This data is used for the Seurat version of the batch correction and integration tutorial on the Galaxy Training Network. The input data was provided by Seurat in the 'Integrative Analysis in Seurat v5' tutorial. The input dataset provided here has been filtered to include only cells for which nFeature_RNA > 1000. The other datasets were produced on Galaxy. The original dataset was published as: Ding, J., Adiconis, X., Simmons, S.K. et al. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nat Biotechnol 38, 737–746 (2020). https://doi.org/10.1038/s41587-020-0465-8.

This record includes training materials associated with the Australian BioCommons workshop 'Single cell RNAseq analysis in R'. This workshop took place over two, 3.5 hour sessions on 26 and 27 October 2023. Event description Analysis and interpretation of single cell RNAseq (scRNAseq) data requires dedicated workflows. In this hands-on workshop we will show you how to perform single cell analysis using Seurat - an R package for QC, analysis, and exploration of single-cell RNAseq data. We will discuss the 'why' behind each step and cover reading in the count data, quality control, filtering, normalisation, clustering, UMAP layout and identification of cluster markers. We will also explore various ways of visualising single cell expression data. This workshop is presented by the Australian BioCommons, Queensland Cyber Infrastructure Foundation (QCIF) and the Monash Genomics and Bioinformatics Platform with the assistance of a network of facilitators from the national Bioinformatics Training Cooperative. Lead trainers: Sarah Williams, Adele Barugahare, Paul Harrison, Laura Perlaza Jimenez Facilitators: Nick Matigan, Valentine Murigneux, Magdalena (Magda) Antczak Infrastructure provision: Uwe Winter Coordinator: Melissa Burke Training materials Materials are shared under a Creative Commons Attribution 4.0 International agreement unless otherwise specified and were current at the time of the event. Files and materials included in this record: Event metadata (PDF): Information about the event including, description, event URL, learning objectives, prerequisites, technical requirements etc. Index of training materials (PDF): List and description of all materials associated with this event including the name, format, location and a brief description of each file. scRNAseq_Schedule (PDF): A breakdown of the topics and timings for the workshop Materials shared elsewhere: This workshop follows the tutorial 'scRNAseq Analysis in R with Seurat' https://swbioinf.github.io/scRNAseqInR_Doco/index.html Slides used to introduce key topics are available via GitHub https://github.com/swbioinf/scRNAseqInR_Doco/tree/main/slides This material is based on the introductory Guided Clustering Tutorial tutorial from Seurat. It is also drawing from a similar workshop held by Monash Bioinformatics Platform Single-Cell-Workshop, with material here.

Necessary datasets to run the Immcantation 10x Tutorial. Below is the description of the files in the data set.

BCR_data_sample1.tsv: data corresponding to the first sample (sample 1) of the two samples analyzed in the 10x tutorial. This is the sample used to show the Change-O steps.

filtered_contig_annotations.csv: filtered contig annotations file for sample 1, output of cellranger vdj.

filtered_contig.fasta: sequence fasta file for sample 1, output of cellranger vdj.

BCR_data.tsv: AIRR rearrangement file containing the data for both samples 1 and 2 used in the 10x tutorial.

BCR.data_08112023.rds: R dataframe object containing the single-cell BCR sequencing data for both samples 1 and 2 used in the 10x tutorial.

GEX.data_08112023.rds: Seurat object containing the single-cell gene expression data used in the 10x tutorial.

Summary: Dendritic cells (DCs) orchestrate innate and adaptive immunity, by translating the sensing of distinct danger signals into the induction of different effector lymphocyte responses, to induce different defense mechanisms suited to face distinct types of threats. Hence, DCs are very plastic, which results from two key characteristics. First, DCs encompass distinct cell types specialized in different functions. Second, each DC type can undergo different activation states, fine-tuning its functions depending on its tissue microenvironment and the pathophysiological context, by adapting the output signals it delivers to the input signals it receives. Hence, to better understand DC biology and harness it in the clinic, we must determine which combinations of DC types and activation states mediate which functions, and how.
To decipher the nature, functions and regulation of DC types and their physiological activation states, one of the methods that can be harnessed most successfully is ex vivo single cell RNA sequencing (scRNAseq). However, for new users of this approach, determining which analytics strategy and computational tools to choose can be quite challenging, considering the rapid evolution and broad burgeoning of the field. In addition, awareness must be raised on the need for specific, robust and tractable strategies to annotate cells for cell type identity and activation states. It is also important to emphasize the necessity of examining whether similar cell activation trajectories are inferred by using different, complementary methods. In this chapter, we take these issues into account for providing a pipeline for scRNAseq analysis and illustrating it with a tutorial reanalyzing a public dataset of mononuclear phagocytes isolated from the lungs of naïve or tumor-bearing mice. We describe this pipeline step-by-step, including data quality controls, dimensionality reduction, cell clustering, cell cluster annotation, inference of the cell activation trajectories and investigation of the underpinning molecular regulation. It is accompanied with a more complete tutorial on Github. We anticipate that this method will be helpful for both wet lab and bioinformatics researchers interested in harnessing scRNAseq data for deciphering the biology of DCs or other cell types, and that it will contribute to establishing high standards in the field.

