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scRNA-seq data analysis tools and papers

MIT License PR's Welcome

Single-cell RNA-seq related tools and genomics data analysis resources. Tools are sorted by publication date, reviews and most recent publications on top. Unpublished tools are listed at the end of each section. Please, contribute and get in touch! See MDmisc notes for other programming and genomics-related notes. See scATAC-seq_notes for scATAC-seq related resources.

Table of content

Awesome

  • single-cell-pseudotime - an overview of single-cell RNA-seq pseudotime estimation algorithms, comprehensive collection of links to software and accompanying papers, by Anthony Gitter

Courses

  • Review of single-cell transcriptomics technologies and analysis steps and software. Sample preparation, scRNA-seq preprocessing, QC, normalization, batch correction, dimensionaliry reduction. Downstream analysis on cell level (clustering, trajectory inference), gene level (differential expression, functional enrichment, network analysis). Table 1 - preprocessing pipelines and tools, brief description. Table 2 - clustering algorithms.
    Paper Nayak, Richa, and Yasha Hasija. “A Hitchhiker’s Guide to Single-Cell Transcriptomics and Data Analysis Pipelines.” Genomics 113, no. 2 (March 2021): 606–19. https://doi.org/10.1016/j.ygeno.2021.01.007.
  • Analysis of single cell RNA-seq data, www.singlecellcourse.org - step-by-step scRNA-seq analysis course. R-based, with code examples, explanations, exercises. From alignment (STAR) and QC (FASTQC) to introduction to R, SingleCellExperiment class, scater object, data exploration (reads, UMI), filtering, normalization (scran), batch effect removal (RUV, ComBat, mnnCorrect, GLM, Harmony), clustering and marker gene identification (SINCERA, SC3, tSNE, Seurat), feature selection (M3Drop::M3DropConvertData, BrenneckeGetVariableGenes), pseudotime analysis (TSCAN, Monocle, diffusion maps, SLICER, Ouija, destiny), imputation (scImpute, DrImpute, MAGIC), differential expression (Kolmogorov-Smirnov, Wilcoxon, edgeR, Monocle, MAST), data integration (scmap, cell-to-cell mapping, Metaneighbour, mnnCorrect, Seurat's canonical correllation analysis). Search for scRNA-seq data (scfind R package), as well as Hemberg group’s public datasets. Seurat chapter. "Ideal" scRNA-seq pipeline. Video lectures.
    Paper Andrews, Tallulah S., Vladimir Yu Kiselev, Davis McCarthy, and Martin Hemberg. "Tutorial: Guidelines for the Computational Analysis of Single-Cell RNA Sequencing Data." https://doi.org/10.1038/s41596-020-00409-w Nature Protocols, December 7, 2020.

Tutorials

Preprocessing pipelines

  • Assessment of 9 preprocessing pipelines (Cell Ranger, Optimus, salmon alevin, kallisto bustools, dropSeqPipe, scPipe, zUMIs, celseq2 and scruff) on 10X and CEL-Seq2 datasets (scmixology and others, 9 datasets total). All pipelines coupled with performant post-processing (normalization, filtering, etc.) produce comparable data quality in terms of clustering/agreement with known cell types. Low-expressed genes are discordant. Details and specific results of each pipeline. GitHub with pre-/postprocessing scripts
    Paper You, Yue, Luyi Tian, Shian Su, Xueyi Dong, Jafar S Jabbari, Peter F Hickey, and Matthew E Ritchie. "Benchmarking UMI-Based Single Cell RNA-Sequencing Preprocessing Workflows" https://doi.org/10.1186/s13059-021-02552-3 Genome Biology. 14 December 2021
  • Single cell current best practices tutorial, GitHub. QC (count depth, number of genes, % mitochondrial), normalization (global, downsampling, nonlinear), data correction (batch, denoising, imputation), feature selection, dimensionality reduction (PCA, diffusion maps, tSNE, UMAP), visualization, clustering (k-means, graph/community detection), annotation, trajectory inference (PAGA, Monocle), differential analysis (DESeq2, EdgeR, MAST), gene regulatory networks. Description of the bigger picture at each step, latest tools, their brief description, references. R-based Scater as the full pipeline for QC and preprocessing, Seurat for downstream analysis, scanpy Python pipeline. Links and refs to other tutorials.
    Paper Luecken, Malte D., and Fabian J. Theis. "Current Best Practices in Single-Cell RNA-Seq Analysis: A Tutorial" https://doi.org/10.15252/msb.20188746 Molecular Systems Biology 15, no. 6 (June 19, 2019)
  • Alevin - end-to-end droplet-based scRNA-seq (10X Genomics) processing pipeline performing cell barcode detection (two-step whitelisting procedure), read mapping, UMI deduplication (parsimonious UMI graphs, PUGs), resolving multimapped reads (EM method to resolve UMI collisions), gene count estimation. Intelligently handles UMI deduplication and multimapped reads, resulting in more accurate gene abundance estimation. Input - sample-demultiplexed FASTQ, output - gene-level UMI counts. Compared against the Cell Ranger, dropEst, STAR and featureCount-based pipelines, UMI-tools, alevin is more accurate and quantifies a greater proportion of sequenced data, especially on combined genomes. Approx. 21X faster than Cell Ranger, low memory requirements, 10-12 threads optimal. C++ implementation, part of Salmon. Alevin documentation, Tutorials that include visualization options.
    Paper Srivastava, Avi, Laraib Malik, Tom Smith, Ian Sudbery, and Rob Patro. "Alevin Efficiently Estimates Accurate Gene Abundances from DscRNA-Seq Data" https://doi.org/10.1186/s13059-019-1670-y Genome Biology, (December 2019)
  • bigSCale - scalable analytical framework to analyze large scRNA-seq datasets, UMIs or counts. Pre-clustering, convolution into iCells, final clustering, differential expression, biomarkers.Correlation metric for scRNA-seq data based on converting expression to Z-scores of differential expression. Robust to dropouts. Matlab implementation. Data, 1847 human neuronal progenitor cells
    Paper Iacono, Giovanni, Elisabetta Mereu, Amy Guillaumet-Adkins, Roser Corominas, Ivon Cuscó, Gustavo Rodríguez-Esteban, Marta Gut, Luis Alberto Pérez-Jurado, Ivo Gut, and Holger Heyn. "BigSCale: An Analytical Framework for Big-Scale Single-Cell Data." https://doi.org/10.1101/gr.230771.117 Genome Research 28, no. 6 (June 2018): 878–90.
  • CALISTA - clustering, lineage reconstruction, transition gene identification, and cell pseudotime single cell transcriptional analysis. Analyses can be all or separate. Uses a likelihood-based approach based on probabilistic models of stochastic gene transcriptional bursts and random technical dropout events, so all analyses are compatible with each other. Input - a matrix of normalized, batch-removed log(RPKM) or log(TPM) or scaled UMIs. Methods detail statistical methodology. Matlab and R version
    Paper Papili Gao N, Hartmann T, Fang T, Gunawan R. [CALISTA: Clustering and LINEAGE Inference in Single-Cell Transcriptional Analysis" https://doi.org/10.3389/fbioe.2020.00018 Frontiers in bioengineering and biotechnology. 2020 Feb 4;8:18.
  • demuxlet - Introduces the ‘demuxlet’ algorithm, which enables genetic demultiplexing, doublet detection, and super-loading for droplet-based scRNA-seq. Recommended approach when samples have distinct genotypes
    Paper Kang, Hyun Min, Meena Subramaniam, Sasha Targ, Michelle Nguyen, Lenka Maliskova, Elizabeth McCarthy, Eunice Wan, et al. "Multiplexed Droplet Single-Cell RNA-Sequencing Using Natural Genetic Variation." https://doi.org/10.1038/nbt.4042 Nature Biotechnology 36, no. 1 (January 2018): 89–94.
  • dropEst - pipeline for pre-processing, mapping, QCing, filtering, and quantifying droplet-based scRNA-seq datasets. Input - FASTQ or BAM, output - an R-readable molecular count matrix. Written in C++
    Paper Petukhov, Viktor, Jimin Guo, Ninib Baryawno, Nicolas Severe, David T. Scadden, Maria G. Samsonova, and Peter V. Kharchenko. "DropEst: Pipeline for Accurate Estimation of Molecular Counts in Droplet-Based Single-Cell RNA-Seq Experiments." https://doi.org/10.1186/s13059-018-1449-6 Genome Biology 19, no. 1 (December 2018): 78.
  • kallistobus - fast pipeline for scRNA-seq processing. New BUS (Barcode, UMI, Set) format for storing and manipulating pseudoalignment results. Includes RNA velocity analysis. Python-based
    Paper Melsted, Páll, A. Sina Booeshaghi, Fan Gao, Eduardo da Veiga Beltrame, Lambda Lu, Kristján Eldjárn Hjorleifsson, Jase Gehring, and Lior Pachter. "Modular and Efficient Pre-Processing of Single-Cell RNA-Seq." https://doi.org/10.1101/673285 Preprint. Bioinformatics, June 17, 2019.
  • PyMINEr - Python-based scRNA-seq processing pipeline. Cell type identification, detection of cell type-enriched genes, pathway analysis, co-expression networks and graph theory approaches to interpreting gene expression. Notes on methods: modified K++ clustering, automatic detection of the number of cell types, co-expression and PPI networks. Input: .txt or .hdf5 files. Detailed analysis of several pancreatic datasets
    Paper Tyler, Scott R., Pavana G. Rotti, Xingshen Sun, Yaling Yi, Weiliang Xie, Michael C. Winter, Miles J. Flamme-Wiese, et al. "PyMINEr Finds Gene and Autocrine-Paracrine Networks from Human Islet ScRNA-Seq." https://doi.org/10.1016/j.celrep.2019.01.063 Cell Reports 26, no. 7 (February 2019): 1951-1964.e8.
  • SEQC - Single-Cell Sequencing Quality Control and Processing Software, a general purpose method to build a count matrix from single cell sequencing reads, able to process data from inDrop, drop-seq, 10X, and Mars-Seq2 technologies.
    Paper Azizi, Elham, Ambrose J. Carr, George Plitas, Andrew E. Cornish, Catherine Konopacki, Sandhya Prabhakaran, Juozas Nainys, et al. "Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment." https://doi.org/10.1016/j.cell.2018.05.060 Cell, June 2018.
  • zUMIs - scRNA-seq processing pipeline that handles barcodes and summarizes UMIs using exonic or exonic + intronic mapped reads (improves clustering, DE detection). Adaptive downsampling of oversequenced libraries. STAR aligner, Rsubread::featureCounts counting UMIs in exons and introns.
    Paper Parekh, Swati, Christoph Ziegenhain, Beate Vieth, Wolfgang Enard, and Ines Hellmann. "ZUMIs - A Fast and Flexible Pipeline to Process RNA Sequencing Data with UMIs." https://doi.org/10.1093/gigascience/giy059 GigaScience 7, no. 6 (01 2018).
  • STAR alignment parameters: –outFilterType BySJout, –outFilterMultimapNmax 100, –limitOutSJcollapsed 2000000 –alignSJDBoverhangMin 8, –outFilterMismatchNoverLmax 0.04, –alignIntronMin 20, –alignIntronMax 1000000, –readFilesIn fastqrecords, –outSAMprimaryFlag AllBestScore, –outSAMtype BAM Unsorted. From Azizi et al., “Single-Cell Map of Diverse Immune Phenotypes in the Breast Tumor Microenvironment.”

Format conversion

  • sceasy - R package to convert different single-cell data formats to each other, supports Seurat, SingleCellExperiment, AnnData, Loom

  • scKirby - R package for automated ingestion and conversion of various single-cell data formats (SingleCellExperiment, SummarizedExperiment, HDF5SummarizedExperiment, Seurat, H5Seurat, anndata, loom, loomR, CellDataSet/monocle, ExpressionSet, and more).

  • zellkonverter - R package for conversion between scRNA-seq objects (the Bioconductor SingleCellExperiment data structure and the Python AnnData-based single-cell analysis environment). Tweet

Visualization pipelines

  • Kana - single-cell analysis in the browser, by Jayaram Kancherla (@jkanche), Aaron Lun (@LTLA). Input - 10X genomics CellRanger's output, matrix or .h5 files. Preprocessing (removal of low-quality cells, Normalization and log-transformation, Modelling of the mean-variance trend across genes), PCA, Clustering (t-SNE/UMAP), Marker detection, custom cluster definition and marker analysis. Works with scATAC-seq data. GitHub, Tweet.

  • cellxgene - An interactive exploratory visualization tool for single-cell transcriptomics data, web and desktop versions. Input - matrix-form datasets, metadata, pre-computed embeddings/clustering. Compatible with Seurat, Scanpy, Bioconductor, scVI GitHub

    Paper Megill, Colin, Bruce Martin, Charlotte Weaver, Sidney Bell, Lia Prins, Seve Badajoz, Brian McCandless, et al. "Cellxgene: A Performant, Scalable Exploration Platform for High Dimensional Sparse Matrices" https://doi.org/10.1101/2021.04.05.438318 Preprint. Systems Biology, April 6, 2021.
  • Granatum - web-based scRNA-seq analysis. list of modules, including plate merging and batch-effect removal, outlier-sample removal, gene-expression normalization, imputation, gene filtering, cell clustering, differential gene expression analysis, pathway/ontology enrichment analysis, protein network interaction visualization, and pseudo-time cell series reconstruction. Twitter.
    Paper Zhu, Xun, Thomas K. Wolfgruber, Austin Tasato, Cédric Arisdakessian, David G. Garmire, and Lana X. Garmire. "Granatum: A Graphical Single-Cell RNA-Seq Analysis Pipeline for Genomics Scientists" https://doi.org/10.1186/s13073-017-0492-3 Genome Medicine 9, no. 1 (December 2017).

  • SCope - Fast visualization tool for large-scale and high dimensional single-cell data in .loom format. R and Python scripts for converting scRNA-seq data to .loom format.

