Benchmarking differential abundance methods for finding condition-specific prototypical cells in multi-sample single-cell datasets.

BACKGROUND: To analyze the large volume of data generated by single-cell technologies and to identify cellular correlates of particular clinical or experimental outcomes, differential abundance analyses are often applied. These algorithms identify subgroups of cells whose abundances change significantly in response to disease progression, or to an experimental perturbation. Despite the effectiveness of differential abundance analyses in identifying critical cell-states, there is currently no systematic benchmarking study to compare their applicability, usefulness, and accuracy in practice across single-cell modalities. RESULTS: Here, we perform a comprehensive benchmarking study to objectively evaluate and compare the benefits and potential downsides of current state-of-the-art differential abundance testing methods. We benchmarked six single-cell testing methods on several practical tasks, using both synthetic and real single-cell datasets. The tasks evaluated include effectiveness in identifying true differentially abundant subpopulations, accuracy in the adequate handling of batch effects, runtime efficiency, and hyperparameter usability and robustness. Based on various evaluation results, this paper gives dataset-specific suggestions for the practical use of differential abundance testing approaches. CONCLUSIONS: Based on our benchmarking study, we provide a set of recommendations for the optimal usage of single-cell DA testing methods in practice, particularly with respect to factors such as the presence of technical noise (for example batch effects), dataset size, and hyperparameter sensitivity.

Carbohydrate-active enzyme annotation in microbiomes using dbCAN.

CAZymes or carbohydrate-active enzymes are critically important for human gut health, lignocellulose degradation, global carbon recycling, soil health, and plant disease. We developed dbCAN as a web server in 2012 and actively maintain it for automated CAZyme annotation. Considering data privacy and scalability, we provide run_dbcan as a standalone software package since 2018 to allow users perform more secure and scalable CAZyme annotation on their local servers. Here, we offer a comprehensive computational protocol on automated CAZyme annotation of microbiome sequencing data, covering everything from short read pre-processing to data visualization of CAZyme and glycan substrate occurrence and abundance in multiple samples. Using a real-world metagenomic sequencing dataset, this protocol describes commands for dataset and software preparation, metagenome assembly, gene prediction, CAZyme prediction, CAZyme gene cluster (CGC) prediction, glycan substrate prediction, and data visualization. The expected results include publication-quality plots for the abundance of CAZymes, CGCs, and substrates from multiple CAZyme annotation routes (individual sample assembly, co-assembly, and assembly-free). For the individual sample assembly route, this protocol takes ∼33h on a Linux computer with 40 CPUs, while other routes will be faster. This protocol does not require programming experience from users, but it does assume a familiarity with the Linux command-line interface and the ability to run Python scripts in the terminal. The target audience includes the tens of thousands of microbiome researchers who routinely use our web server. This protocol will encourage them to perform more secure, rapid, and scalable CAZyme annotation on their local computer servers.

scLKME: A Landmark-based Approach for Generating Multi-cellular Sample Embeddings from Single-cell Data.

Single-cell technologies enable high-dimensional profiling of individual cells, therefore offering profound insights into subtle variation between specialized cell-types. However, translating the multitude of nuanced cellular profiles into meaningful per-sample representations is challenging due to heterogeneous cellular composition across individual profiled samples. To compute informative per-sample representations, we developed scLKME, a novel approach that uses a landmark-based kernel mean embedding method to convert multi-sample single-cell data into compact per-sample embeddings. Treating each sample as a distribution over cells, scLKME identifies landmarks across samples and maps these distributions into a reproducing kernel Hilbert space. Overall, scLKME outperforms state-of-the-art techniques in robustness, efficiency, accuracy, and practical usefulness of sample embeddings. Its application on a CyTOF dataset profiling immune responses in preterm birth highlighted its capacity to accurately identify patient-specific variations correlating with gestational age, suggesting broad applicability to multi-sample single-cell datasets with complex experimental designs. scLKME is available as an open-sourced python package at https://github.com/CompCy-lab/scLKME.

Benchmarking differential abundance methods for finding condition-specific prototypical cells in multi-sample single-cell datasets.