Data:

1. negative_cDC1_relative_signatures.csv : Negative signatures for performing Connectivity Map (cMAP) Analysis

2. positive_cDC1_relative_signatures.csv : Positive signatures for performing Connectivity Map (cMAP) Analysis

The demo datasets for scMetabolismThe demo data is the dataset of Peripheral Blood Mononuclear Cells (PBMC) from 10X Genomics open access dataset (~2,700 single cells, also used by Seurat tutorial).

ObjectiveTo guide animal experiments, we investigated the similarities and differences between humans and mice in aging and Alzheimer’s disease (AD) at the single-nucleus RNA sequencing (snRNA-seq) or single-cell RNA sequencing (scRNA-seq) level.MethodsMicroglia cells were extracted from dataset GSE198323 of human post-mortem hippocampus. The distributions and proportions of microglia subpopulation cell numbers related to AD or age were compared. This comparison was done between GSE198323 for humans and GSE127892 for mice, respectively. The Seurat R package and harmony R package were used for data analysis and batch effect correction. Differentially expressed genes (DEGs) were identified by FindMarkers function with MAST test. Comparative analyses were conducted on shared genes in DEGs associated with age and AD. The analyses were done between human and mouse using various bioinformatics techniques. The analysis of genes in DEGs related to age was conducted. Similarly, the analysis of genes in DEGs related to AD was performed. Cross-species analyses were conducted using orthologous genes. Comparative analyses of pseudotime between humans and mice were performed using Monocle2.Results(1) Similarities: The proportion of microglial subpopulation Cell_APOE/Apoe shows consistent trends, whether in AD or normal control (NC) groups in both humans and mice. The proportion of Cell_CX3CR1/Cx3cr1, representing homeostatic microglia, remains stable with age in NC groups across species. Tuberculosis and Fc gamma R-mediated phagocytosis pathways are shared in microglia responses to age and AD across species, respectively. (2) Differences: IL1RAPL1 and SPP1 as marker genes are more identifiable in human microglia compared to their mouse counterparts. Most genes of DEGs associated with age or AD exhibit different trends between humans and mice. Pseudotime analyses demonstrate varying cell density trends in microglial subpopulations, depending on age or AD across species.ConclusionsMouse Apoe and Cell_Apoe maybe serve as proxies for studying human AD, while Cx3cr1 and Cell_Cx3cr1 are suitable for human aging studies. However, AD mouse models (App_NL_G_F) have limitations in studying human genes like IL1RAPL1 and SPP1 related to AD. Thus, mouse models cannot fully replace human samples for AD and aging research.

The data is derived from the 3k PBMC data used in scanpy & Seurat tutorials. In comes in the AnnData h5ad format.

Processed 3k PBMCs from a Healthy Donor from 10x Genomics, available at https://scanpy.readthedocs.io/en/stable/generated/scanpy.datasets.pbmc3k_processed.html Original 10X data available at http://cf.10xgenomics.com/samples/cell-exp/1.1.0/pbmc3k/pbmc3k_filtered_gene_bc_matrices.tar.gz from this website: https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k

The changes made to the original scanpy.datasets.pbmc3k_processed() data are described in this github issue: https://github.com/scverse/scverse-tutorials/issues/51

See jupyter notebook for details.

Remark: For trajectory inference discussion for that dataset, see paper: https://www.mdpi.com/1099-4300/22/11/1274 "Minimum Spanning vs. Principal Trees for Structured Approximations of Multi-Dimensional Datasets Alexander Chervov, Jonathan Bac and Andrei Zinovyev

For cell cycle analysis see: https://arxiv.org/abs/2208.05229 "Computational challenges of cell cycle analysis using single cell transcriptomics" Alexander Chervov, Andrei Zinovyev

Data and Context

Data - results of single cell RNA sequencing, i.e. rows - correspond to cells, columns to genes (or vice versa). value of the matrix shows how strong is "expression" of the corresponding gene in the corresponding cell. https://en.wikipedia.org/wiki/Single-cell_transcriptomics

See tutorials: https://scanpy.readthedocs.io/en/stable/tutorials.html ("Scanpy" - main Python package to work with scRNA-seq data). Or https://satijalab.org/seurat/ "Seurat" - "R" package

Particular data: Gene expressions count matrix. Single cell RNA sequencing data. 447 cells , 24748 genes Mouse Liver Hepatoblast in vivo.