  • singleCellTK - R/Shiny package for an interactive scRNA-Seq analysis. Input, raw counts in SingleCellExperiment. Analysis: filtering raw results, clustering, batch correction, differential expression, pathway enrichment, and scRNA-Seq study design.

  • scDataviz - single cell data vizualization and downstream analyses, by Kevin Blighe

  • scOrange - visual pipeline builder for an in-depth analysis and visualization of scRNA-seq data. Works with 10X data, tab-delimited. Filtering, preprocessiong, differential gene expression, marker analysis, enrichment analysis, batch removal, clustering, tSNE. Screenshots, Short video tutorials. Python-based, Conda-installable. GitHub

  • scCustomize - an R package, Collection of functions created and/or curated to aid in the visualization and analysis of single-cell data. Extends Seurat, Liger visualization, helper functions to enhance analysis of Seurat objects.

  • UCSC Single Cell Browser - Python pipeline and Javascript scatter plot library for single-cell datasets. Pre-process an expression matrix by filtering, PCA, nearest-neighbors, clustering, t-SNE and UMAP and formats them for cbBuild. Demo that includes several landmark datasets

Quality control

  • miQC - data-driven identification of cells with high mitochondrial content (likely, dead cells) from scRNA-seq data. Joint statistical model the proportion of reads mapping to mtDNA genes and the number of detected genes, EM for parameter estimation (flexmix). Tested on various datasets processed with CellRanged and salon alevin - improves removal of compromised cells as compared with hard threshold. Bioconductor R package, integrates with scater.
    Paper Hippen, Ariel A., Matias M. Falco, Lukas M. Weber, Erdogan Pekcan Erkan, Kaiyang Zhang, Jennifer Anne Doherty, Anna Vähärautio, Casey S. Greene, and Stephanie C. Hicks. "MiQC: An Adaptive Probabilistic Framework for Quality Control of Single-Cell RNA-Sequencing Data" https://doi.org/10.1371/journal.pcbi.1009290 PLOS Computational Biology, (August 24, 2021)
  • doubletD - doublet detection in single-cell DNA-seq data. doublets in scRNA-seq data have a characteristic variant allele frequency spectrum due to increased copy number and allelic dropout. A maximum likelihood approach with a closed-form solution - stats in Methods. Simulated and real data, outperforms SCG, Scrublet, robust to the presence of CNAs, mixture of two cell types. Python3 implementation.
    Paper Weber, Leah L, Palash Sashittal, and Mohammed El-Kebir. "DoubletD: Detecting Doublets in Single-Cell DNA Sequencing Data" https://doi.org/10.1093/bioinformatics/btab266 Bioinformatics, (August 4, 2021)
  • DropletUtils - Provides a number of utility functions for handling single-cell (RNA-seq) data from droplet technologies such as 10X Genomics. This includes data loading, identification of cells from empty droplets, removal of barcode-swapped pseudo-cells, and downsampling of the count matrix.
    Paper Lun ATL, Riesenfeld S, Andrews T, Dao T, Gomes T, participants in the 1st Human Cell Atlas Jamboree, Marioni JC (2019). "EmptyDrops: distinguishing cells from empty droplets in droplet-based single-cell RNA sequencing data" https://doi.org/10.1186/s13059-019-1662-y Genome Biol.
  • DoubletFinder - doublet detection using gene expression data. Simulates artificial doublets, incorporate them into existing scRNA-seq data. Integrates with Seurat (Figure 1). Three input parameters (the expected number of doublets, the number of artificial doublets pN, the neighborhood size pN), need to be tailored to data with different number of cell types and magnitudes of transcriptional heterogeneity. Bimodality Coefficient maximization to select pN. Benchmarked against ground-truth scRNA-seq datasets. Not optimal for homogeneous data.
    Paper -McGinnis, Christopher S., Lyndsay M. Murrow, and Zev J. Gartner. "DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors" https://doi.org/10.1016/j.cels.2019.03.003 Cell Systems 8, no. 4 (April 2019)
  • celloline - A pipeline to remove low quality single cell files. Figure 2 - 20 biological and technical features used for filtering. High mitochondrial genes = broken cells.
    Paper Ilicic, Tomislav, Jong Kyoung Kim, Aleksandra A. Kolodziejczyk, Frederik Otzen Bagger, Davis James McCarthy, John C. Marioni, and Sarah A. Teichmann. "Classification of Low Quality Cells from Single-Cell RNA-Seq Data" https://doi.org/10.1186/s13059-016-0888-1 Genome Biology 17 (February 17, 2016)

Normalization

  • SCnorm - normalization for single-cell data. Quantile regression to estimate the dependence of transcript expression on sequencing depth for every gene. Genes with similar dependence are then grouped, and a second quantile regression is used to estimate scale factors within each group. Within-group adjustment for sequencing depth is then performed using the estimated scale factors to provide normalized estimates of expression. Good statistical methods description.
    Paper Bacher, Rhonda, Li-Fang Chu, Ning Leng, Audrey P Gasch, James A Thomson, Ron M Stewart, Michael Newton, and Christina Kendziorski. "SCnorm: Robust Normalization of Single-Cell RNA-Seq Data" https://doi.org/10.1038/nmeth.4263 Nature Methods 14, no. 6 (April 17, 2017)

Integration, Batch correction

  • MOFA2 - Multi-Omics Factor Analysis v2 (MOFA+), a statistical framework for the integration of single-cell multi-modal data. Reconstructs a low-dimensional representation of the data using variational inference (a stochastic variant parallelizable on GPU, 20-fold speed increase). Supports sparsity constraints, allowing to jointly model variation across multiple sample groups and data modalities. Infers K latent factors with associated feature weight matrices (per data modality, Figure 1a) that can be used for clustering, trajectory inference, variance decomposition etc. Input - multiple datasets measuring non-overlapping modalities, cells grouped by experiments, batches, or conditions. Python and R implementation.
    Paper Argelaguet, Ricard, Damien Arnol, Danila Bredikhin, Yonatan Deloro, Britta Velten, John C. Marioni, and Oliver Stegle. “MOFA+: A Statistical Framework for Comprehensive Integration of Multi-Modal Single-Cell Data.” Genome Biology 21, no. 1 (December 2020): 111. https://doi.org/10.1186/s13059-020-02015-1.
  • CarDEC - Count adapted regularized Deep Embedded Clustering, a joint deep learning model that simultaneously clusters, denoises, corrects for multiple batch effects in gene expression space (Figure 1). Outperforms scVI, DCA, MNN, scDeepCluster. Separately treats highly and lowly variable genes. Improves integration of omics generated by multiple technologies, pseudotime reconstruction.
    Paper Lakkis, Justin, David Wang, Yuanchao Zhang, Gang Hu, Kui Wang, Huize Pan, Lyle Ungar, Muredach P. Reilly, Xiangjie Li, and Mingyao Li. “A Joint Deep Learning Model for Simultaneous Batch Effect Correction, Denoising and Clustering in Single-Cell Transcriptomics.” Preprint. Bioinformatics, September 25, 2020. https://doi.org/10.1101/2020.09.23.310003
  • iCellR - batch correction in scRNA-seq data. Combined Coverage Correction Alignment (CCCA) and Combined Principal Component Alignment (CPCA). CCCA - PCA into 30 dimensions, for each cell, take k=10 nearest neighbors, average gene expression, thus imputing the adjusted matrix. CPCA skips imputation, instead PCs themselves get averaged. Similar performance. Tested on nine PBMC datasets provided by the Broad institute to test batch effect. Outperforms MAGIC. Data in text and .rda formats.
    Paper Khodadadi-Jamayran, Alireza, Joseph Pucella, Hua Zhou, Nicole Doudican, John Carucci, Adriana Heguy, Boris Reizis, and Aristotelis Tsirigos. "ICellR: Combined Coverage Correction and Principal Component Alignment for Batch Alignment in Single-Cell Sequencing Analysis" https://doi.org/10.1101/2020.03.31.019109 Preprint. Bioinformatics, April 1, 2020
  • BERMUDA - Batch Effect ReMoval Using Deep Autoencoders, for scRNA-seq data. Requires batches to share at least one common cell type. Five step framework: 1) preprocessing, 2) clustering of cells in each batch individually, 3) identifying similar cell clusters across different batches, 4) removing batch effect by training an autoencoder, 5) further analysis of batch-corrected data. Tested on simulated (splatter) and experimental (10X genomics) data.
    Paper Wang, Tongxin, Travis S. Johnson, Wei Shao, Zixiao Lu, Bryan R. Helm, Jie Zhang, and Kun Huang. "BERMUDA: A Novel Deep Transfer Learning Method for Single-Cell RNA Sequencing Batch Correction Reveals Hidden High-Resolution Cellular Subtypes" https://doi.org/10.1186/s13059-019-1764-6 Genome Biology 20, no. 1 (December 2019).
  • BBKNN (batch balanced k nearest neighbours) - batch correction for scRNA-seq data. Neighborhood graphs, balanced across all batches of the data, separately for each batch, that are merged. Main assumption (as in mnnCorrect) - at least some cells of the same type exist across batches. Preserves data structure allowing subsequent embedding, trajectory reconstruction. Python, compatible with SCANPY, very fast.
    Paper Polański, Krzysztof, Matthew D Young, Zhichao Miao, Kerstin B Meyer, Sarah A Teichmann, and Jong-Eun Park. "BBKNN: Fast Batch Alignment of Single Cell Transcriptomes" https://doi.org/10.1093/bioinformatics/btz625 Bioinformatics, August 10, 2019
  • LIGER - R package for integrating and analyzing multiple single-cell datasets, across conditions, technologies (scRNA-seq and methylation), or species (human and mouse). Integrative nonnegative matrix factorization (W and H matrices), dataset-specific and shared patterns (metagenes, matrix H). Graphs of factor loadings onto these patterns (shared factor neighborhood graph), then comparing patterns. Alignment and agreement metrics to assess performance, LIGER outperforms Seurat on agreement. Analysis of published blood cells, brain. Human/mouse brain data.
    Paper Welch, Joshua D., Velina Kozareva, Ashley Ferreira, Charles Vanderburg, Carly Martin, and Evan Z. Macosko. "Single-Cell Multi-Omic Integration Compares and Contrasts Features of Brain Cell Identity" https://doi.org/10.1016/j.cell.2019.05.006 Cell 177, no. 7 (June 13, 2019)
  • scMerge - R package for batch effect removal and normalizing of multipe scRNA-seq datasets. fastRUVIII batch removal method. Tested on 14 datasets, compared with scran, MNN, ComBat, Seurat, ZINB-WaVE using Silhouette, ARI - better separation of clusters, pseudotime reconstruction.
    Paper Lin, Yingxin, Shila Ghazanfar, Kevin Wang, Johann A. Gagnon-Bartsch, Kitty K. Lo, Xianbin Su, Ze-Guang Han, et al. "ScMerge: Integration of Multiple Single-Cell Transcriptomics Datasets Leveraging Stable Expression and Pseudo-Replication" https://doi.org/10.1101/393280 September 12, 2018.
  • MNN - mutual nearest neighbors method for single-cell batch correction. Assumptions: MNN exist between batches, batch is orthogonal to the biology. Cosine normalization, Euclidean distance, a pair-specific barch-correction vector as a vector difference between the expression profiles of the paired cells using selected genes of interest and hypervariable genes. Supplementary note 5 - algorithm. mnnCorrect function in the scran package. Code for paper.
    Paper Haghverdi, Laleh, Aaron T L Lun, Michael D Morgan, and John C Marioni. "Batch Effects in Single-Cell RNA-Sequencing Data Are Corrected by Matching Mutual Nearest Neighbors" https://doi.org/10.1038/nbt.4091 Nature Biotechnology, April 2, 2018.

  • scLVM - a modelling framework for single-cell RNA-seq data that can be used to dissect the observed heterogeneity into different sources and remove the variation explained by latent variables. Can correct for the cell cycle effect. Applied to naive T cells differentiating into TH2 cells.

    Paper Buettner, Florian, Kedar N Natarajan, F Paolo Casale, Valentina Proserpio, Antonio Scialdone, Fabian J Theis, Sarah A Teichmann, John C Marioni, and Oliver Stegle. "Computational Analysis of Cell-to-Cell Heterogeneity in Single-Cell RNA-Sequencing Data Reveals Hidden Subpopulations of Cells" https://doi.org/10.1038/nbt.3102 Nature Biotechnology 33, no. 2 (March 2015)

    Buettner, Florian, Naruemon Pratanwanich, Davis J. McCarthy, John C. Marioni, and Oliver Stegle. "F-ScLVM: Scalable and Versatile Factor Analysis for Single-Cell RNA-Seq" https://doi.org/10.1186/s13059-017-1334-8 Genome Biology 18, no. 1 (December 2017) - f-scLVM - factorial single-cell latent variable model guided by pathway annotations to infer interpretable factors behind heterogeneity. PCA components are annotated by correlated genes and their enrichment in pathways. Docomposition of the original gene expression matrix to a sum of annotated, unannotated, and confounding components. Applied to their own naive T to TH2 cells, mESCs, reanalyzed 3005 neuronal cells. Simulated data. https://github.com/bioFAM/slalom

Imputation

Assessment of 18 scRNA-seq imputation methods (model-based, smooth-based, deep learning, matrix decomposition). Similarity of scRNA- and bulk RNA-seq profiles (Spearman), differential expression (MAST and Wilcoxon), clustering (k-means, Louvain), trajectory reconstruction (Monocle 2, TSCAN), didn't test velocity. scran for normalization. Imputation methods improve correlation with bulk RNA-seq, but have minimal effect on downstream analyses. MAGIC, kNN-smoothing, SAVER perform well overall. Plate- and droplet-derived scRNA-seq cell line data, Additional File 4), Summary table of the functionality of all imputation methods, Additional File 5.

