal over conservation of biological variation, or vice versa, our goal is to provide a bird's eye view of the trade-offs between these two conflicting objectives. Specifically, using the well established concept of Pareto front from economics and engineering, we seek to learn the entire trade-off curve between conservation of biological variation and removal of batch effects. RESULTS: A multi-objective optimisation technique known as Pareto multi-task learning (Pareto MTL) is used to obtain the Pareto front between conservation of biological variation and batch effect removal. Our results indicate Pareto MTL can obtain a better Pareto front than the naive scalarization approach typically encountered in the literature. In addition, we propose to measure batch effect by applying a neural-network based estimator called Mutual Information Neural Estimation (MINE) and show benefits over the more standard maximum mean discrepancy measure. CONCLUSION: The Pareto front between conservation of biological variation and batch effect removal is a valuable tool for researchers in computational biology. Our results demonstrate the efficacy of applying Pareto MTL to estimate the Pareto front in conjunction with applying MINE to measure the batch effect.

NanoSplicer: accurate identification of splice junctions using Oxford Nanopore sequencing.

MOTIVATION: Long-read sequencing methods have considerable advantages for characterizing RNA isoforms. Oxford Nanopore sequencing records changes in electrical current when nucleic acid traverses through a pore. However, basecalling of this raw signal (known as a squiggle) is error prone, making it challenging to accurately identify splice junctions. Existing strategies include utilizing matched short-read data and/or annotated splice junctions to correct nanopore reads but add expense or limit junctions to known (incomplete) annotations. Therefore, a method that could accurately identify splice junctions solely from nanopore data would have numerous advantages. RESULTS: We developed 'NanoSplicer' to identify splice junctions using raw nanopore signal (squiggles). For each splice junction, the observed squiggle is compared to candidate squiggles representing potential junctions to identify the correct candidate. Measuring squiggle similarity enables us to compute the probability of each candidate junction and find the most likely one. We tested our method using (i) synthetic mRNAs with known splice junctions and (ii) biological mRNAs from a lung-cancer cell-line. The results from both datasets demonstrate NanoSplicer improves splice junction identification, especially when the basecalling error rate near the splice junction is elevated. AVAILABILITY AND IMPLEMENTATION: NanoSplicer is available at https://github.com/shimlab/NanoSplicer and archived at https://doi.org/10.5281/zenodo.6403849. Data is available from ENA: ERS7273757 and ERS7273453. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

Integrating quantitative information from ChIP-chip experiments into motif finding.

Identifying binding locations of transcription factors (TFs) within long segments of noncoding DNA is a challenging task. Recent chromatin immunoprecipitation on microarray (ChIP-chip) experiments utilizing tiling arrays are especially promising for this task since they provide high-resolution genome-wide maps of the interactions between the TFs and the DNA. Data from these experiments are invaluable for characterizing DNA recognition profiles (regulatory motifs) of TFs. A 2-step paradigm is commonly used for performing motif searches based on ChIP-chip data. First, candidate bound sequences that are in the order of 500-1000 bp are identified from ChIP-chip data. Then, motif searches are performed among these sequences. These 2 steps are typically carried out in a disconnected fashion in the sense that the quantitative nature of the ChIP-chip information is ignored in the second step. More specifically, all bound regions are assumed to equally likely have the motif(s), and the motifs are assumed to reside at any position of the bound regions with equal probability. We develop a conditional two-component mixture (CTCM) model that relaxes both these common assumptions by adaptively incorporating ChIP-chip information. The performances of the new and existing methods are compared using simulated data and ChIP-chip data from recently available ENCODE studies (Consortium, 2004). These studies indicate that CTCM efficiently utilizes the information available in the ChIP-chip experiments and has superior sensitivity and specificity especially when the motif of interest has low abundance among the ChIP-chip bound regions and/or low information content.