NathanSkene / liger

R package for integrating and analyzing multiple single-cell datasets

LIGER (Linked Inference of Genomic Experimental Relationships)

LIGER (liger) is a package for integrating and analyzing multiple single-cell datasets, developed and maintained by the Macosko lab. It relies on integrative non-negative matrix factorization to identify shared and dataset-specific factors.

Check out our Cell paper for a more complete description of the methods and analyses. To access data used in our SN and BNST analyses, visit our study on the Single Cell Portal.

LIGER can be used to compare and contrast experimental datasets in a variety of contexts, for instance:

• Across experimental batches
• Across individuals
• Across sex
• Across tissues
• Across species (e.g., mouse and human)
• Across modalities (e.g., scRNAseq and spatial transcriptomics data, scMethylation, or scATAC-seq)

Once multiple datasets are integrated, the package provides functionality for further data exploration, analysis, and visualization. Users can:

• Identify clusters
• Find significant shared (and dataset-specific) gene markers
• Compare clusters with previously identified cell types
• Visualize clusters and gene expression using t-SNE and UMAP

We have also designed LIGER to interface with existing single-cell analysis packages, including Seurat.

Usage

For usage examples and guided walkthroughs, check the vignettes directory of the repo.

• Basic commands tutorial

• Walkthrough with two PBMC datasets

Installation

liger is written in R and has a few other system requirements (Java) and recommended packages (umap in Python). To install the most recent development version, follow these instructions:

1. Install R (>= 3.4)
2. Install Rstudio (recommended)
3. Make sure you have Java installed in your machine. Check by typing java -version into Terminal or Command Prompt.
4. Use the following R commands.

install.packages('devtools')
library(devtools)
install_github('MacoskoLab/liger')

Additional Installation Steps for MacOS (recommended before step 4)

Installing RcppArmadillo on R>=3.4 requires Clang >= 4 and gfortran-6.1. Follow the instructions below if you have R version 3.4.0-3.4.4. These instructions (using clang4) may also be sufficient for R>=3.5 but for newer versions of R, it's recommended to follow the instructions in this post.

1. Install gfortran as suggested here
2. Download clang4 from this page
3. Uncompress the resulting zip file and type into Terminal (sudo if needed):

mv /path/to/clang4/ /usr/local/ 

4. Create .R/Makevars file containing following:

# The following statements are required to use the clang4 binary
CC=/usr/local/clang4/bin/clang
CXX=/usr/local/clang4/bin/clang++
CXX11=/usr/local/clang4/bin/clang++
CXX14=/usr/local/clang4/bin/clang++
CXX17=/usr/local/clang4/bin/clang++
CXX1X=/usr/local/clang4/bin/clang++
LDFLAGS=-L/usr/local/clang4/lib

For example, use the following Terminal commands:

cd ~
mkdir .R
cd .R 
nano Makevars

Paste in the required text above and save with Ctrl-X.

Running liger with Docker

If installing natively is difficult, you can run liger through our Docker image (available publically), which also comes with Rstudio and Seurat (v2) installed.

1. Install Docker.
2. Run the following in terminal:

docker run -d -p 8787:8787 docker.io/vkozareva/sc-liger:latest

3. Type http://localhost:8787 in any browser and enter "rstudio" as the username and password when prompted. liger and all of its dependencies are already installed in this environment.

If you wish to access local files in this container (mounting to /data) modify the command as follows:

docker run -d -v /path/to/local/directory:/data -p 8787:8787 docker.io/vkozareva/sc-liger:latest

Note that you will have to stop the container if you wish to allocate port 8787 to another application later on. Further Docker documentation can be found here.

Detailed Instructions for FIt-SNE Installation for use in runTSNE (recommended for large datasets)

Using FIt-SNE is recommended for computational efficiency when using runTSNE on very large datasets. Installing and compiling the necessary software requires the use of git, FIt-SNE, and FFTW. For a basic overview of installation, visit this page.

Basic installation for most Unix machines can be achieved with the following commands after downloading the latest version of FFTW from here. In the fftw directory, run:

./configure
make
make install

(Additional instructions if necessary). Then in desired directory:

git clone https://github.com/KlugerLab/FIt-SNE.git
cd FIt-SNE
g++ -std=c++11 -O3  src/sptree.cpp src/tsne.cpp src/nbodyfft.cpp  -o bin/fast_tsne -pthread -lfftw3 -lm
pwd

Use the output of pwd as the fitsne.path parameter in runTSNE.

Note that the above instructions require root access. To install into a specified folder (such as your home directory) on a server, use the --prefix option:

./configure --prefix=<install_dir>
make
make install
git clone https://github.com/KlugerLab/FIt-SNE.git
cd FIt-SNE
g++ -std=c++11 -O3  src/sptree.cpp src/tsne.cpp src/nbodyfft.cpp  -I<install_dir>/include/ -L<install_dir>/lib/ -o bin/fast_tsne -pthread -lfftw3 -lm
pwd