Paper Hou, Wenpin, Zhicheng Ji, Hongkai Ji, and Stephanie C. Hicks. "A Systematic Evaluation of Single-Cell RNA-Sequencing Imputation Methods" https://doi.org/10.1186/s13059-020-02132-x Genome Biology 21, no. 1 (December 2020)

  • Deepimpute - scRNA-seq imputation using deep neural networks. Sub-networks, each processes up to 512 genes needed to be imputed. Four layers: Input - dense (ReLU activation) - 20% dropout - output. MSE as loss function. Outperforms MAGIC, DrImpute, ScImpute, SAVER, VIPER, and DCA on multiple metrics (PCC, several clustering metrics). Using 9 datasets.
    Paper Arisdakessian, Cédric, Olivier Poirion, Breck Yunits, Xun Zhu, and Lana X. Garmire. "DeepImpute: An Accurate, Fast, and Scalable Deep Neural Network Method to Impute Single-Cell RNA-Seq Data" https://doi.org/10.1186/s13059-019-1837-6 Genome Biology 20, no. 1 (December 2019)
  • ENHANCE, an algorithm that denoises single-cell RNA-Seq data by first performing nearest-neighbor aggregation and then inferring expression levels from principal components. Variance-stabilizing normalization of the data before PCA. Implements its own simulation procedure for simulating sampling noise. Outperforms MAGIC, SAVER, ALRA. Python, and R implementations.
    Paper Wagner, Florian, Dalia Barkley, and Itai Yanai. "ENHANCE: Accurate Denoising of Single-Cell RNA-Seq Data" https://doi.org/10.1101/655365 Preprint. Bioinformatics, June 3, 2019.
  • scHinter - imputation for small-size scRNA-seq datasets. Three modules: voting-based ensemble distance for learning cell-cell similarity, a SMOTE-based random interpolation module for imputing dropout events, and a hierarchical model for multi-layer random interpolation. RNA-seq blog.
    Paper Ye, Pengchao, Wenbin Ye, Congting Ye, Shuchao Li, Lishan Ye, Guoli Ji, and Xiaohui Wu. "ScHinter: Imputing Dropout Events for Single-Cell RNA-Seq Data with Limited Sample Size" https://doi.org/10.1093/bioinformatics/btz627 Bioinformatics, August 8, 2019.
  • netNMF-sc - scRNA-seq nonnegative matrix factorization for imputation and dimensionality reduction for improved clustering. Uses gene-gene interaction network to constrain W gene matrix on prior knowledge (graph regularized NMF). Added penalization for dropouts. Tested on simulated and experimental data, compared with several imputation and clustering methods.
    Paper Elyanow, Rebecca, Bianca Dumitrascu, Barbara E Engelhardt, and Benjamin J Raphael. "NetNMF-Sc: Leveraging Gene-Gene Interactions for Imputation and Dimensionality Reduction in Single-Cell Expression Analysis" https://doi.org/10.1101/544346 BioRxiv, February 8, 2019.
  • scRMD - dropout imputation in scRNA-seq via robust matrix decomposition into true expression matrix (further decomposed into a matrix of means and gene's random deviation from its mean) minus dropout matrix plus error matrix. A function to estimate the matrix of means and dropouts. Comparison with MAGIC, scImpute.
    Paper Chen, Chong, Changjing Wu, Linjie Wu, Yishu Wang, Minghua Deng, and Ruibin Xi. "ScRMD: Imputation for Single Cell RNA-Seq Data via Robust Matrix Decomposition" https://doi.org/10.1101/459404 November 4, 2018
  • netSmooth - network diffusion-based method that uses priors for the covariance structure of gene expression profiles to smooth scRNA-seq experiments. Incorporates prior knowledge (i.e. protein-protein interaction networks) for imputation. Note that dropout applies to whole transcriptome. Compared with MAGIC, scImpute. Improves clustering, biological interpretation.
    Paper Ronen, Jonathan, and Altuna Akalin. "NetSmooth: Network-Smoothing Based Imputation for Single Cell RNA-Seq" https://doi.org/10.12688/f1000research.13511.3 F1000Research 7 (July 10, 2018)
  • DCA - A deep count autoencoder network to denoise scRNA-seq data. Zero-inflated negative binomial model. Current approaches - scimpute, MAGIC, SAVER. Benchmarking by increased correlation between bulk and scRNA-seq data, between protein and RNA levels, between key regulatory genes, better DE concordance in bulk and scRNA-seq, improved clustering.
    Paper Eraslan, Gökcen, Lukas M. Simon, Maria Mircea, Nikola S. Mueller, and Fabian J. Theis. "Single Cell RNA-Seq Denoising Using a Deep Count Autoencoder" https://doi.org/10.1101/300681 April 13, 2018.
  • kNN-smoothing of scRNA-seq data, aggregates information from similar cells, improves signal-to-noise ratio. Based on observation that gene expression in technical replicates are Poisson distributed. Freeman-Tukey transform to minimize variability of low expressed genes. Tested using real and simulated data. Improves clustering, PCA, Selection of k is critical, discussed.
    Paper Wagner, Florian, Yun Yan, and Itai Yanai. "K-Nearest Neighbor Smoothing for High-Throughput Single-Cell RNA-Seq Data" https://doi.org/10.1101/217737 BioRxiv, April 9, 2018.

  • scimpute - imputation of scRNA-seq data. Methodology: 1) Determine K subpopulations using PCA, remove outliers; 2) Mixture model of gene i in subpopulation k as gamma and normal distributions, estimate dropout probability d; 3) Impute dropout values by splitting the subpopulation into A (dropout larger than threshold t) and B (smaller). Information from B is used to impute A. Better than MAGIC, SAVER.

    Paper Li, Wei Vivian, and Jingyi Jessica Li. "An Accurate and Robust Imputation Method ScImpute for Single-Cell RNA-Seq Data" https://doi.org/10.1038/s41467-018-03405-7 Nature Communications 9, no. 1 (08 2018)

  • LATE - Learning with AuToEncoder to imputescRNA-seq data. TRANSLATE (TRANSfer learning with LATE) uses reference (sc)RNA-seq dataset to learn initial parameter estimates. TensorFlow implementation for GPU and CPU. ReLu as an activation function. Various optimization techniques. Comparison with MAGIC, scVI, DCA, SAVER. Links to data.

    Paper Badsha, Md. Bahadur, Rui Li, Boxiang Liu, Yang I. Li, Min Xian, Nicholas E. Banovich, and Audrey Qiuyan Fu. "Imputation of Single-Cell Gene Expression with an Autoencoder Neural Network" https://doi.org/10.1101/504977 BioRxiv, January 1, 2018
  • MAGIC - Markov Affinity-based Graph Imputation of Cells. Only ~5-15% of scRNA-seq data is non-zero, the rest are drop-outs. Use the diffusion operator to discover the manifold structure and impute gene expression. Detailed methods description. In real (bone marrow and retinal bipolar cells) and synthetic datasets, Imputed scRNA-seq data clustered better, enhances gene interactions, restores expression of known surface markers, trajectories. scRNA-seq data is preprocessed by library size normalization and PCA (to retain 70% of variability). Comparison with SVD-based low-rank data approximation (LDA) and Nuclear-Norm-based Matrix Completion (NNMC). GitHub.
    Paper Van Dijk, David, Roshan Sharma, Juozas Nainys, Kristina Yim, Pooja Kathail, Ambrose J. Carr, Cassandra Burdziak et al. "Recovering gene interactions from single-cell data using data diffusion." Cell 174, no. 3 (2018): 716-729. https://doi.org/10.1016/j.cell.2018.05.061

Dimensionality reduction

  • MultiMAP dimensionality reduction algorithm. Works with dataset-specific features (does not require features to be shared across datasets, e.g., 20K-gene scRNA-seq and 100K-peak scATAC-seq datasets). Generalizes the UMAP algorithm to data with different dimensions, constructs a nonlinear manifold, constructs a joint graph on the manifold (MultiGraph), cross-entropy minimization to optimize the low-dimensional embedding of the manifold and data. Allows to specify the influence of each dataset on the embedding. Tested on synthetic and experimental data, including spatial transcriptomics datasets, outperforms Seurat, LIGER, iNMF, Conos, GLUER, significantly faster and scalable.
    Paper Jain, M.S., Polanski, K., Conde, C.D. et al. MultiMAP: dimensionality reduction and integration of multimodal data. Genome Biol 22, 346 (2021). https://doi.org/10.1186/s13059-021-02565-y
  • Poincare maps for two-dimensional scRNA-seq data representation. Preserves local and global distances, hierarchy, the center of the Poincare disk can be considered as a root node. Three-step procedure: 1) k-nearest-neighbor graph, 2) global geodesic distances from the kNN graph, 3) two-dimensional embeddings in the Poincare disk with hyperbolic distances preserve the inferred geodesic distances. Compared with t-SNE, UMAP, PCA, Monocle 2, SAUCIE and several other visualization and lineage detection methods. Two metrics to compare embeddings, Qlocal and Qglobal. References to several public datasets used for reanalysis.
    Paper Klimovskaia, Anna, David Lopez-Paz, Léon Bottou, and Maximilian Nickel. "Poincaré Maps for Analyzing Complex Hierarchies in Single-Cell Data" https://doi.org/10.1038/s41467-020-16822-4 Nature Communications 11, no. 1 (December 2020)
  • scHPF - single-cell hierarchical Poisson Factorization for discovering patterns of gene expressions and cells. A Bayesian factorization method, does not require normalization, explicitly models sparsity across cells and genes. Compared with PCA, NMF, FA, ZIFA, ZINB-WaVE on three datasets, it better captures statistical and biological properties of scRNA-seq data. Python implementation.
    Paper Levitin, Hanna Mendes, Jinzhou Yuan, Yim Ling Cheng, Francisco JR Ruiz, Erin C Bush, Jeffrey N Bruce, Peter Canoll, et al. "De Novo Gene Signature Identification from Single‐cell RNA‐seq with Hierarchical Poisson Factorization" https://doi.org/10.15252/msb.2018855 Molecular Systems Biology 15, no. 2 (February 2019)
  • SAUCIE - deep neural network with regularization on layers to improve interpretability. Denoising, batch removal, imputation, visualization of low-dimensional representation. Extensive comparison on simulated and real data.
    Paper Amodio, Matthew, David van Dijk, Krishnan Srinivasan, William S Chen, Hussein Mohsen, Kevin R Moon, Allison Campbell, et al. "Exploring Single-Cell Data with Deep Multitasking Neural Networks" https://doi.org/10.1101/237065 August 27, 2018.
  • CIDR - Clustering through Imputation and Dimensionality Reduction. Impute dropouts. Explicitly deconvolve Euclidean distance into distance driven by complete, partially complete, and dropout pairs. Principal Coordinate Analysis.
    Paper Lin, Peijie, Michael Troup, and Joshua W. K. Ho. "CIDR: Ultrafast and Accurate Clustering through Imputation for Single-Cell RNA-Seq Data" https://doi.org/10.1186/s13059-017-1188-0 Genome Biology 18, no. 1 (December 2017).
  • VASC - deep variational autoencoder for scRNA-seq data for dimensionality reduction and visualization. Tested on twenty datasets vs PCA, tSNE, ZIFA, and SIMLR. Four metrics to assess clustering performance: NMI (normalized mutual information score), ARI (adjusted rand index), HOM (homogeneity) and COM (completeness). No filtering, only log transformation. Keras implementation. Datasets.
    Paper Wang, Dongfang, and Jin Gu. "VASC: Dimension Reduction and Visualization of Single Cell RNA Sequencing Data by Deep Variational Autoencoder" https://doi.org/10.1101/199315 October 6, 2017.
  • ZINB-WAVE - Zero-inflated negative binomial model for normalization, batch removal, and dimensionality reduction. Extends the RUV model with more careful definition of "unwanted" variation as it may be biological. Good statistical derivations in Methods. Refs to real and simulated scRNA-seq datasets.
    Paper Risso, Davide, Fanny Perraudeau, Svetlana Gribkova, Sandrine Dudoit, and Jean-Philippe Vert. "ZINB-WaVE: A General and Flexible Method for Signal Extraction from Single-Cell RNA-Seq Data" https://doi.org/10.1101/125112 BioRxiv, January 1, 2017.
  • RobustAutoencoder - Autoencoder and robust PCA for gene expression representation, robust to outliers. Main idea - split the input data X into two parts, L (reconstructed data) and S (outliers and noise). Grouped "l2,1" norm - an l2 regularizer within a group and then an l1 regularizer between groups. Iterative procedure to obtain L and S. TensorFlow implementation.
    Paper Zhou, Chong, and Randy C. Paffenroth. "Anomaly Detection with Robust Deep Autoencoders" https://doi.org/10.1145/3097983.3098052 In Proceedings of the 23rd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining - KDD ’17, 665–74. Halifax, NS, Canada: ACM Press, 2017.
  • ZIFA - Zero-inflated dimensionality reduction algorithm for single-cell data. Single-cell dimensionality reduction. Model dropout rate as double exponential, give less weights to these counts. EM algorithm that incorporates imputation step for the expected gene expression level of drop-outs.
    Paper Pierson, Emma, and Christopher Yau. "ZIFA: Dimensionality Reduction for Zero-Inflated Single-Cell Gene Expression Analysis" https://doi.org/10.1186/s13059-015-0805-z Genome Biology 16 (November 2, 2015)