Modern single-cell data analysis relies on statistical testing (e.g. differential expression testing) to identify genes or proteins that are up-or down-regulated in relation to cell-types or clinical outcomes. However, existing algorithms for such statistical testing are often limited by technical noise and cellular heterogeneity, which lead to false-positive results. To constrain the analysis to a compact and phenotype-related cell population, differential abundance (DA) testing methods were employed to identify subgroups of cells whose abundance changed significantly in response to disease progression, or experimental perturbation. Despite the effectiveness of DA testing algorithms of identifying critical cell-states, there are no systematic benchmarking or comparative studies to compare their usages in practice. Herein, we performed the first comprehensive benchmarking study to objectively evaluate and compare the benefits and potential downsides of current state-of-the-art DA testing methods. We benchmarked six DA testing methods on several practical tasks, using both synthetic and real single-cell datasets. The task evaluated include, recognizing true DA subpopulations, appropriate handing of batch effects, runtime efficiency, and hyperparameter usability and robustness. Based on various evaluation results, this paper gives dataset-specific suggestions for the usage of DA testing methods.

COBRAC: a fast implementation of convex biclustering with compression.

SUMMARY: Biclustering is a generalization of clustering used to identify simultaneous grouping patterns in observations (rows) and features (columns) of a data matrix. Recently, the biclustering task has been formulated as a convex optimization problem. While this convex recasting of the problem has attractive properties, existing algorithms do not scale well. To address this problem and make convex biclustering a practical tool for analyzing larger data, we propose an implementation of fast convex biclustering called COBRAC to reduce the computing time by iteratively compressing problem size along with the solution path. We apply COBRAC to several gene expression datasets to demonstrate its effectiveness and efficiency. Besides the standalone version for COBRAC, we also developed a related online web server for online calculation and visualization of the downloadable interactive results. AVAILABILITY AND IMPLEMENTATION: The source code and test data are available at https://github.com/haidyi/cvxbiclustr or https://zenodo.org/record/4620218. The web server is available at https://cvxbiclustr.ericchi.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AcrDB: a database of anti-CRISPR operons in prokaryotes and viruses.

CRISPR-Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR-Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR-Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.

dbCAN-PUL: a database of experimentally characterized CAZyme gene clusters and their substrates.

PULs (polysaccharide utilization loci) are discrete gene clusters of CAZymes (Carbohydrate Active EnZymes) and other genes that work together to digest and utilize carbohydrate substrates. While PULs have been extensively characterized in Bacteroidetes, there exist PULs from other bacterial phyla, as well as archaea and metagenomes, that remain to be catalogued in a database for efficient retrieval. We have developed an online database dbCAN-PUL (http://bcb.unl.edu/dbCAN_PUL/) to display experimentally verified CAZyme-containing PULs from literature with pertinent metadata, sequences, and annotation. Compared to other online CAZyme and PUL resources, dbCAN-PUL has the following new features: (i) Batch download of PUL data by target substrate, species/genome, genus, or experimental characterization method; (ii) Annotation for each PUL that displays associated metadata such as substrate(s), experimental characterization method(s) and protein sequence information, (iii) Links to external annotation pages for CAZymes (CAZy), transporters (UniProt) and other genes, (iv) Display of homologous gene clusters in GenBank sequences via integrated MultiGeneBlast tool and (v) An integrated BLASTX service available for users to query their sequences against PUL proteins in dbCAN-PUL. With these features, dbCAN-PUL will be an important repository for CAZyme and PUL research, complementing our other web servers and databases (dbCAN2, dbCAN-seq).

AcrFinder: genome mining anti-CRISPR operons in prokaryotes and their viruses.