Paper: Hepatology. 2017 Nov;66(5):1387-1401. doi: 10.1002/hep.29353. Epub 2017 Sep 29. A single-cell transcriptomic analysis reveals precise pathways and regulatory mechanisms underlying hepatoblast differentiation Li Yang 1 2 , Wei-Hua Wang 1 2 , Wei-Lin Qiu 1 3 , Zhen Guo 1 , Erfei Bi 4 , Cheng-Ran Xu 1

Data: GSE90047 https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE90047 Downloaded from: https://cytotrace.stanford.edu/#shiny-tab-dataset_download

Related datasets:

Other single cell RNA seq datasets can be found on kaggle: Look here: https://www.kaggle.com/alexandervc/datasets Or search kaggle for "scRNA-seq"

Inspiration

Single cell RNA sequencing is important technology in modern biology, see e.g. "Eleven grand challenges in single-cell data science" https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-1926-6

Also see review : Nature. P. Kharchenko: "The triumphs and limitations of computational methods for scRNA-seq" https://www.nature.com/articles/s41592-021-01171-x

Search scholar.google "challenges in single cell rna sequencing" https://scholar.google.fr/scholar?q=challenges+in+single+cell+rna+sequencing&hl=en&as_sdt=0&as_vis=1&oi=scholart gives many interesting and highly cited articles

(Cited 968) Computational and analytical challenges in single-cell transcriptomics Oliver Stegle, Sarah A. Teichmann, John C. Marioni Nat. Rev. Genet., 16 (3) (2015), pp. 133-145 https://www.nature.com/articles/nrg3833

Challenges in unsupervised clustering of single-cell RNA-seq data https://www.nature.com/articles/s41576-018-0088-9 Review Article 07 January 2019 Vladimir Yu Kiselev, Tallulah S. Andrews & Martin Hemberg Nature Reviews Genetics volume 20, pages273–282 (2019)

Challenges and emerging directions in single-cell analysis https://link.springer.com/article/10.1186/s13059-017-1218-y Published: 08 May 2017 Guo-Cheng Yuan, Long Cai, Michael Elowitz, Tariq Enver, Guoping Fan, Guoji Guo, Rafael Irizarry, Peter Kharchenko, Junhyong Kim, Stuart Orkin, John Quackenbush, Assieh Saadatpour, Timm Schroeder, Ramesh Shivdasani & Itay Tirosh Genome Biology volume 18, Article number: 84 (2017)

Single-Cell RNA Sequencing in Cancer: Lessons Learned and Emerging Challenges https://www.sciencedirect.com/science/article/pii/S1097276519303569 Molecular Cell Volume 75, Issue 1, 11 July 2019, Pages 7-12 Journal home page for Molecular Cell

`bcr_phylo_tutorial.zip` is used in the Reconstruction and analysis of B-cell lineage trees from single cell data using Immcantation tutorial.

`immcantation-BCR-Seurat-tutorial.zip` is used in the Integration of BCR and GEX data tutorial.

IFN-beta treated and control PBMCs from 8 donorstwo groups of PBMCs from Kang et al. 2017 (https://www.nature.com/articles/nbt.4042), analyzed using the Seurat sample alignment strategy as explained in the tutorial at https://satijalab.org/seurat/v2.4/immune_alignment.html and then converted to h5ad format* ~14000 cells* IFN-treated and unstimulated PBMCs from 8 donors* donor identities determined using demuxlet (see GSE96583)

This archive contains 10x matrix data (processed with cellranger) used for the Introduction to single cell RNAseq analysis workshop taught by the Danish National Sandbox for Health Data Science. The course repo can be found on Github.

Data.zip contains 6 runs on Spermatogonia development. 3 from healthy individuals and 3 from azoospermic individuals. Data has been already preprocessed using cellranger and can be loaded using Seurat (R) or scanpy (python).

