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      Single Cell RNA-Seq Analysis

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Introduction and Lectures
Intro to the Workshop and Core
Schedule
What is Bioinformatics/Genomics?
Experimental Design and Cost Estimation
Support
Slack
Zoom
Cheat Sheets
Software and Links
Scripts
Prerequisites
CLI
R
Data Reduction
Files and Filetypes
Project setup
Generating Expression Matrix
scRNAseq Analysis
Prepare scRNAseq Analysis
scRNAseq Analysis - PART1
scRNAseq Analysis - PART2
scRNAseq Analysis - PART3
scRNAseq Analysis - PART4
scRNAseq Analysis - PART5
scRNAseq Analysis - PART6
scRNAseq Analysis - PART7
ETC
Closing thoughts
Workshop Photos
Github page
Biocore website

Create a new RStudio project

Open RStudio and create a new project, for more info see Using-Projects:

File > New Project > New Directory > New Project

Name the new directory (e.g. Differential_Expression), and check “use renv with this project” if present.

Learn more about renv.

Run the following commands to set some options and make sure the packages Seurat, ggplot2, dplyr, limma, and topGO are installed (if not install it), and then load them and verify they all loaded correctly.

In the R console run the following commands:

if (!requireNamespace("BiocManager", quietly = TRUE)){
    install.packages("BiocManager")
}

if (!any(rownames(installed.packages()) == "rmarkdown")){
  BiocManager::install("rmarkdown")
}

if (!any(rownames(installed.packages()) == "tinytex")){
  BiocManager::install("tinytex")
}

if (!any(rownames(installed.packages()) == "Seurat")){
  BiocManager::install("Seurat")
}

if (!any(rownames(installed.packages()) == "hdf5r")){
  BiocManager::install("hdf5r")
}

if (!any(rownames(installed.packages()) == "knitr")){
  BiocManager::install("knitr")
}

if (!any(rownames(installed.packages()) == "kableExtra")){
  BiocManager::install("kableExtra")
}

if (!any(rownames(installed.packages()) == "ggplot2")){
  BiocManager::install("ggplot2")
}

if (!any(rownames(installed.packages()) == "dplyr")){
  BiocManager::install("dplyr")
}

if (!any(rownames(installed.packages()) == "reshape2")){
  BiocManager::install("reshape2")
}

if (!any(rownames(installed.packages()) == "biomaRt")){
  BiocManager::install("biomaRt")
}

if (!any(rownames(installed.packages()) == "org.Hs.eg.db")){
  BiocManager::install("org.Hs.eg.db")
}

if (!any(rownames(installed.packages()) == "limma")){
  BiocManager::install("limma")
}

if (!any(rownames(installed.packages()) == "topGO")){
  BiocManager::install("topGO")
}

if (!any(rownames(installed.packages()) == "remotes")){
  utils::install.packages("remotes")
}

if (!any(rownames(installed.packages()) == "ape")){
  utils::install.packages("ape")
}

if (!any(rownames(installed.packages()) == "DoubletFinder")){
  remotes::install_github('chris-mcginnis-ucsf/DoubletFinder')
}

## All of these should now load without error.

library(rmarkdown)
library(tinytex)
library(Seurat)
library(hdf5r)
library(knitr)
library(kableExtra)
library(ggplot2)
library(dplyr)
library(reshape2)
library(biomaRt)
library(limma)
library(topGO)
library(org.Hs.eg.db)

sessionInfo()

Download the template Markdown workshop document PART1 and open it.

In the R console run the following command to download part 1 of data analysis

download.file("https://raw.githubusercontent.com/ucdavis-bioinformatics-training/2022-December-Single-Cell-RNA-Seq-Analysis/main/data_analysis/scRNA_Workshop-PART1.Rmd", "scRNA_Workshop-PART1.Rmd")

Download the data for the workshop, extract it.

In the R console run the following command to download and extract the dataset (Little over 160MB file).

options(timeout=3000)
download.file("https://bioshare.bioinformatics.ucdavis.edu/bioshare/download/feb28v7lew62um4/expression_data_cellranger.zip", "expression_data_cellranger.zip")
system("unzip expression_data_cellranger.zip") # works in Linux and Mac, not sure about Windows

This way of downloading the file might be very slow (could take > 1hr). So, the recommended way to download the file is to use scp on Mac/Windows Powershell, or Filezilla/WinSCP on Windows.

scp username@tadpole.genomecenter.ucdavis.edu:/share/workshop/scRNA_workshop/cellranger.outs/expression_data_cellranger.zip ./

If the system command didn’t work in extracting the zip file, navigate to the folder you downloaded the data in and manually unzip the archive file. Please make sure you see three folders: A001-C-007, A001-C-104 and B001-A-301. Make sure “scRNA_Workshop-PART1.Rmd” file is in the same location.

Edit the file YAML portion

Please open the downloaded “scRNA_Workshop-PART1.Rmd” in RStudio. The top YAML (YAML ain’t markup language) portion of the doc tells RStudio how to parse the document.

---
title: "Introduction to Single Cell RNAseq Part 1"
author: your_name
date: current_date
output:
    html_notebook: default
    html_document: default
---

Your RStudio should look something like this

RStudio

Now spend a few minutes navigating through our data. How may samples are there? Find the hdf5 file and the matrix files. View the HTML files and let’s discuss.