Clustering

  • BAMM-SC - scRNA-seq clustering. A Bayesian hierarchical Dirichlet multinomial mixture model, accounts for batch effect, operates on raw counts. Outperforms K-means, TSCAN, Seurat corrected for batch using MNN or CCA in simulated and experimental settings.
    Paper Sun, Zhe, Li Chen, Hongyi Xin, Yale Jiang, Qianhui Huang, Anthony R. Cillo, Tracy Tabib, et al. "A Bayesian Mixture Model for Clustering Droplet-Based Single-Cell Transcriptomic Data from Population Studies" https://doi.org/10.1038/s41467-019-09639-3 Nature Communications 10, no. 1 (December 2019)
  • Spectrum - a spectral clustering method for single- or multi-omics datasets. Self-tuning kernel that adapts to local density of the graph. Tensor product graph data integration method. Implementation of fast spectral clustering method (single dataset only). Finds optimal number of clusters using eigenvector distribution analysis. References to previous methods. Excellent methods description. Compared with M3C, CLEST, PINSplus, SNF, iClusterPlus, CIMLR, MUDAN. GitHub.
    Paper John, Christopher R., David Watson, Michael R. Barnes, Costantino Pitzalis, and Myles J. Lewis. "Spectrum: Fast Density-Aware Spectral Clustering for Single and Multi-Omic Data" https://doi.org/10.1093/bioinformatics/btz704 Bioinformatics (Oxford, England), September 10, 2019
  • PanoView - scRNA-seq iterative clustering in an evolving 3D PCA space, Ordering Local Maximum by Convex hull (OLMC) to identify clusters of varying density. PCA on most variable genes, finding most optimal largest cluster within first 3 PCs, repeat PCA for the remaining cells etc. Tested on multiple simulated and experimental scRNA-seq datasets, compared with 9 methods, the Adjusted Rand Index as performance metric.
    Paper Hu, Ming-Wen, Dong Won Kim, Sheng Liu, Donald J. Zack, Seth Blackshaw, and Jiang Qian. "PanoView: An Iterative Clustering Method for Single-Cell RNA Sequencing Data" https://doi.org/10.1371/journal.pcbi.1007040 Edited by Qing Nie. PLOS Computational Biology 15, no. 8 (August 30, 2019)
  • TooManyCells - divisive hierarchical spectral clustering of scRNA-seq data. Uses truncated singular vector decomposition to bipartition the cells. Newman-Girvain modularity Q to assess whether bipartition is significant or should be stopped. BirchBeer visualization. Outperforms Phenograph, Seurat, Cellranger, Monocle, the latter is second in performance. Excels for rare populations. Normalization marginally affects performance.
    Paper Schwartz, Gregory W, Jelena Petrovic, Maria Fasolino, Yeqiao Zhou, Stanley Cai, Lanwei Xu, Warren S Pear, Golnaz Vahedi, and Robert B Faryabi. "TooManyCells Identifies and Visualizes Relationships of Single-Cell Clades" https://doi.org/10.1101/519660 BioRxiv, January 13, 2019.
  • scClustViz - assessment of scRNA-seq clustering using differential expression (Wilcoxon test) as a guide. Testing for two differences: difference in detection rate (dDR) and log2 gene expression ratio (logGER). Two hypothesis testing: one cluster vs. all, each cluster vs. another cluster. accepts SincleCellExperiment and Seurat objects (log2-transformed data), needs a data frame with different cluster assignments. Analysis within R, save as RData, visualize results in R Shiny app.
    Paper Innes, BT, and GD Bader. "ScClustViz - Single-Cell RNAseq Cluster Assessment and Visualization" https://doi.org/10.12688/f1000research.16198.2 F1000Research 7, no. 1522 (2019).
  • SHARP - an ensemble random projection (RP)-based algorithm. Scalable, allows for clustering of 1.3 million cells (splitting the matrix into blocks, RP on each, then weighted ensemble clustering. Outperforms SC3, SIMLR, hierarchical clustering, tSNE + k-means. Tested on 17 public datasets. Robust to dropouts. Compatible with (UMI-based) counts (per million), FPKM/RPKM, TPM. Methods detailing four algorithm steps (data partition, RP, weighted ensemble clustering, similarity-based meta-clustering).
    Paper Wan, Shibiao, Junil Kim, and Kyoung Jae Won. "SHARP: Single-Cell RNA-Seq Hyper-Fast and Accurate Processing via Ensemble Random Projection" https://doi.org/10.1101/461640 Preprint. Bioinformatics, November 4, 2018
  • Performance evaluation of 14 scRNA-seq clustering algorithms using nine experimental and three simulated datasets. SC3 and Seurat perform best overall. Normalized Shannon entropy, adjusted Rand index for performance evaluation. Ensemble clustering doesn't help. R scripts and a data package for clustering benchmarking with preprocessed and experimental scRNA-seq datasets.
    Paper Duò, Angelo, Mark D. Robinson, and Charlotte Soneson. "A Systematic Performance Evaluation of Clustering Methods for Single-Cell RNA-Seq Data" https://doi.org/10.12688/f1000research.15666.2 F1000Research 7 (September 10, 2018)
  • clusterExperiment R package for scRNA-seq data visualization. Resampling-based Sequential Ensemble Clustering (RSEC) method. clusterMany - makeConsensus - makeDendrogram - mergeClusters pipeline. Biomarker detection by differential expression analysis between clusters.
    Paper Risso, Davide, Liam Purvis, Russell B. Fletcher, Diya Das, John Ngai, Sandrine Dudoit, and Elizabeth Purdom. "ClusterExperiment and RSEC: A Bioconductor Package and Framework for Clustering of Single-Cell and Other Large Gene Expression Datasets" https://doi.org/10.1371/journal.pcbi.1006378 Edited by Aaron E. Darling. PLOS Computational Biology 14, no. 9 (September 4, 2018)
  • PHATE (Potential of Heat-diffusion for Affinity-based Transition Embedding) - low-dimensional embedding, denoising, and visualization, applicable to scRNA-seq, microbiome, SNP, Hi-C (as affinity matrices) and other data. Preserves biological structures and branching better than PCA, tSNE, diffusion maps. Robust to noise and subsampling. Detailed methods description and graphical representation of the algorithm. Tweetorial.
    Paper Moon, Kevin R., David van Dijk, Zheng Wang, Scott Gigante, Daniel Burkhardt, William Chen, Antonia van den Elzen, et al. "Visualizing Transitions and Structure for Biological Data Exploration" https://doi.org/10.1101/120378 June 28, 2018.
  • scVAE - Variational auroencoder frameworks for modelling raw RNA-seq counts, denoising the data to improve biologically plausible grouping in scRNA-seq data. Improvement in Rand index.
    Paper Grønbech, Christopher Heje, Maximillian Fornitz Vording, Pascal N Timshel, Casper Kaae Sønderby, Tune Hannes Pers, and Ole Winther. "ScVAE: Variational Auto-Encoders for Single-Cell Gene Expression Data" https://doi.org/10.1101/318295 May 16, 2018.
  • Conos - clustering of scRNA-seq samples by joint graph construction. Seurat or pagoda2 for data preprocessing, selection of hypervariable genes, initial clustering (KNN, or dimensionality reduction), then joint clustering. R package.
    Paper Barkas, Nikolas, Viktor Petukhov, Daria Nikolaeva, Yaroslav Lozinsky, Samuel Demharter, Konstantin Khodosevich, and Peter V Kharchenko. “Wiring Together Large Single-Cell RNA-Seq Sample Collections.” BioRxiv, January 1, 2018.
  • MetaCell - partitioning scRNA-seq data into metacells - disjoint and homogeneous/compact groups of cells exhibiting only sampling variance. Most variable genes to cell-to-cell similarity matrix (PCC on to Knn similarity graph that is partitioned by bootstrapping to obtain subgraphs. Tested on several 10X datasets.
    Paper Baran, Yael, Arnau Sebe-Pedros, Yaniv Lubling, Amir Giladi, Elad Chomsky, Zohar Meir, Michael Hoichman, Aviezer Lifshitz, and Amos Tanay. "MetaCell: Analysis of Single Cell RNA-Seq Data Using k-NN Graph Partitions" https://doi.org/10.1101/437665 BioRxiv, January 1, 2018
  • SIMLR - scRNA-seq dimensionality reduction, clustering, and visualization based on multiple kernel-learned distance metric. Comparison with PCA, t-SNE, ZIFA. Seven datasets. R and Matlab implementation.
    Paper Wang, Bo, Junjie Zhu, Emma Pierson, Daniele Ramazzotti, and Serafim Batzoglou. "Visualization and Analysis of Single-Cell RNA-Seq Data by Kernel-Based Similarity Learning" https://doi.org/10.1101/460246 Nature Methods 14, no. 4 (April 2017)
  • SC3 - single-cell clustering. Multiple clustering iterations, consensus matrix, then hierarhical clustering. Benchmarking against other methods.
    Paper Kiselev, Vladimir Yu, Kristina Kirschner, Michael T Schaub, Tallulah Andrews, Andrew Yiu, Tamir Chandra, Kedar N Natarajan, et al. "SC3: Consensus Clustering of Single-Cell RNA-Seq Data" https://doi.org/10.1038/nmeth.4236 Nature Methods 14, no. 5 (March 27, 2017)
  • PhenoGraph - discovers subpopulations in scRNA-seq data. High-dimensional space is modeled as a nearest-neighbor graph, then the Louvain community detection algorithm. No assumptions about the size, number, or form of subpopulations.
    Paper Levine, Jacob H., Erin F. Simonds, Sean C. Bendall, Kara L. Davis, El-ad D. Amir, Michelle D. Tadmor, Oren Litvin, et al. "Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells That Correlate with Prognosis" https://doi.org/10.1016/j.cell.2015.05.047 Cell 162, no. 1 (July 2015)
  • SNN-Cliq - shared nearest neighbor clustering of scRNA-seq data, represented as a graph. Similarity between two data points based on the ranking of their shared neighborhood. Automatically determine the number of clusters, accomodates different densities and shapes. Compared with K-means and DBSCAN using Purity, Adjusted Rand Indes, F1-score. Matlab, Python, R implementation.
    Paper Xu, Chen, and Zhengchang Su. "Identification of Cell Types from Single-Cell Transcriptomes Using a Novel Clustering Method" https://doi.org/10.1093/bioinformatics/btv088 Bioinformatics (Oxford, England) 31, no. 12 (June 15, 2015)
  • celda - CEllular Latent Dirichlet Allocation. Simultaneous clustering of cells into subpopulations and genes into transcriptional states. Tutorials. No preprint yet.

Spatial inference

  • brainmapr - R package to infer spatial location of neuronal subpopulations within the developing mouse brain by integrating single-cell RNA-seq data with in situ RNA patterns from the Allen Developing Mouse Brain Atlas.

  • Seurat - single-cell RNA-seq for spatial cellular localization, using in situ RNA patterns as a reference. Impute landmark genes, relate them to the reference map. Tutorial, and Dave Tang notes.

    Paper Satija, Rahul, Jeffrey A. Farrell, David Gennert, Alexander F. Schier, and Aviv Regev. "Spatial Reconstruction of Single-Cell Gene Expression Data" https://doi.org/10.1038/nbt.3192 Nature Biotechnology 33, no. 5 (May 2015)

Time, trajectory inference

  • CellRank - single-cell fate mapping combining trajectory inference and RNA velocity directionality (scVelo), accounting for the stochastic nature of fate decisions and uncertainty in velocity vectors. Velocity alone is insufficient. Detects the initial, terminal and intermediate cell states and computes a global map of fate potentials. State transitions are modeled using a Markov chain. Stability index to automatically identify terminal states. Outperforms Palantir, STEMNET and FateID in diverse scenarious (development, regeneration, reprogramming, disease), fast, less memory, scalable. Input - (imputed) gene count matrix and velocity matrix (any vector field). Python, installable in Conda environment, Jupyter notebooks. Tutorial, Code to reproduce the results. Tweet by Dana Pe'er.
    Paper Lange, Marius, Volker Bergen, Michal Klein, Manu Setty, Bernhard Reuter, Mostafa Bakhti, Heiko Lickert, et al. “CellRank for Directed Single-Cell Fate Mapping.” Nature Methods, January 13, 2022. https://doi.org/10.1038/s41592-021-01346-6
  • Tempora - pseudotime reconstruction for scRNA-seq data, considering time series information. Matrix of scRNA-seq gene expression to clusters to pathways (single-cell enrichment), to graph (ARACNE), incorporate time scores, refine cell types. Outperforms Monocle2, TSCAN.
    Paper Tran, Thinh N., and Gary D. Bader. "Tempora: Cell Trajectory Inference Using Time-Series Single-Cell RNA Sequencing Data" https://doi.org/10.1101/846907 Preprint. Bioinformatics, November 18, 2019.
  • Slingshot - Inferring multiple developmental lineages from single-cell gene expression. Clustering by gene expression, then inferring cell lineage as an ordered set of clusters -minimum spanning tree through the clusters using Mahalanobis distance. Initial state and terminal state specification. Principal curves to draw a path through the gene expression space of each lineage.
    Paper Street, Kelly, Davide Risso, Russell B Fletcher, Diya Das, John Ngai, Nir Yosef, Elizabeth Purdom, and Sandrine Dudoit. "Slingshot: Cell Lineage and Pseudotime Inference for Single-Cell Transcriptomics" https://doi.org/10.1186/s12864-018-4772-0 BioRxiv, January 1, 2017.
  • SCITE - a stochastic search algorithm to identify the evolutionary history of a tumor from mutation patterns in scRNA-seq data. MCMC to compute the maximum-likelihood mutation history. Accounts for noise and dropouts. Input - Boolean mutation matrix, output - maximum-likelihood-inferred mutation tree. Compared with Kim & Simon approach, BitPhylogeny.
    Paper Jahn, Katharina, Jack Kuipers, and Niko Beerenwinkel. "Tree Inference for Single-Cell Data" https://doi.org/10.1186/s13059-016-0936-x Genome Biology 17, no. 1 (December 2016)
  • DPT - diffusion pseudotime, arrange cells in the pseudotemporal order. Random-walk-based distance that is computed based on Euclidean distance in the diffusion map space. Weighted nearest neighborhood of the data, probabilities of transitioning to each other cell using random walk, DTP is the euclidean distance between the two vectors, stored in a transition matrix. Robust to noise and sparsity. Method compared with Monocle, Wishbone, Wanderlust.
    Paper Haghverdi, Laleh, Maren Büttner, F. Alexander Wolf, Florian Buettner, and Fabian J. Theis. "Diffusion Pseudotime Robustly Reconstructs Lineage Branching" https://doi.org/10.1038/nmeth.3971 Nature Methods 13, no. 10 (2016)
  • TSCAN - pseudo-time reconstruction for scRNA-seq. Clustering first, then minimum spanning tree over cluster centers. Cells are projected to the tree (PCA) to determine their pseudo-time and order. R package that includes GUI.
    Paper Ji, Zhicheng, and Hongkai Ji. "TSCAN: Pseudo-Time Reconstruction and Evaluation in Single-Cell RNA-Seq Analysis" https://doi.org/10.1093/nar/gkw430 Nucleic Acids Research 44, no. 13 (27 2016)
  • Wishbone - ordering scRNA-seq along bifurcating developmental trajectories. nearest-heighbor graphs to capture developmental distances using shortest paths. Solves short-circuits by low-dimensional projection using diffusion maps. Waypoints as guides for building the trajectory. Detailed and comprehensive Methods description. Supersedes Wanderlust. Comparison with SCUBA, Monocle.
    Paper Setty, Manu, Michelle D. Tadmor, Shlomit Reich-Zeliger, Omer Angel, Tomer Meir Salame, Pooja Kathail, Kristy Choi, Sean Bendall, Nir Friedman, and Dana Pe’er. "Wishbone Identifies Bifurcating Developmental Trajectories from Single-Cell Data" https://doi.org/10.1038/nbt.3569 Nature Biotechnology 34, no. 6 (2016)
  • cellTree - hierarchical tree inference and visualization. Latent Dirichlet Allocation (LDA). Cells are analogous to text documents, discretized gene expression levels replace word frequencies. The LDA model represents topic distribution for each cell, analogous to low-dimensional embedding of the data where similarity is measured with chi-square distance. Fast and precise.
    Paper duVerle, David A., Sohiya Yotsukura, Seitaro Nomura, Hiroyuki Aburatani, and Koji Tsuda. "CellTree: An R/Bioconductor Package to Infer the Hierarchical Structure of Cell Populations from Single-Cell RNA-Seq Data" https://doi.org/10.1186/s12859-016-1175-6 BMC Bioinformatics 17, no. 1 (December 2016).
  • Monocle - Temporal ordering of single cell gene expression profiles. Independent Component Analysis to reduce dimensionality, Minimum Spanning Tree on the reduced representation and the longest path through it.
    Paper Trapnell, Cole, Davide Cacchiarelli, Jonna Grimsby, Prapti Pokharel, Shuqiang Li, Michael Morse, Niall J. Lennon, Kenneth J. Livak, Tarjei S. Mikkelsen, and John L. Rinn. "The Dynamics and Regulators of Cell Fate Decisions Are Revealed by Pseudotemporal Ordering of Single Cells" https://doi.org/10.1038/nbt.2859 Nature Biotechnology 32, no. 4 (April 2014)
  • SCUBA - single-cell clustering using bifurcation analysis. Cells may differentiate in a monolineage manner or may differentiate into multiple cell lineages, which is the bifurcation event - two new lineages. Methods. Matlab code.
    Paper Marco, Eugenio, Robert L. Karp, Guoji Guo, Paul Robson, Adam H. Hart, Lorenzo Trippa, and Guo-Cheng Yuan. "Bifurcation Analysis of Single-Cell Gene Expression Data Reveals Epigenetic Landscape" https://doi.org/10.1073/pnas.1408993111 Proceedings of the National Academy of Sciences of the United States of America 111, no. 52 (December 30, 2014)