Remark: See paper: https://arxiv.org/abs/2208.05229 results on cell cycle analysis discussed there. "Computational challenges of cell cycle analysis using single cell transcriptomics" Alexander Chervov, Andrei Zinovyev

Data and Context

Data - results of single cell RNA sequencing, i.e. rows - correspond to cells, columns to genes (or vice versa). value of the matrix shows how strong is "expression" of the corresponding gene in the corresponding cell. https://en.wikipedia.org/wiki/Single-cell_transcriptomics

See tutorials: https://scanpy.readthedocs.io/en/stable/tutorials.html ("Scanpy" - main Python package to work with scRNA-seq data). Or https://satijalab.org/seurat/ "Seurat" - "R" package

Particular data: Paper: CENP-A overexpression promotes distinct fates in human cells, depending on p53 status Daniel Jeffery, Alberto Gatto, Katrina Podsypanina, Charlène Renaud-Pageot, Rebeca Ponce Landete, Lorraine Bonneville, Marie Dumont, Daniele Fachinetti & Geneviève Almouzni https://www.nature.com/articles/s42003-021-01941-5

Data: https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-9861/

Related datasets:

Other single cell RNA seq datasets can be found on kaggle: Look here: https://www.kaggle.com/alexandervc/datasets Or search kaggle for "scRNA-seq"

Inspiration

Single cell RNA sequencing is important technology in modern biology, see e.g. "Eleven grand challenges in single-cell data science" https://genomebiology.biomedcentral.com/articles/10.1186/s13059-020-1926-6

Also see review : Nature. P. Kharchenko: "The triumphs and limitations of computational methods for scRNA-seq" https://www.nature.com/articles/s41592-021-01171-x

Search scholar.google "challenges in single cell rna sequencing" https://scholar.google.fr/scholar?q=challenges+in+single+cell+rna+sequencing&hl=en&as_sdt=0&as_vis=1&oi=scholart gives many interesting and highly cited articles

(Cited 968) Computational and analytical challenges in single-cell transcriptomics Oliver Stegle, Sarah A. Teichmann, John C. Marioni Nat. Rev. Genet., 16 (3) (2015), pp. 133-145 https://www.nature.com/articles/nrg3833

Challenges in unsupervised clustering of single-cell RNA-seq data https://www.nature.com/articles/s41576-018-0088-9 Review Article 07 January 2019 Vladimir Yu Kiselev, Tallulah S. Andrews & Martin Hemberg Nature Reviews Genetics volume 20, pages273–282 (2019)

Challenges and emerging directions in single-cell analysis https://link.springer.com/article/10.1186/s13059-017-1218-y Published: 08 May 2017 Guo-Cheng Yuan, Long Cai, Michael Elowitz, Tariq Enver, Guoping Fan, Guoji Guo, Rafael Irizarry, Peter Kharchenko, Junhyong Kim, Stuart Orkin, John Quackenbush, Assieh Saadatpour, Timm Schroeder, Ramesh Shivdasani & Itay Tirosh Genome Biology volume 18, Article number: 84 (2017)

Single-Cell RNA Sequencing in Cancer: Lessons Learned and Emerging Challenges https://www.sciencedirect.com/science/article/pii/S1097276519303569 Molecular Cell Volume 75, Issue 1, 11 July 2019, Pages 7-12 Journal home page for Molecular Cell

This is a single cell transcriptomics dataset containing roughly 3,000 PBMCs. The original data was downloaded from the Seurat 3k PBMC tutorial: https://satijalab.org/seurat/v3.0/pbmc3k_tutorial.html. We reprocessed the dataset using the Besca package (https://github.com/bedapub/besca).

Datasets for ShinyCell2 Example Applications, which include:

spatial_brain.rds: Example spatial transcriptomics dataset of sagital mouse brain slices generated using the 10x Visium v1 chemistry, processed using the Seurat spatial pipeline (https://satijalab.org/seurat/articles/spatial_vignette)

multimodal_pbmc.rds: Example CITE-seq dataset of PBMC reference containing 162,000 PBMC cells measured with 228 antibodies (https://satijalab.org/seurat/articles/multimodal_reference_mapping.html)

ArchR-ProjHeme.tar.gz: Example scATAC-seq dataset of bone marrow and peripheral blood mononuclear cells, which is used as the tutorial dataset for the ArchR pipeline (https://www.archrproject.com/articles/Articles/tutorial.html). As ArchR objects are stored in a directory containing many files, the entire folder is tarred and compressed here.

signac_pbmc.rds: Example scATAC-seq dataset of PBMC provided by 10x Genomics, which is used as the tutorial dataset for the signac pipeline (https://stuartlab.org/signac/articles/pbmc_vignette.html). Signac objects store the full list of all unique fragments across all single cells in a separate fragment file, uploaded as signac_pbmc_fragments.tsv.gz here

This repository hosts example data for reproducible analysis of intra- and intercellular signaling in single cell RNA sequencing (scRNAseq) data based on transcription factor (TF) activation. We demonstrate analysis using dominoSignal on the 10X Genomics Peripheral Blood Mononuclear Cells (PBMC) data set of 2,700 cells PBMC3K. scRNA-seq data is preprocessed following the Satija Lab's Guided Clustering Tutorial. Quantification of TF activation is conducted using pySCENIC. For more details on how this analysis is conducted, please refer to the vignettes in the dominoSignal package.