Scanorama

Overview

Scanorama enables batch-correction and integration of heterogeneous scRNA-seq data sets, which is described in the paper "Efficient integration of heterogeneous single-cell transcriptomes using Scanorama" by Brian Hie, Bryan Bryson, and Bonnie Berger. This repository contains the Scanorama source code as well as scripts necessary for reproducing the results in the paper.

Scanorama is designed to be used in scRNA-seq pipelines downstream of noise-reduction methods, including those for imputation and highly-variable gene filtering. The results from Scanorama integration and batch correction can then be used as input to other tools for scRNA-seq clustering, visualization, and analysis.

Tools for data sketching can also greatly accelerate Scanorama integration, as described in the paper "Geometric sketching compactly summarizes the single-cell transcriptomic landscape" and implemented here.

API example usage

Here is example usage of Scanorama in Python:

# List of data sets (matrices of cells-by-genes):
datasets = [ list of scipy.sparse.csr_matrix or numpy.ndarray ]
# List of gene lists:
genes_list = [ list of list of string ]

import scanorama

# Integration.
integrated, genes = scanorama.integrate(datasets, genes_list)

# Batch correction.
corrected, genes = scanorama.correct(datasets, genes_list)

# Integration and batch correction.
integrated, corrected, genes = scanorama.correct(datasets, genes_list, return_dimred=True)

Parameter documentation for each method is in the Scanorama source code at the top of scanorama/scanorama.py.

There are also wrappers that make it easy to use Scanorama with scanpy's AnnData object:

# List of data sets:
adatas = [ list of scanpy.AnnData ]

import scanorama

# Integration.
integrated = scanorama.integrate_scanpy(adatas)

# Batch correction.
corrected = scanorama.correct_scanpy(adatas)

# Integration and batch correction.
integrated, corrected = scanorama.correct_scanpy(adatas, return_dimred=True)

You can also call Scanorama from R using the reticulate package (tested with R version 3.5.1 and reticulate version 1.10):

# List of data sets (matrices of cells-by-genes):
datasets <- list( list of matrix )
# List of gene lists:
genes_list <- list( list of list of string )

library(reticulate)
scanorama <- import('scanorama')

# Integration.
integrated.data <- scanorama$integrate(datasets, genes_list)

# Batch correction.
corrected.data <- scanorama$correct(datasets, genes_list, return_dense=TRUE)

# Integration and batch correction.
integrated.corrected.data <- scanorama$correct(datasets, genes_list,
                                               return_dimred=TRUE, return_dense=TRUE)

Note that reticulate has trouble returning sparse matrices, so you should set the return_dense flag to TRUE (which returns the corrected data as R matrix objects) when attempting to use Scanorama's correct() method in R. This will increase memory usage, however, especially for very large data sets.

Installation

Setup

You should be able to download Scanorama using pip:

pip install scanorama

If for some reason this doesn't work, you can also install from within the Scanorama repository:

git clone https:/brianhie/scanorama.git
cd scanorama/
python setup.py install --user

If you are running inside an anaconda environment, first install annoy by doing:

conda install -c conda-forge python-annoy

Examples from paper

Data set download

All of the data used in our study (around 4 GB) can be downloaded from http://scanorama.csail.mit.edu/data.tar.gz. Download and unpack this data with the command:

wget http://scanorama.csail.mit.edu/data.tar.gz
tar xvf data.tar.gz

A smaller version of the data (around 720 MB), including 26 heterogeneous data sets, can be similarly downloaded from http://scanorama.csail.mit.edu/data_light.tar.gz.

Data processing

The script bin/process.py can handle two file formats. The first is a tab-delimited table format where the columns correspond to cells and the rows correspond to genes. A sample file looks something like:

gene	cell_a	cell_b
gene_1	10	10
gene_2	20	20

The second is a sparse matrix format used by 10X Genomics (example here). This format has a directory where one file has a list of gene names (genes.tsv) and one file has a list of the nonzero transcript counts at certain gene/cell coordinates (matrix.mtx).

To ensure a consistent data format, the examples first processes these raw files and saves them in .npz files along with some related metadata. To generate these files, run the command:

python bin/process.py conf/panorama.txt

The corresponding .npz files will be saved in the data/ directory.

New files can be processed by feeding them into bin/process.py via the command line or a configuration file, or by modifying the data_names variables at the top of bin/config.py.

Panorama stitching

Toy data sets

For a good illustration of how Scanorama works, we can integrate three toy data sets: 293T cells, Jurkat cells, and a 50:50 293T:Jurkat mixture. To integrate these data sets, run:

python bin/293t_jurkat.py

By default, this prints a log reporting the alignments the algorithm has found between data sets and saves visualization images to a file in the repository's top-level directory.

26 data sets

We can also stitch a much larger number of cells from many more datsets. To do this, run

python bin/integration_panorama.py conf/panorama.txt

to integrate the data sets or

python bin/panorama.py conf/panorama.txt

to batch correct the data sets as well. The collection of data sets to be integrated is specified in the config file conf/panorama.txt. Default parameters are listed at the top of scanorama/scanorama.py.

By default, this script will output a verbose log as it finds alignments and applies batch correction. At the end, it will automatically save t-SNE visualized images of the integrated result. The numpy matrices containing the batch-corrected data sets are also available (in memory) to integrate with other single cell pipelines and packages.

Runtime performance and memory requirements

Scanorama runs on multiple cores to speed up its computation; here are some instructions to check if Python is making use of the benefits from multicore processing. Aligning and batch-correcting 105,476 cells across 26 data sets should complete in around 15 minutes with the process running on 10 cores. The memory usage should be under 8 GB for integration and under 26 GB for batch correction.

Note that the gradient descent portion of the t-SNE visualization step can take a very long time (a few hours) and require a lot of memory (around 30 GB) on more than 100k cells. Other methods for accelerating t-SNE could be used in place of the t-SNE implementation used in this pipeline, such as a faster C++ implementation of t-SNE, Multicore-TSNE, or net-SNE, a version of t-SNE that uses a neural network to reduce the time required for the gradient descent optimization procedure.

Additional analyses from paper

Scripts for performing additional analyses of the data are also available in the bin/ directory.

Scanorama implementation

For those interested in the algorithm implementation, scanorama/scanorama.py is the main file that handles the mutual nearest neighbors-based matching, batch correction, and panorama assembly.

Troubleshooting

• Make sure the input matrices are cells-by-genes, not the transpose.

• For large data set integration under memory constraints (e.g., if you run into a MemoryError), try lowering the batch_size parameter to improve memory usage and try sketch-based acceleration using the sketch parameter to integrate() to improve both memory usage and runtime.

• Some users report "Illegal instruction" or "Segfault" errors using the most recent versions of the annoy package; Scanorama is tested with annoy version 1.11.5 on Ubuntu 18.04.

• For the example scripts, be sure to run bin/process.py first, although this is not necessary if you are using Scanorama through the API.

Questions

For questions about the pipeline and code, contact [email protected]. We will do our best to provide support, address any issues, and keep improving this software. And do not hesitate to submit a pull request and contribute!