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.
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).
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.
install.packages("BiocManager")
BiocManager::install(c("AnnotationHub", "BiocParallel", "BiocSingular",
"DropletUtils", "PCAtools", "batchelor",
"bluster", "cluster", "clustree", "dynamicTreeCut",
"edgeR", "ensembldb", "ggplot2", "igraph",
"patchwork", "pheatmap", "scater", "scran", "tidyverse"))
For Cellranger, you will need to use a Linux machine. The instructions below are based on the installation instructions from 10x Genomics.
Create a directory for your software. In this example, we will use $HOME/software
:
mkdir -p $HOME/software
cd $HOME/software
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"
Unpack the downloaded file:
tar -xzvf cellranger-9.0.0.tar.gz
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.
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!