SingleCell_RNAseq_Mar25

Introduction to single-cell RNA-seq data analysis

27th, 28th, 31st March 2025 || 09:30 - 17:00

In-person at the Craik Marshall training room (map)

Instructors

Outline

This workshop is aimed at biologists interested in learning how to perform standard single-cell RNA-seq analyses.

This will focus on the droplet-based assay by 10X genomics and include running the accompanying cellranger pipeline to align reads to a genome reference and count the number of read per gene, reading the count data into R, quality control, normalisation, data set integration, clustering and identification of cluster marker genes, as well as differential expression and abundance analyses. You will also learn how to generate common plots for analysis and visualisation of gene expression data, such as TSNE, UMAP and violin plots.

Prerequisites

**Some basic experience of using a UNIX/LINUX command line is assumed**

**Some R knowledge is assumed and essential. Without it, you will struggle on this course.** If you are not familiar with the R statistical programming language we strongly encourage you to work through an introductory R course before attempting these materials. We recommend our Introduction to R course.

Data

Schedule

PDF of materials: if you want a PDF version of the materials go to the “Print” option on your browser and select “Print to PDF” (all major browsers have this functionality).

Day 1

Day 2

Day 3

Extended Materials

Software Installation

You can make use of the computer environment provided for the course, which is ready for use and have the necessary data & software installed. However, if you want to run the analysis on your own computer, you can follow these instructions.

R and Bioconductor packages

Cellranger

For Cellranger, you will need to use a Linux machine. The instructions below are based on the installation instructions from 10x Genomics.

  1. Create a directory for your software. In this example, we will use $HOME/software:

     mkdir -p $HOME/software
     cd $HOME/software
    
  2. Download cellranger from 10xgenomics. You will need to register with 10x, and they will then provide you with a link to download a .tar.gz archive file. You can use wget to download the file:

     wget -O cellranger-9.0.0.tar.gz "YOUR-LINK-HERE"
    
  3. Unpack the downloaded file:

     tar -xzvf cellranger-9.0.0.tar.gz
    
  4. Add the Cellranger directory to your $PATH, allowing you to use the cellranger command from any location:

     export PATH=$HOME/software/cellranger-9.0.0:$PATH
    

    You may want to add this command to your $HOME/.bashrc for convenience.

Acknowledgments:

Much of the material in this course has been derived from the demonstrations found in OSCA book and the Hemberg Group course materials. Additional material concerning miloR has been based on the demonstration from the Marioni Lab.

The materials have been contributed to by many individuals over the last 2 years, including:

Abigail Edwards, Ashley D Sawle, Chandra Chilamakuri, Kamal Kishore, Stephane Ballereau, Zeynep Kalendar Atak, Hugo Tavares, Jon Price, Katarzyna Kania, Roderik Kortlever, Adam Reid, Tom Smith, Jiawei Wang

Apologies if we have missed anyone!