Anti-CRISPR (Acr) proteins encoded by (pro)phages/(pro)viruses have a great potential to enable a more controllable genome editing. However, genome mining new Acr proteins is challenging due to the lack of a conserved functional domain and the low sequence similarity among experimentally characterized Acr proteins. We introduce here AcrFinder, a web server (http://bcb.unl.edu/AcrFinder) that combines three well-accepted ideas used by previous experimental studies to pre-screen genomic data for Acr candidates. These ideas include homology search, guilt-by-association (GBA), and CRISPR-Cas self-targeting spacers. Compared to existing bioinformatics tools, AcrFinder has the following unique functions: (i) it is the first online server specifically mining genomes for Acr-Aca operons; (ii) it provides a most comprehensive Acr and Aca (Acr-associated regulator) database (populated by GBA-based Acr and Aca datasets); (iii) it combines homology-based, GBA-based, and self-targeting approaches in one software package; and (iv) it provides a user-friendly web interface to take both nucleotide and protein sequence files as inputs, and output a result page with graphic representation of the genomic contexts of Acr-Aca operons. The leave-one-out cross-validation on experimentally characterized Acr-Aca operons showed that AcrFinder had a 100% recall. AcrFinder will be a valuable web resource to help experimental microbiologists discover new Anti-CRISPRs.

GDASC: a GPU parallel-based web server for detecting hidden batch factors.

SUMMARY: We developed GDASC, a web version of our former DASC algorithm implemented with GPU. It provides a user-friendly web interface for detecting batch factors. Based on the good performance of DASC algorithm, it is able to give the most accurate results. For two steps of DASC, data-adaptive shrinkage and semi-non-negative matrix factorization, we designed parallelization strategies facing convex clustering solution and decomposition process. It runs more than 50 times faster than the original version on the representative RNA sequencing quality control dataset. With its accuracy and high speed, this server will be a useful tool for batch effects analysis. AVAILABILITY AND IMPLEMENTATION: http://bioinfo.nankai.edu.cn/gdasc.php. CONTACT: zhanghan@nankai.edu.cn. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

dbCAN-seq: a database of carbohydrate-active enzyme (CAZyme) sequence and annotation.

Carbohydrate-active enzyme (CAZymes) are not only the most important enzymes for bioenergy and agricultural industries, but also very important for human health, in that human gut microbiota encode hundreds of CAZyme genes in their genomes for degrading various dietary and host carbohydrates. We have built an online database dbCAN-seq (http://cys.bios.niu.edu/dbCAN_seq) to provide pre-computed CAZyme sequence and annotation data for 5,349 bacterial genomes. Compared to the other CAZyme resources, dbCAN-seq has the following new features: (i) a convenient download page to allow batch download of all the sequence and annotation data; (ii) an annotation page for every CAZyme to provide the most comprehensive annotation data; (iii) a metadata page to organize the bacterial genomes according to species metadata such as disease, habitat, oxygen requirement, temperature, metabolism; (iv) a very fast tool to identify physically linked CAZyme gene clusters (CGCs) and (v) a powerful search function to allow fast and efficient data query. With these unique utilities, dbCAN-seq will become a valuable web resource for CAZyme research, with a focus complementary to dbCAN (automated CAZyme annotation server) and CAZy (CAZyme family classification and reference database).

Detecting hidden batch factors through data-adaptive adjustment for biological effects.

Motivation: Batch effects are one of the major source of technical variations that affect the measurements in high-throughput studies such as RNA sequencing. It has been well established that batch effects can be caused by different experimental platforms, laboratory conditions, different sources of samples and personnel differences. These differences can confound the outcomes of interest and lead to spurious results. A critical input for batch correction algorithms is the knowledge of batch factors, which in many cases are unknown or inaccurate. Hence, the primary motivation of our paper is to detect hidden batch factors that can be used in standard techniques to accurately capture the relationship between gene expression and other modeled variables of interest. Results: We introduce a new algorithm based on data-adaptive shrinkage and semi-Non-negative Matrix Factorization for the detection of unknown batch effects. We test our algorithm on three different datasets: (i) Sequencing Quality Control, (ii) Topotecan RNA-Seq and (iii) Single-cell RNA sequencing (scRNA-Seq) on Glioblastoma Multiforme. We have demonstrated a superior performance in identifying hidden batch effects as compared to existing algorithms for batch detection in all three datasets. In the Topotecan study, we were able to identify a new batch factor that has been missed by the original study, leading to under-representation of differentially expressed genes. For scRNA-Seq, we demonstrated the power of our method in detecting subtle batch effects. Availability and implementation: DASC R package is available via Bioconductor or at https://github.com/zhanglabNKU/DASC. Contact: zhanghan@nankai.edu.cn or zhandonl@bcm.edu. Supplementary information: Supplementary data are available at Bioinformatics online.