Networks

  • Benchmarking of 11 scRNA-seq network inference methods. Top performers (PEARSON, PIDC, MERLIN, SCENIC), middle (Inferelator, SCODE, LEAP, Scribe) and bottom (knnDREMI, SILGGM). Simple correlation works well. Imputation did not benefit network inference, Human, mouse, yeast data, using scRNA-seq and bulk data (minimal performance differences). Brief description of methods, gold standard, evaluation metrics.
    Paper Stone, Matthew, Sunnie Grace McCalla, Alireza Fotuhi Siahpirani, Viswesh Periyasamy, Junha Shin, and Sushmita Roy. "Identifying Strengths and Weaknesses of Methods for Computational Network Inference from Single Cell RNA-Seq Data" https://doi.org/10.1101/2021.06.01.446671 Preprint. Bioinformatics, June 2, 2021.
  • PAGA - graph-like representation of scRNA-seq data. The kNN graph is partitioned using Louvain community detection algorithm, discarding spurious edged (denoising). Much faster than UMAP. Part of Scanpy pipeline.
    Paper Wolf, F. Alexander, Fiona K. Hamey, Mireya Plass, Jordi Solana, Joakim S. Dahlin, Berthold Göttgens, Nikolaus Rajewsky, Lukas Simon, and Fabian J. Theis. "PAGA: Graph Abstraction Reconciles Clustering with Trajectory Inference through a Topology Preserving Map of Single Cells" https://doi.org/10.1186/s13059-019-1663-x Genome Biology 20, no. 1 (March 19, 2019)
  • SCIRA - infer tissue-specific regulatory networks using large-scale bulk RNA-seq, estimate regulatory activity. SEPIRA uses a greedy partial correlation framework to infer a regulatory network from GTeX data, TF-specific regulons used as target profiles in a linear regression model framework. Compared against SCENIC. Works even for small cell populations. Tested on three scRNA-seq datasets. A part of SEPIRA R package.
    Paper Wang, Ning, and Andrew E Teschendorff. "Leveraging High-Powered RNA-Seq Datasets to Improve Inference of Regulatory Activity in Single-Cell RNA-Seq Data" https://doi.org/10.1101/553040 BioRxiv, February 22, 2019.
  • GraphDDP - combines user-guided clustering and transition of differentiation processes between clusters. Shortcomings of PCA, MDS, t-SNE. Tested on several datasets to improve interpretability of clustering, compared with other methods (Monocle2, SPRING, TSCAN). Detailed methods.
    Paper Costa, Fabrizio, Dominic Grün, and Rolf Backofen. "GraphDDP: A Graph-Embedding Approach to Detect Differentiation Pathways in Single-Cell-Data Using Prior Class Knowledge" https://doi.org/10.1038/s41467-018-05988-7 Nature Communications 9, no. 1 (December 2018)
  • SINCERA - identification of major cell types, the corresponding gene signatures and transcription factor networks. Pre-filtering (expression filter, cell specificity filter) improves inter-sample correlation and decrease inter-sample distance. Normalization: per-sample z-score, then trimmed mean across cells. Clustering (centered Pearson for distance, average linkage), and other metrics, permutation to assess clustering significance. Functional enrichment, cell type enrichment analysis, identification of cell signatures. TF networks and their parameters (disruptive fragmentation centrality, disruptive connection centrality, disruptive distance centrality). Example analysis of mouse lung cells at E16.5, Fluidigm, 9 clusters, comparison with SNN-Cliq, scLVM, SINGuLAR Analysis Toolset. Web-site: https://research.cchmc.org/pbge/sincera.html, GitHub, Data.
    Paper Guo, Minzhe, Hui Wang, S. Steven Potter, Jeffrey A. Whitsett, and Yan Xu. "SINCERA: A Pipeline for Single-Cell RNA-Seq Profiling Analysis" https://doi.org/10.1371/journal.pcbi.1004575 PLoS Computational Biology 11, no. 11 (November 2015)

RNA velocity

Differential expression

  • waddR - R package for testind distributional differences, using 2-Wasserstein distance, decomposed into change in location, shape, size, proportion of zeros (CMH test). Methods for one/multiple replicates per condition. Compared with scDD, SigEMD. Fast. In real data, detects similar genes to edgeR plus additional revealing new biology. GitHub.
    Paper Schefzik, Roman, Julian Flesch, and Angela Goncalves. "Fast Identification of Differential Distributions in Single-Cell RNA-Sequencing Data with WaddR" https://doi.org/10.1093/bioinformatics/btab226 Bioinformatics, 01 April 2021
  • singleCellHaystack - prediction of differentially expressed genes in scRNA-seq data in a multi-dimensional space, without explicit clustering. Grid in a multi-dimensional space, estimation of a reference distribution, comparing gene distribution at each grid point vs. reference using Kullback-Leibler divergence. Permutation to estimate significance. Compared with DEsingle, EMDo,ocs. scDD, edgeR, Monocle2, Seurat on synthetic (Splatter) and experimental scRNA-seq data (Tabula Muris), including spatial transcriptomics. Input - binarized expression matrix. Very fast. An R package, GitHub.
    Paper Vandenbon, Alexis, and Diego Diez. "A Clustering-Independent Method for Finding Differentially Expressed Genes in Single-Cell Transcriptome Data" https://doi.org/10.1038/s41467-020-17900-3 Nature Communications 11, no. 1 (2020): 1–10.
  • DEsingle - an R package for detecting three types of differentially expressed genes in scRNA-seq data. Using Zero Inflated Negative Binomial distribution to distinguish true zeros from dropouts. Differential expression status (difference in the proportion of real zeros), DE abundance (typical DE without proportion of zeros change), DE general (both DE and proportion of zeros change). The majority of information is in supplementary material.
    Paper Miao, Zhun, Ke Deng, Xiaowo Wang, and Xuegong Zhang. "DEsingle for Detecting Three Types of Differential Expression in Single-Cell RNA-Seq Data" https://doi.org/10.1093/bioinformatics/bty332 Edited by Bonnie Berger. Bioinformatics 34, no. 18 (September 15, 2018)
  • scDD R package to identify differentially expressed genes in single cell RNA-seq data. Accounts for unobserved data. Four types of differential expression (DE, DP, DM, DB, see paper).
    Paper Korthauer, Keegan D., Li-Fang Chu, Michael A. Newton, Yuan Li, James Thomson, Ron Stewart, and Christina Kendziorski. "A Statistical Approach for Identifying Differential Distributions in Single-Cell RNA-Seq Experiments" https://doi.org/10.1186/s13059-016-1077-y Genome Biology 17, no. 1 (December 2016).
  • MAST - scRNA-seq DEG analysis. CDR - the fraction of genes that are detectably expressed in each cell - added to the hurdle model that explicitly parameterizes distributions of expressed and non-expressed genes. Generalized linear model, log2(TPM+1), Gaussian. Regression coeffs are estimated using Bayesian approach. Variance shrinkage, gamma distribution.
    Paper Finak, Greg, Andrew McDavid, Masanao Yajima, Jingyuan Deng, Vivian Gersuk, Alex K. Shalek, Chloe K. Slichter, et al. "MAST: A Flexible Statistical Framework for Assessing Transcriptional Changes and Characterizing Heterogeneity in Single-Cell RNA Sequencing Data" https://doi.org/10.1186/s13059-015-0844-5 Genome Biology 16 (December 10, 2015)
  • SCDE - Stochasticity of gene expression, high drop-out rate. A mixture model of two processes - detected expression and drop-out failure modeled as low-magnitude Poisson. Drop-out rate depends on the expected expression and can be approximated by logistic regression.
    Paper Kharchenko, Peter V., Lev Silberstein, and David T. Scadden. "Bayesian Approach to Single-Cell Differential Expression Analysis" https://doi.org/10.1038/nmeth.2967 Nature Methods 11, no. 7 (July 2014)

Differential abundance

  • Milo - an R package for differential abundance testing on scRNA-seq data between two groups or multiple conditions. Building a graph on the first 40 components of PCA, defining neighborhoods using a graph sampling algorithm. Each neighborhood (partially overlapping, in contrast to discrete clustering) contains cells from different conditions - differential abundance is tested using a negative binomial GLM. Tested on simulated datasets (dyntoy), a time course of mouse thymic epithelial cells development, liver cirrhosis analysis. Replicated datasets needed, batch corrected. Competitors: DA-seq, Cydar. Code to reproduce results for the paper.
    Paper Dann, Emma, Neil C. Henderson, Sarah A. Teichmann, Michael D. Morgan, and John C. Marioni. "Milo: Differential Abundance Testing on Single-Cell Data Using k-NN Graphs" https://doi.org/10.1101/2020.11.23.393769 BioRxiv, January 1, 2020

CNV

  • CaSpER - identification of CNVs from RNA-seq data, bulk and single-cell (full-transcript only, like SMART-seq). Utilized multi-scale smoothed global gene expression profile and B-allele frequency (BAF) signal profile, detects concordant shifts in signal using a 5-state HMM (homozygous deletion, heterozygous deletion, neutral, one-copy-amplification, high-copy-amplification). Reconstructs subclonal CNV architecture for scRNA-seq data. Tested on GBM scRNA-seq, TCGA, other. Compared with HoneyBADGER. R code and tutorials.
    Paper Serin Harmanci, Akdes, Arif O. Harmanci, and Xiaobo Zhou. "CaSpER Identifies and Visualizes CNV Events by Integrative Analysis of Single-Cell or Bulk RNA-Sequencing Data" https://doi.org/10.1038/s41467-019-13779-x Nature Communications 11, no. 1 (December 2020)
  • CNV estimation algorithm in scRNA-seq data - moving 100-gene window, deviation of expression from the chromosome average. Details in Methods.
    Paper Tirosh, Itay, Andrew S. Venteicher, Christine Hebert, Leah E. Escalante, Anoop P. Patel, Keren Yizhak, Jonathan M. Fisher, et al. "Single-Cell RNA-Seq Supports a Developmental Hierarchy in Human Oligodendroglioma" https://doi.org/10.1038/nature20123 Nature 539, no. 7628 (November 2016)
  • infercnv - Inferring copy number alterations from tumor single cell RNA-Seq data, as compared with a set of reference normal cells. Positional expression intensity comparison. Documentation.

