visium

A quarto workflow to analyse and visualise 10x Visium Spatial Transcriptomics data

Contents

• Overview
• Installation
• Usage
• Contributing
• Authors
• Tests
• Acknowledgements
• License

Overview

Visium is a quarto workflow to process and analyse 10x Visium Spatial transcriptomic data from the 10x Genomics platform. It is compatible with both FFPE and Fresh frozen protocols and utilises the following key analysis frameworks:

• Seurat
• OSCA

Installation

Please ensure you also have make and R version 4.0 or greater installed.

1. Install Renv

   Visium and all of its dependencies can be installed via the renv package manager. In each workflow renv::restore() will restore the project state from the renv.lock file.

   For more details on renv please follow this link.

   The first thing we install is renv which requires yaml to parse dependencies within Quarto Markdown files:

   $ R
   
   R version 4.2.0 (2022-04-22) -- "Vigorous Calisthenics"
   Copyright (C) 2022 The R Foundation for Statistical Computing
   Platform: x86_64-pc-linux-gnu (64-bit)
   
   R is free software and comes with ABSOLUTELY NO WARRANTY.
   You are welcome to redistribute it under certain conditions.
   Type 'license()' or 'licence()' for distribution details.
   
   R is a collaborative project with many contributors.
   Type 'contributors()' for more information and
   'citation()' on how to cite R or R packages in publications.
   
   Type 'demo()' for some demos, 'help()' for on-line help, or
   'help.start()' for an HTML browser interface to help.
   Type 'q()' to quit R.
   
   > install.packages("yaml")
   > install.packages("renv")

2. Install quarto

   As this workflow is written as a number of quarto documents we next we need to install Quarto by following the link:

   We can then install the .deb file and test quarto install:

   $ sudo dpkg -i quarto-0.9.640-linux-amd64.deb
   $ quarto -V # Test version and install

   See the following link for more details on rendering quarto documents .qmd

Usage

1. Create sample table

   $ vim data/sample_table.csv # containing sample meta data 

   This table should look something like the following and must contain these headers:

   sample	image	slide	group	area	index	files	protocol
   V1_Breast_Cancer_Block_A_Section_1	V1_Breast_Cancer_Block_A_Section_1_image	V19L29-097	slide1	B1	T1T2-F10	V1_Breast_Cancer_Block_A_Section_1_filtered_feature_bc_matrix.h5	FF
   V1_Breast_Cancer_Block_A_Section_2	V1_Breast_Cancer_Block_A_Section_2_image	V19L29-098	slide2	B1	T1T2-H10	V1_Breast_Cancer_Block_A_Section_2_filtered_feature_bc_matrix.h5	FF

2. Fill in paramaters

   These can be filled in at the top of each workbook e.g. for 01-data-loading:

        ---
        title: "Data loading"
        params:
            prefix:
            - "data/"
            marker:
            - "ACTA2"
            sample.sheet:
            - "V1_Breast_Cancer.csv"
        ---

   or supplied upon render using the -P flag:

3. Construct your make file

   $ vim Makefile # Edit/Comment out .qmd files not in use.

4. Test make file

   $ make -n

5. Run make file

   $ make

Alternatively this quarto workflow may be rendered in separate parts using RStudio

Support

If you need help, open an issue with one of the following labels:

• help wanted (extra attention is needed)
• question (further information is requested)

Feedback

If you have any suggestions, open an issue with one of the following labels:

• documentation (improvements or additions to documentation)
• enhancement (new feature or request)

Contributing

To contribute to Visium, clone this repository locally and commit your code on a separate branch. Please generate unit tests for your code and run a linter before opening a pull request.

You can find more details in the Contributing guide.

Participation in this project is subject to a Code of Conduct.

Authors

Visium was developed by Ben Southgate and James Ashmore.

If you would like to be added to this list, please open a pull request with your contribution.

Citation

Used By

Visium is used by the following companies and institutes:

• Zifornd

If you would like to be added to this list, please open a pull request with your information.

Acknowledgements

This work was based primarily on the Seurat workflow for analysis of spatial transcriptomics data. Please see below for relevant citations:

Hao Y, Hao S, Andersen-Nissen E, Mauck WM 3rd, Zheng S, Butler A, Lee MJ, Wilk AJ, Darby C, Zager M, Hoffman P, Stoeckius M, Papalexi E, Mimitou EP, Jain J, Srivastava A, Stuart T, Fleming LM, Yeung B, Rogers AJ, McElrath JM, Blish CA, Gottardo R, Smibert P, Satija R. Integrated analysis of multimodal single-cell data. Cell. 2021 Jun 24;184(13):3573-3587.e29. doi: 10.1016/j.cell.2021.04.048. Epub 2021 May 31. PMID: 34062119; PMCID: PMC8238499.

Stuart T, Butler A, Hoffman P, Hafemeister C, Papalexi E, Mauck WM 3rd, Hao Y, Stoeckius M, Smibert P, Satija R. Comprehensive Integration of Single-Cell Data. Cell. 2019 Jun 13;177(7):1888-1902.e21. doi: 10.1016/j.cell.2019.05.031. Epub 2019 Jun 6. PMID: 31178118; PMCID: PMC6687398.

Hafemeister, C., Satija, R. Normalization and variance stabilization of single-cell RNA-seq data using regularized negative binomial regression. Genome Biol 20, 296 (2019). https://doi.org/10.1186/s13059-019-1874-1

Amezquita, R.A., Lun, A.T.L., Becht, E. et al. Orchestrating single-cell analysis with Bioconductor. Nat Methods 17, 137–145 (2020). https://doi.org/10.1038/s41592-019-0654-x

The workflow was motivated by the following projects:

• Seurat
• OSCA
• 10XGenomics/spaceranger

The documentation was informed by the following articles:

• easiest way to create a readme
• writing a friendly readme
• writing well for the web

License

Visium is licensed under the MIT license.
Copyright © 2022, Zifo rnd