Annotation, subpopulation identification

  • CellTypist - machine learning tool for precise cell type annotation, immune cell types. Trained on 20 tissues with harmonized cell type labels, hierarchy of 45 cell types. L2-regularized logistic regression, machine learning framework, gradient descent, 30 epoch. Scanpy pipeline, batch correction using bbknn, markers detection using rbcde. GitHub.
    Paper Domínguez Conde, C, C Xu, Lb Jarvis, T Gomes, Sk Howlett, Db Rainbow, O Suchanek, et al. "Cross-Tissue Immune Cell Analysis Reveals Tissue-Specific Adaptations and Clonal Architecture in Humans" https://doi.org/10.1101/2021.04.28.441762 Preprint. Immunology, April 28, 2021.
  • tricycle - transfer learning approach to learn cell cycle PCA projections from a reference dataset and project new data on it. Combining the biology of the cell cycle, the mathematical properties of PCA of unimodal periodicity of genes associated with cell cycle. Tweet.
    Paper Zheng, Shijie C., Genevieve Stein-O’Brien, Jonathan J. Augustin, Jared Slosberg, Giovanni A. Carosso, Briana Winer, Gloria Shin, Hans T. Bjornsson, Loyal A. Goff, and Kasper D. Hansen. "[Universal prediction of cell cycle position using transfer learning" https://doi.org/10.1101/2021.04.06.438463 bioRxiv April 11, 2021
  • Azimuth - Mapping query scRNA-seq dataset to multimodal references and assigning cell types. Supervised principal component analysis to identify a projection of the query dataset that maximally captures the structure defined by the WNN graph. Combined with the anchor-based framework, allows projection on the previously defined reference UMAP visualization. Human PBMC, motor cortex, pancreas, mouse motor cortex references. Online apps.
    Paper Hao, Yuhan, Stephanie Hao, Erica Andersen-Nissen, William M. Mauck, Shiwei Zheng, Andrew Butler, Maddie Jane Lee, et al. "Integrated Analysis of Multimodal Single-Cell Data" https://doi.org/10.1101/2020.10.12.335331 Preprint. Genomics, October 12, 2020.
  • MARS - a meta-learning approach for identifying known and new cell types in scRNA-seq data. Constructs a meta-dataset from experiments with annotated cell types (used to learn the cell type landmarks in the embedding space) and an unannotated experiment (mathed to the embedded landmarks). The embedding space and objective function are defined such that cells (annotated and unannotated) embed close to their cell-type landmarks, cell type landmarks are most distinct. Autoencoder with 1000 and 100 neurons, input - all 22.9K genes. Applied to the Tabula Muris Senis dataset, several others. Significantly outperform ScVi, SIMLR, Scanpy and Seurat on adjusted Rand index, adjusted MI and other metrics.
    Paper Brbić, Maria, Marinka Zitnik, Sheng Wang, Angela O. Pisco, Russ B. Altman, Spyros Darmanis, and Jure Leskovec. "MARS: Discovering Novel Cell Types across Heterogeneous Single-Cell Experiments" https://doi.org/10.1038/s41592-020-00979-3 Nature Methods, October 19, 2020.
  • Garnett - annotating cells in scRNA-seq data. Hierarchy of cell types and their markers should be pre-defined using a markup language. A classifier is trained to classify additional datasets. Trained on cells from one organisms, can be applied to different organisms. Pre-trained classifiers available. R-based.
    Paper Pliner, Hannah A., Jay Shendure, and Cole Trapnell. "Supervised Classification Enables Rapid Annotation of Cell Atlases" https://doi.org/10.1038/s41592-019-0535-3 Nature Methods 16, no. 10 (October 2019)
  • CellAssign - R package for scRNA-seq cell type inference. Probabilistic graphical model to assign cell type probabilities to single cells using known marker genes (binarized matrix), including "unassigned" categorization. Insensitive to batch- or sample-specific effects. Outperforms Seurat, SC3, PhenoGraph, densityCut, dynamicTreeCut, scmap-cluster, correlation-based methods, SCINA. Applied to delineate the composition of the tumor microenvironment. Built using TensorFlow.
    Paper Zhang, Allen W., Ciara O’Flanagan, Elizabeth A. Chavez, Jamie L. P. Lim, Nicholas Ceglia, Andrew McPherson, Matt Wiens, et al. "Probabilistic Cell-Type Assignment of Single-Cell RNA-Seq for Tumor Microenvironment Profiling" https://doi.org/10.1038/s41592-019-0529-1 Nature Methods, September 9, 2019.
  • scPopCorn - subpopulation identification across scRNA-seq experiments. Identifies shared and unique subpopulations. Joint network of two graphs. First, graphs are built for each experiment using co-expression to identify subpopulations. Second, the corresponsence of the identified subpopulations is refined using Google's PageRank algorithm to identify subpopulations. Compared with Seurat alignment + Louvain, mutual nearest neighbor (MNN) method, and MNN + Louvain. Several assessment metrics. Tested on pancreatic, kidney cells, healthy brain and glioblastoma scRNA-seq data. Sankey diagrams showing how subpopulation assignment change.
    Paper Wang, Yijie, Jan Hoinka, and Teresa M. Przytycka. "Subpopulation Detection and Their Comparative Analysis across Single-Cell Experiments with ScPopCorn" https://doi.org/10.1016/j.cels.2019.05.007 Cell Systems 8, no. 6 (June 2019)
  • Single-Cell Signature Explorer - gene signature (~17,000 from MSigDb, KEGG, Reactome) scoring (sum of UMIs in in a gene signature over the total UMIs in a cell) for single cells, and visualization on top of a t-SNE plot. Optional Noise Reduction (Freeman-Tuckey transform to stabilize technical noise). Four consecutive tools (Go language, R/Shiny). Comparison with Seurat's Cell CycleScore module and AUCell from SCENIC. Very fast.
    Paper Pont, Frédéric, Marie Tosolini, and Jean Jacques Fournié. "Single-Cell Signature Explorer for Comprehensive Visualization of Single Cell Signatures across ScRNA-Seq Data Sets" https://doi.org/10.1101/621805 Preprint. Bioinformatics, April 29, 2019.
  • TooManyCells - divisive hierarchical spectral clustering of scRNA-seq data. Uses truncated singular vector decomposition to bipartition the cells. Newman-Girvain modularity Q to assess whether bipartition is significant or should be stopped. BirchBeer visualization. Outperforms Phenograph, Seurat, Cellranger, Monocle, the latter is second in performance. Excels for rare populations. Normalization marginally affects performance.
    Paper Schwartz, Gregory W, Jelena Petrovic, Maria Fasolino, Yeqiao Zhou, Stanley Cai, Lanwei Xu, Warren S Pear, Golnaz Vahedi, and Robert B Faryabi. "TooManyCells Identifies and Visualizes Relationships of Single-Cell Clades" https://doi.org/10.1101/519660 BioRxiv, January 13, 2019.
  • VISION - functional annotation of scRNA-seq data using gene signatures (Geary's C statistics), unsupervised and supervised. Operates downstream of dimensionality reduction, clustering. A continuation of FastProject.
    Paper DeTomaso, David, Matthew Jones, Meena Subramaniam, Tal Ashuach, Chun J Ye, and Nir Yosef. "Functional Interpretation of Single-Cell Similarity Maps" https://doi.org/10.1101/403055 August 29, 2018.

Cell markers

  • UCell - an R package for gene signature enrichment in scRNA-seq data based on the Mann-Whitney U statistics. Integrates with the Seurat pipeline. Annotates each cell with signature enrichments. Requires gene lists as signature definitions.
    Paper Andreatta, Massimo, and Santiago J. Carmona. “UCell: Robust and Scalable Single-Cell Gene Signature Scoring.” Computational and Structural Biotechnology Journal 19 (2021): 3796–98. https://doi.org/10.1016/j.csbj.2021.06.043.

Phylogenetic inference

  • OncoNEM (oncogenetic nested effects model) - tumor evolution inference from single cell data from somatic SNPs of single cells. Identifies homogeneous subpopulations and infers their genotypes and phylogenetic tree. Probabilistically accounts for noise in the observed genotypes, allele dropouts, unobserved subpopulations. Input - binary genotype matrix, false positive and negative rates. Output - inferred tumor subpopulations, evolutionary tree, posterior probabilities of mutations. Assessed in simulation studies, outperforms similar methods. Robust to the selection of parameters.
    Paper Ross, Edith M., and Florian Markowetz. "OncoNEM: Inferring Tumor Evolution from Single-Cell Sequencing Data" https://doi.org/10.1186/s13059-016-0929-9 Genome Biology, (December 2016)

Immuno-analysis

  • TCR-BCR-seq-analysis - T/B cell receptor sequencing analysis notes by Ming Tang

  • Overview of B-cell receptor development/affinity maturation, sequencing B-cell repertoire, clonal lineage assignment and clustering, somatic hypermutation analysis, challenges.

    Paper Hoehn, Kenneth B., Anna Fowler, Gerton Lunter, and Oliver G. Pybus. “The Diversity and Molecular Evolution of B-Cell Receptors during Infection.” Molecular Biology and Evolution 33, no. 5 (May 2016): 1147–57. https://doi.org/10.1093/molbev/msw015.
  • DALI - R-package for the analysis of single-cell TCR/BCR data and scRNA-seq (10X Genomics) in the Seurat ecosystem. Read10X_vdj reads Cellranger multi data, Interactive_VDJ launches Shiny app. Input - scRNA-seq Seurat object (.rds), and vdj data. Demo data.
    Paper Verstaen, Kevin, Inés Lammens, Jana Roels, Yvan Saeys, Bart N Lambrecht, Niels Vandamme, and Stijn Vanhee. “DALI (Diversity AnaLysis Interface): A Novel Tool for the Integrated Analysis of Multimodal Single Cell RNAseq Data and Immune Receptor Profiling.” Preprint. Bioinformatics, December 7, 2021. https://doi.org/10.1101/2021.12.07.471549.
  • CellPhoneDB - database and a tool for cell-cell communication analysis. Contains ligands, receptors, their interactions (978 proteins, 1396 interactions). Input - annotated scRNA-seq data, also protein expression, . Permutation-based comparison of mean expression of receptor-ligand coding genes. Examples how to reformat a Seurat object, scanpy adata. The paper contains full tutorial on using the cellphonedb Python package.
    Paper Efremova, Mirjana. "CellPhoneDB: Inferring Cell–Cell Communication from Combined Expression of Multi-Subunit Ligand–Receptor Complexes" https://doi.org/10.1038/s41596-020-0292-x NATURE PROTOCOLS, (2020)
  • scRepertoire - R package for 10x Genomics Chromium Immune Profiling for both T-cell receptor (TCR) and immunoglobulin (Ig) enrichment data. Clonotype analysis: cdr3 distribution, clonotype calling, proportion, repertoire overlap, diversity. Comparison between samples. Rich visualization capabilities. Tweet.
    Paper Borcherding, N, NL Bormann, and G Kraus. “ScRepertoire: An R-Based Toolkit for Single-Cell Immune Receptor Analysis [Version 2; Peer Review: 2 Approved].” F1000Research 9, no. 47 (2020). https://doi.org/10.12688/f1000research.22139.2.

  • enclone - Accurate and user-friendly computational tool for clonal grouping to study the adaptive immune system. Analyzes 10x Genomics Chromium Single Cell V(D)J data containing B cell receptor (BCR) and T cell receptor (TCR) RNA sequences. GitHub

  • immunarch - Exploration of Single-cell and Bulk T-cell/Antibody Immune Repertoires in R

  • Immcantation - a start-to-finish analytical ecosystem for high-throughput AIRR-seq (adaptive immune receptor repertoire) datasets. Beginning from raw reads, Python and R packages are provided for pre-processing, population structure determination, and repertoire analysis (pRESTO, Change-O, Alakazam, SHazaM, TIgGER, SCOPer, dowser, RDI, RAbHIT, IgPhyML, sumrep). Pipelines for various technologies, inclusing 10X Genomics. Tutorials. Introduction to B cell repertoire analysis using the Immcantation framework and the Jupyter notebook. 10x Genomics VDJ Sequence Analysis Tutorial with the Docker image

Simulation

  • scDesign - scRNA-seq data simulator and statistical framework to access experimental design for differential gene expression analysis. Gamma-Normal mixture model better fits scRNA-seq data, accounts for dropout events (Methods describe step-wise statistical derivations). Single- or double-batch sequencing scenarios. Comparable or superior performance to simulation methods splat, powsimR, scDD, Lun et al. method. DE tested using t-test. Applications include DE methods evaluation, dimensionality reduction testing.
    Paper Li, Wei Vivian, and Jingyi Jessica Li. "A Statistical Simulator ScDesign for Rational ScRNA-Seq Experimental Design" https://doi.org/10.1093/bioinformatics/btz321 Bioinformatics 35, no. 14 (July 15, 2019)

Power

  • SCOPIT - Shiny app for estimating the number of cells that must be sequenced to observe cell types in a single-cell sequencing experiment. By Alexander Davis

  • How many cells do we need to sample so that we see at least n cells of each type. By Satija's lab.

  • scPower - an R package for power calculation for single-cell RNA-seq studies. Estimates power of differential expression and eQTLs using zero-inflated negative binomial distribution. Also, power to detect rare cell types. Figure 1 shows the dependence among experimental design parameters. Tested on several datasets, generalizes well across technologies. GitHub and Shiny app.

    Paper Schmid, Katharina T., Barbara Höllbacher, Cristiana Cruceanu, Anika Böttcher, Heiko Lickert, Elisabeth B. Binder, Fabian J. Theis, and Matthias Heinig. "ScPower Accelerates and Optimizes the Design of Multi-Sample Single Cell Transcriptomic Studies" https://doi.org/10.1038/s41467-021-26779-7 Nature Communications, (December 2021)
  • powsimR - an R package for simulating scRNA-seq datasets and assess performance of differential analysis methods. Supports Poisson, Negative Binomial, and zero inflated NB, or estimates parameters from user-provided data. Simulates differential expression with pre-defined fold changes, estimates power, TPR, FDR, sample size, and for the user-provided dataset.
    Paper Vieth, Beate, Christoph Ziegenhain, Swati Parekh, Wolfgang Enard, and Ines Hellmann. "PowsimR: Power Analysis for Bulk and Single Cell RNA-Seq Experiments" https://doi.org/10.1093/bioinformatics/btx435 Edited by Ivo Hofacker. Bioinformatics 33, no. 21 (November 1, 2017)

Benchmarking

  • CellBench - an R package for benchmarking of scRNA-seq analysis pipelines. Simulated datasets using mixtures of either cells of RNA from five cancer cell lines, dilution series, ERCC spike-in controls. Four technologies. Methods: normalization, imputation, clustering, trajectory analysis, data integration. Evaluation metrics: silhouette width, correlations, others. Best performers: Normalization - Linnorm, scran, scone; Imputation - kNN, DrImpute; Clustering - all methods are OK, Seurat performs well; Trajectory - Slingshot and Monocle2. Processed datasets used for the analysis, GitHub.
    Paper Tian, Luyi, Xueyi Dong, Saskia Freytag, Kim-Anh Lê Cao, Shian Su, Abolfazl JalalAbadi, Daniela Amann-Zalcenstein, et al. "Benchmarking Single Cell RNA-Sequencing Analysis Pipelines Using Mixture Control Experiments" https://doi.org/10.1038/s41592-019-0425-8 Nature Methods, May 27, 2019.

Deep learning

  • scScope - for extracting informative representations, clustering and analysing cell type composition. Recurrent neural network architecture, variable number of recurrent steps (at step = 1, the architecture is standard autoencoder). Tested on simulated data (Splatter, SIMLR) and four experimental scRNA-seq dtasets, at different sparsity levels, rare subpopulation fractions. Compared with PCA, ZINB-WaVE, scVI, others. Multi-GPU Python, TensorFlow, numpy, scikit-learn implementation.
    Paper Deng, Yue, Feng Bao, Qionghai Dai, Lani F. Wu, and Steven J. Altschuler. “Scalable Analysis of Cell-Type Composition from Single-Cell Transcriptomics Using Deep Recurrent Learning.” Nature Methods, March 18, 2019. https://doi.org/10.1038/s41592-019-0353-7
  • Solo - semi-supervised deep learning for doublet identification. Variational autoencoder (scVI) followed by a classifier to detect doublets. Compared with Scrubled and DoubletFinder, improves area under the precision-recall curve.
    Paper Bernstein, Nicholas J., Nicole L. Fong, Irene Lam, Margaret A. Roy, David G. Hendrickson, and David R. Kelley. "Solo: Doublet Identification in Single-Cell RNA-Seq via Semi-Supervised Deep Learning" https://doi.org/10.1016/j.cels.2020.05.010 Cell Systems 11, no. 1 (July 2020)
  • scover - de novo identification of regulatory motifs and their cell type-specific importance from scRNA-seq or scATAC-seq data. Shallow convolutional neural network on one-hot encoded sequence data, k-fold training and selecting most optimal network, extracting motifs from convolutional filters, cluster them, matching with motifs, associating with peak strength/gene expression. application for human kidney scRNA-seq data, Tabula Muris, mouse cerebral cortex SNARE-seq data. Docs, Tweet.
    Paper Hepkema, Jacob, Nicholas Keone Lee, Benjamin J Stewart, Siwat Ruangroengkulrith, Varodom Charoensawan, Menna R Clatworthy, and Martin Hemberg. "Predicting the Impact of Sequence Motifs on Gene Regulation Using Single-Cell Data" https://doi.org/10.1101/2020.11.26.400218
  • SAVER-X - denoising scRNA-seq data using deep autoencoder with a Bayesian model. Decomposes the variation into three components: 1) predictable, 2) unpredictable, 3) technical noise. Pretrained on the Human Cell Atlas project, 10X Genomics immune cells, allows for human-mouse cross-species learning. Improves clustering and the detection of differential genes. Outperforms downsampling, MAGIC, DCA, scImpute.
    Paper Littmann, Maria, Katharina Selig, Liel Cohen-Lavi, Yotam Frank, Peter Hönigschmid, Evans Kataka, Anja Mösch, et al. "Validity of Machine Learning in Biology and Medicine Increased through Collaborations across Fields of Expertise" https://doi.org/10.1038/s42256-019-0139-8 Nature Machine Intelligence, January 13, 2020.
  • scVI - low-dimensional representation of scRNA-seq data used for batch correction, imputation, clustering, differential expression. Deep neural networks to approximate the distribution that underlie observed expression values. Zero-inflated negative binomial distribution conditioned on the batch annotation and unobserved random variables. Compared with DCA, ZINB-WAVE on simulated and real large and small datasets. Perspective by Way & Greene.
    Paper Lopez, Romain, Jeffrey Regier, Michael B Cole, Michael Jordan, and Nir Yosef. "Bayesian Inference for a Generative Model of Transcriptome Profiles from Single-Cell RNA Sequencing" https://doi.org/10.1101/292037 September 23, 2018.

Spatial transcriptomics

  • NovoSpaRc Python package and protocol for reconstruction of spatial positioning of scRNA-seq data. Structural correspondence hypothesis (optimal transport), cells in physical proximity share similar expression profiles. Reference atlas is optional and improves the spatial reconstruction. Input: gene expression matrix and a target space (1D, 2D, or 3D coordinates of the physical space, defaults available). Reference atlas expression is optional. Calculates three cost matrices (cell-cell, location-location, reference atlas), outputs a transport matrix (probabilistic mapping of cells onto the target locations) and the inferred gene expression over the target space. Table 1 - comparison with Seurat, DistMap, Perler, Tangram, CSOmap. Application demo, Python code. GitHub.

    Paper Moriel, Noa, Enes Senel, Nir Friedman, Nikolaus Rajewsky, Nikos Karaiskos, and Mor Nitzan. “NovoSpaRc: Flexible Spatial Reconstruction of Single-Cell Gene Expression with Optimal Transport.” Nature Protocols 16, no. 9 (September 2021): 4177–4200. https://doi.org/10.1038/s41596-021-00573-7.

    Nitzan, Mor, Nikos Karaiskos, Nir Friedman, and Nikolaus Rajewsky. “Gene Expression Cartography.” Nature 576, no. 7785 (December 5, 2019): 132–37. https://doi.org/10.1038/s41586-019-1773-3.

  • SpatialExperiment - R/Bioconductor package providing data infrastructure (S4 class) for spatial transcriptomics data. Intro into spot-based and molecule-based spatial transcriptomics technologies. read10xVisium() reads in SpaceRanger-processed data. Example datasets and visualization tools in the STexampleData, TENxVisiumData, and ggspavis packages.
    Paper Righelli, Dario, Lukas M Weber, Helena L Crowell, Brenda Pardo, Leonardo Collado-Torres, Shila Ghazanfar, Aaron TL Lun, Stephanie C Hicks, and Davide Risso. “SpatialExperiment: Infrastructure for Spatially Resolved Transcriptomics Data in R Using Bioconductor.” Preprint. Bioinformatics, January 27, 2021. https://doi.org/10.1101/2021.01.27.428431.
  • Review of spatial single-cell transcriptomics technologies. Categorized as 1)microdissection-based, 2) in situ hybridization, 3) in situ sequencing, 4) in situ capturing, 5) in silico reconstruction. Timeline (Figure 1), summary of technologies (Table 1), details of each technology, studies, illustrations.
    Paper Asp, Michaela, Joseph Bergenstråhle, and Joakim Lundeberg. "Spatially Resolved Transcriptomes—Next Generation Tools for Tissue Exploration" https://doi.org/10.1002/bies.201900221 BioEssays, May 4, 2020
  • spatialLIBD - an R package for 10X Visium spatial transcriptomics data manipulation and visualization. Can handle multiple samples, in contrast to Loupe and Giotto pipelines. Includes demo data. Integrates with Bioconductor via SpatialExperiment class. Shiny app.
    Paper Pardo, Brenda, Abby Spangler, Lukas M Weber, Stephanie C Hicks, Andrew E Jaffe, Keri Martinowich, and Kristen R Maynard. "SpatialLIBD: An R/Bioconductor Package to Visualize Spatially-Resolved Transcriptomics Data" https://doi.org/10.1101/2021.04.29.440149 biorXiv, April 30, 2021
  • Giotto - an R framework for spatial transcriptomics analysis pipeline. Two modules, analysis and visualization. Custom S4 data structure. QC, preprocessing, feature selection, dimensionality reduction, clustering, marker gene identification, spatial grid and neighborhood networks HMRF for spatial gene expression patterns detection. Visualization using tSNE, physical, physicalsimple panels (figures in the paper). Applied to the seqFISH+ dataset with 10K genes profiled in 913 cells.
    Paper Dries R, Zhu Q, Dong R, Eng CH, Li H, Liu K, Fu Y, Zhao T, Sarkar A, Bao F, George RE. "[Giotto: a toolbox for integrative analysis and visualization of spatial expression data. Genome biology" https://doi.org/10.1186/s13059-021-02286-2 08 March 2021

Technology

  • Collections of library structure and sequence of popular single cell genomic methods from Sarah Teichmann's group. GitHub

  • SPARC (Single-Cell Protein And RNA Co-profiling) - combines scRNA-seq (improved Smart-seq2 protocol) with proximity extension assays (PEA) to simultaneously measure global mRNA and 89 intracellular proteins in individual cells. Description of PEA - a proximity-based assay that require two binding events to generate a DNA reporter molecule (Figure 1). Applied to human embryonic stem cells following directed neural induction (from 0h to 48h). mRNA data highly correlate with regular Smart-seq2 data, not so with protein expression. Temporal reconstruction using mRNA and protein-based profiles produces similar results, suggesting that protein changes follow mRNA changes. Raw and processed data at SciLifeLab Data Repository. GitHub.

    Paper Reimegård, Johan, Marcel Tarbier, Marcus Danielsson, Jens Schuster, Sathishkumar Baskaran, Styliani Panagiotou, Niklas Dahl, Marc R. Friedländer, and Caroline J. Gallant. “A Combined Approach for Single-Cell MRNA and Intracellular Protein Expression Analysis.” Communications Biology 4, no. 1 (December 2021): 624. https://doi.org/10.1038/s42003-021-02142-w
  • SHARE-seq - simultaneous profiling of scRNA-seq and sc-ATAC-seq from the same cells. Built upon SPLiT-seq, a combinatorial indexing method. Confirmed by separate scRNA-seq and scATAC-seq datasets. Chromatin opening precedes transcriptional activation.
    Paper Ma, Sai, Bing Zhang, Lindsay M. LaFave, Andrew S. Earl, Zachary Chiang, Yan Hu, Jiarui Ding, et al. "Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin" https://doi.org/10.1016/j.cell.2020.09.056 Cell, (November 2020)
  • Drop-seq technology - single cells encapsulated in lipid droplets with nanoparticles with cell- and UMI barcodes. Barcoding strategy, "split-and-pool" synthesis cycles to synthesize 12bp cell barcodes, then 8bp UMI synthesis (Figure 1). Majority of droplets are empty, doublets depend on initial cell concentration. Example on a mixture of 589 human HEK and 412 mouse 3T3 cells. Expression profiles from 49,300 retinal cells profiled using Drop-seq. 13,155 largest libraries, reduce dimensionality by PCA to 32 components (decided by permutation), tSNE for visualization. 39 clusters matched to known cell types. GEO GSE63473.
    Paper Macosko, Evan Z., Anindita Basu, Rahul Satija, James Nemesh, Karthik Shekhar, Melissa Goldman, Itay Tirosh, et al. "Highly Parallel Genome-Wide Expression Profiling of Individual Cells Using Nanoliter Droplets" https://doi.org/10.1016/j.cell.2015.05.002 Cell, (May 21, 2015)

10X Genomics

10X QC

Data

  • CellBench data, single cell RNA-seq benchmarking, R SingleCellExperiment object.

  • SingleCellExperiment data, human and mouse, brain, embryo development, embryo stem cells, hematopoietic stem cells, pancreas, retina, other tissues. GitHub

  • 12 scRNA-seq datasets (Tabula Muris, Microwell-Seq, Baron pancreas, Xin pancreas, Segerstolpe pancreas, Murano pancreas, Zheng PBMC, Darminis brain, Zeisel brain, Tasic cortex) processed in the SingleCellNet study.

  • Pan-cancer portal of genomic blueprint of stromal cell heterogeneity using scRNA-seq ( from lung, colorectal, ovary, and breast cancer and normal tissues.68 stromal cell populations, 46 are shared and 22 are unique. Clustering and characterization of each cluster - marker genes, functional enrichment, transcription factors (SCENIC), trajectory reconstruction (Monocle). Table S5 - markers of cells present in stroma. A web portal for exploring gene expression in each cell subtype, comparison of conditions, raw data is not available. Visualization using SCope.

    Paper Qian, Junbin, Siel Olbrecht, Bram Boeckx, Hanne Vos, Damya Laoui, Emre Etlioglu, Els Wauters, et al. "A Pan-Cancer Blueprint of the Heterogeneous Tumor Microenvironment Revealed by Single-Cell Profiling" https://doi.org/10.1038/s41422-020-0355-0 Cell Research, June 19, 2020.
  • SCPortalen - database of scRNA-seq datasets from the International Nucleotide Sequence Database Collaboration (INSDC). Manually curated datasets with metadata,QC's, processed, PCA and tSNE coordinates, FPKM gene expression.
    Paper Abugessaisa, Imad, Shuhei Noguchi, Michael Böttcher, Akira Hasegawa, Tsukasa Kouno, Sachi Kato, Yuhki Tada, et al. "SCPortalen: Human and Mouse Single-Cell Centric Database" https://doi.org/10.1093/nar/gkx949 Nucleic Acids Research 46, no. D1 (04 2018)
  • scEiaD - scRNA-seq data of the eye. 1.2 million single-cell back of the eye transcriptomes across 28 studies, 18 publications, and 3 species.

Human

Cancer

  • 3CA Curated Cancer Cell Atlas - Collected, annotated and analyzed cancer scRNA-seq datasets. Expression matrix, cell/gene names, copy number alterations, UMAP coordinates. Tweet

  • CancerSEA - cancer scRNA-seq studies. Download individual studies, as well as gene signatures (from Angiogenesis, DNA damage to EMT, metastasis, etc.)

  • scTIME Portal - a database and an exploration/analysis portal for single cell transcriptomes of tumor immune microenvironment. Cell clusters, expression of selected genes, data/image download. Links to other portals/databases.

  • Multi-omics single-cell analysis of breast cancer. >130K scRNA-seq across 11 ER+, 5 HER2+ and 10 TNBC primary breast tumors. Immunophenotyping by CIRE-seq. 10X Visium Spatial transcriptomics. SCSubtype signatures - subtype classification (Basal, Her2E, LumA, LumB), Supplementary Table 4. Recurrent gene modules (GMs) driving neoplastic cell heterogeneity. Supplementary Table 5 - gene lists for 7 GMs. DScore (BIRC5, CCNB1, CDC20, NUF2, CEP55, NDC80, MKI67, PTTG1, RRM2, TYMS and UBE2C) and proliferation score. The cytotoxic gene list containing effector cytotoxic proteins (GZMA, GZMB, GZMH, GZMK, GZMM, GNLY, PRF1 and FASLG) and cytotoxic T cell activation markers (IFNG, TNF, IL2R and IL2). Bioinformatics methods: inferCNV, Stereoscope, CIBERSORTx, Monocle 2, CITE-seq-Count, DWLS. Code for all tools on GitHub. Supplementary Tables, Processed data, GEO GSE176078, Spatially resolved transcriptomics data.

    Paper Wu, S.Z., Al-Eryani, G., Roden, D.L. et al. "A Single-Cell and Spatially Resolved Atlas of Human Breast Cancers" https://doi.org/10.1038/s41588-021-00911-1 Nature Genetics 53 (September 2021)
  • Interactive mammary cell gene expression atlas - Integrated 50K mouse and 24K human mammary epithelial cell atlases, scRNA-seq. Consensus lineage trajectory - embryonic stem cells differentiate into three epithelial lineages (Basal, luminal hormone-sensing L-Hor, luminal alveolar L-Alv). Integration of four public and one new datasets. Harmony, LIGER, scALIGN for integration. STREAM for lineage tracing. ssGSVA for gene set enrichment. Supplementary Data: Supplementary Data 4 - mouse gene signatures of MaSC (mammary stem cells), Basal, LA-Pro (Luminal Alveloar progenitors), L-Alv, LH-Pro (Luminal Hormone-sensing), L-Hor. Supplementary Data 10 - mouse/human-specific and common stem/basal/Alv/Hor lineage genes.
    Paper Saeki, Kohei, Gregory Chang, Noriko Kanaya, Xiwei Wu, Jinhui Wang, Lauren Bernal, Desiree Ha, Susan L. Neuhausen, and Shiuan Chen. "Mammary Cell Gene Expression Atlas Links Epithelial Cell Remodeling Events to Breast Carcinogenesis" https://doi.org/10.1038/s42003-021-02201-2 Communications Biology, (December 2021)
  • scRNA-seq of breast cancer, four women, human mammary epithelial cells. Marker-free algorithm (LandSCENT) that identifies stem-like bipotent state, characterized by YBX1 and ENO1, two modulators of breast cancer risk. Source data 6B - 12- and 72 gene signature of bipotent state, basal-like. GEO GSE113197 - scRNA-seq data, annotated.
    Paper Chen, Weiyan, Samuel J. Morabito, Kai Kessenbrock, Tariq Enver, Kerstin B. Meyer, and Andrew E. Teschendorff. "Single-Cell Landscape in Mammary Epithelium Reveals Bipotent-like Cells Associated with Breast Cancer Risk and Outcome" https://doi.org/10.1038/s42003-019-0554-8 Communications Biology, (December 2019)
  • scRNA-seq of >25K normal human breast epithelial cells from seven individuals. Three cell populations, one basal and two luminal (secretory L1 and hormone-responsive L2). Within luminal L1, three cell states (milk production, secretory, epithelial keratin expression), but the combined analysis reports L1_1 and L1_2 signatures. Fluidigm, 10X Genomics, Seurat, Monocle analyses. GitHub, GEO GSE113197. Supplementary Data 2 - myeloepithelial gene signature to stratify basal cells into either "Basal" or "Myeloepithelial" grouping; scRNA-seq derived "Basal", "Basal_myoepithelial", "L1_1", "L1_2", "L_2", "Unclassified" signatures; Metabric-derived LumA and LumB signatures.
    Paper Nguyen, Quy H., Nicholas Pervolarakis, Kerrigan Blake, Dennis Ma, Ryan Tevia Davis, Nathan James, Anh T. Phung, et al. "Profiling Human Breast Epithelial Cells Using Single Cell RNA Sequencing Identifies Cell Diversity" https://doi.org/10.1038/s41467-018-04334-1 Nature Communications 9, no. 1 (December 2018)

Mouse

  • scRNA-seq of aging. >50,000 cells from kidney, lung, and spleen in young (7 months) and aged (22-23 months) mice. Transcriptional variation using difference from the median. Cell-cell heterogeneity using the Euclidean distance from centroids. Aging trajectories derived from NMF embedding. Cell type identification by neural network trained on Tabula Muris. ln(CPM + 1) UMIs used for all analyses. Visualization, downloadable data, code, pre-trained network.
    Paper Kimmel, Jacob C, Lolita Penland, Nimrod D Rubinstein, David G Hendrickson, David R Kelley, and Adam Z Rosenthal. "A Murine Aging Cell Atlas Reveals Cell Identity and Tissue-Specific Trajectories of Aging" https://doi.org/10.1101/657726 BioRxiv, January 1, 2019
  • Single-cell ATAC-seq, approx. 100,000 single cells from 13 adult mouse tissues. Two sequence platforms, good concordance. Filtered data assigned into 85 clusters. Genes associated with the corresponding ATAC sites (Cicero for identification). Differential accessibility. Motif enrichment (Basset CNN). GWAS results enrichment. All data and metadata are available for download as text or rds format.
    Paper Cusanovich, Darren A., Andrew J. Hill, Delasa Aghamirzaie, Riza M. Daza, Hannah A. Pliner, Joel B. Berletch, Galina N. Filippova, et al. "A Single-Cell Atlas of In Vivo Mammalian Chromatin Accessibility" https://doi.org/10.1016/j.cell.2018.06.052 Cell 174, no. 5 (August 2018)

  • Buettner, Florian, Kedar N Natarajan, F Paolo Casale, Valentina Proserpio, Antonio Scialdone, Fabian J Theis, Sarah A Teichmann, John C Marioni, and Oliver Stegle. "Computational Analysis of Cell-to-Cell Heterogeneity in Single-Cell RNA-Sequencing Data Reveals Hidden Subpopulations of Cells" Nature Biotechnology 33, no. 2 (March 2015).

    • [data/scLVM/nbt.3102-S7.xlsx] - Uncorrected and cell-cycle corrected expression values (81 cells x 7073 genes) for T-cell data. Includes cluster assignment to naive T cells vs. TH2 cells (GATA3 high marker). Source
    • [data/scLVM/nbt.3102-S8.xlsx] - Corrected and uncorrected expression values for the newly generated mouse ESC data. 182 samples x 9571 genes. Source

  • Zeisel, A., Munoz-Manchado, A.B., Codeluppi, S., Lonnerberg, P., La Manno, G., Jureus, A., Marques, S., Munguba, H., He, L., Betsholtz, C., et al. (2015). Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA- seq. Science 347, 1138–1142. - 3,005 single cells from the hippocampus and cerebral cortex of mice. GEO, Web, and more on this site.

    • [data/Brain/Zeisel_2015_TableS1.xlsx] - Table S1 - gene signatures for Ependymal, Oligodendrocyte, Microglia, CA1 Pyramidal, Interneuron, Endothelial, S1 Pyramidal, Astrocyte, Mural cells. Source
    • [data/Brain/expression_mRNA_17-Aug-2014.txt] - 19,972 genes x 3005 cells. Additional rows with class annotations to interneurons, pyramidal SS, pyramidal CA1, oligodendrocytes, microglia, endothelial-mural, astrocytes_ependymal, further subdivided into 47 subclasses. Source

Brain

  • Cell-type specificity of schizophrenia SNPs judged by enrichment in expressed genes. scRNA-seq custom data collection. Difference between schizophrenia and neurological disorders.

    • data/Brain_cell_type_gene_expression.xlsx - Supplementary Table 4 - Specificity values for Karolinska scRNA-seq superset. Specificity represents the proportion of the total expression of a gene found in one cell type as compared to that in all cell types (i.e., the mean expression in one cell type divided by the mean expression in all cell types). Gene X cell type matrix. Level 1 (core cell types) and level 2 (extended collection of cell types) data. Major Depressive Disorder Working Group of the Psychiatric Genomics Consortium et al., "Genetic Identification of Brain Cell Types Underlying Schizophrenia" https://doi.org/10.1038/s41588-018-0129-5 Nature Genetics 50, no. 6 (June 2018)

  • Drop-seq scRNA-seq data of 690,000 cells from 9 regions of adult mouse brain. Independent Component Analysis (ICA). ICs grouped into 565 transcriptionally distinct clusters (323 neuronal) corresponding to biological signals using network-based clustering. DropViz visualization, data download in CSV, RData, with annotations.

    Paper Saunders, Arpiar, Evan Z. Macosko, Alec Wysoker, Melissa Goldman, Fenna M. Krienen, Heather de Rivera, Elizabeth Bien, et al. "Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain" https://doi.org/10.1016/j.cell.2018.07.028 Cell 174, no. 4 (09 2018)

  • Single-cell RNA-seq of human neuronal cell types. Dimensionality reduction, clustering, WGCNA, defining cell type-specific signatures, comparison with other signatures (Zeng, Miller). Supplementary material. Wired story. Controlled access data on dbGAP, summarized matrix with annotations

  • Single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq) of >60K cells from various parts of human adult brain. Resolving subpopulations, integrating the datasets, predicting one modality from another using the GBM classifier, integration with GWAS signal. Detailed methods, Data processing using Pagoda2, Seurat, LIGER, Monocle, Code, Data, Supplementary Table 3 - neuronal subpopulation-specific gene lists.

    Paper Lake, Blue B, Song Chen, Brandon C Sos, Jean Fan, Gwendolyn E Kaeser, Yun C Yung, Thu E Duong, et al. "Integrative Single-Cell Analysis of Transcriptional and Epigenetic States in the Human Adult Brain" https://doi.org/10.1038/nbt.4038 Nature Biotechnology 36, no. 1 (December 11, 2017)
  • Single cell brain transcriptomics, human. Fluidigm C1 platform. Healthy cortex cells (466 cells) containing: Astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), neurons, microglia, and vascular cells. Single cells clustered into 10 clusters, their top 20 gene signatures are in Supplementary Table S3. Raw data.
    • [data/Brain/TableS3.txt] - top 20 cell type-specific genes
    • [data/Brain/TableS3_matrix.txt] - genes vs. cell types with 0/1 indicator variables. Darmanis, S., Sloan, S.A., Zhang, Y., Enge, M., Caneda, C., Shuer, L.M., Hayden Gephart, M.G., Barres, B.A., and Quake, S.R. (2015). A survey of human brain transcriptome diversity at the single cell level. Proc. Natl. Acad. Sci.

Links

Papers

  • Stuart, Tim, Andrew Butler, Paul Hoffman, Christoph Hafemeister, Efthymia Papalexi, William M. Mauck, Yuhan Hao, Marlon Stoeckius, Peter Smibert, and Rahul Satija. "Comprehensive Integration of Single-Cell Data" Cell, (June 2019) - Seurat v.3 paper. Integration of multiple scRNA-seq and other single-cell omics (spatial transcriptomics, scATAC-seq, immunophenotyping), including batch correction. Anchors as reference to harmonize multiple datasets. Canonical Correlation Analysis (CCA) coupled with Mutual Nearest Neighborhoors (MNN) to identify shared subpopulations across datasets. CCA to reduce dimensionality, search for MNN in the low-dimensional representation. Shared Nearest Neighbor (SNN) graphs to assess similarity between two cells. Outperforms scmap. Extensive validation on multiple datasets (Human Cell Atlas, STARmap mouse visual cortex spatial transcriptomics. Tabula Muris, 10X Genomics datasets, others in STAR methods). Data normalization, variable feature selection within- and between datasets, anchor identification using CCA (methods), their scoring, batch correction, label transfer, imputation. Methods correspond to details of each Seurat function. Preprocessing of real single-cell data. GitHub with code for the paper

  • The single cell studies database, over 1000 studies. Main database, Tweet by Valentine Svensson

  • scATACdb - list of scATAC-seq studies, Google Sheet by Caleb Lareau

  • Journal club on single-cell multimodal data technology and analysis - Data science seminar led by Levi Waldron

  • Review of manifold learning-based methods for denoising scRNA-seq data, revealing gene interactions, extracting pseudotime progressions with model fitting, visualizing the cellular state space via dimensionality reduction, and clustering. Manifold = a mathematical construct that represents a locally-Euclidean smoothly varying space. Modeling single-cell data as a manifold: 1. a graph based on local affinities such as the minimal spanning tree or a k- nearest neighbors (nn); 2. data diffusion. Applications: 1. Denoising and gene interactions (Seurat, ZIFA, CIDR, PCoA, scIMPUTE, SAVER, MAGIC, DREMI); 2. Pseudotime (Monocle, Wishbone, Wanderlust, Diffusion Pseudotime); 3. Dimensionality reduction (t-SNE, diffusion map, PHATE); 4. Density estimation and clustering (k-nn graph, PhenoGraph). Future directions in manifold learning.

    Paper Moon, Kevin R., Jay S. Stanley, Daniel Burkhardt, David van Dijk, Guy Wolf, and Smita Krishnaswamy. “Manifold Learning-Based Methods for Analyzing Single-Cell RNA-Sequencing Data.” Current Opinion in Systems Biology 7 (February 2018): 36–46. https://doi.org/10.1016/j.coisb.2017.12.008.
  • Review of single-cell sequencing technologies, individual and combined, technical details of each. Combinatorial indexing. Genomic DNA, methylomes, histone modifications, open chromatin, 3D genomics, proteomics, spatial transcriptomics. Table 1 - multiomics technologies, summary. Areas of application, in cancer and cell atlases. Future development, e.g., single-cell metabolomics.
    Paper Chappell, Lia, Andrew J. C. Russell, and Thierry Voet. "Single-Cell (Multi)Omics Technologies" https://doi.org/10.1146/annurev-genom-091416-035324 Annual Review of Genomics and Human Genetics 19 (31 2018)

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A list of scRNA-seq analysis tools

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