Repository files navigation

• Introduction
• Basics of using conos
• Tutorials
  • Conos walkthrough
  • Adjustment of alignment strength with conos
  • Integration with Scanpy
  • Integrating RNA-seq and ATAC-seq with conos
  • Running RNA velocity on a Conos object
• Installation
  • Running conos via Docker
• References

• What is conos?Conos is an R package to wire together large collections of single-cell RNA-seq datasets, which allows for both the identification of recurrent cell clusters and the propagation of information between datasets in multi-sample or atlas-scale collections. It focuses on the uniform mapping of homologous cell types across heterogeneous sample collections. For instance, users could investigate a collection of dozens of peripheral blood samples from cancer patients combined with dozens of controls, which perhaps includes samples of a related tissue such as lymph nodes.

• How does it work?Conos applies one of many error-prone methods to align each pair of samples in a collection, establishing weighted inter-sample cell-to-cell links. The resulting joint graph can then be analyzed to identify subpopulations across different samples. Cells of the same type will tend to map to each other across many such pairwise comparisons, forming cliques that can be recognized as clusters (graph communities).

  Conos processing can be divided into three phases:

  • Phase 1: Filtering and normalization Each individual dataset in the sample panel is filtered and normalized using standard packages for single-dataset processing: eitherpagoda2 orSeurat. Specifically, Conos relies on these methods to perform cell filtering, library size normalization, identification of overdispersed genes and, in the case of pagoda2, variance normalization. (Conos is robust to variations in the normalization procedures, but it is recommended that all of the datasets be processed uniformly.)
  • Phase 2: Identify multiple plausible inter-sample mappings Conos performs pairwise comparisons of the datasets in the panel to establish an initial error-prone mapping between cells of different datasets.
  • Phase 3: Joint graph construction These inter-sample edges from Phase 2 are then combined with lower-weight intra-sample edges during the joint graph construction. The joint graph is then used for downstream analysis, including community detection and label propagation. For a comprehensive description of the algorithm, please refer to ourpublication.

• What does it produce?In essence, conos will take a large, potentially heterogeneous panel of samples and will produce clustering grouping similar cell subpopulations together in a way that will be robust to inter-sample variation:
  

• What are the advantages over existing alignment methods?Conos is robust to heterogeneity of samples within a collection, as well as noise. The ability to resolve finer subpopulation structure improves as the size of the panel increases.

Given a list of individual processed samples (pl), conos processing can be as simple as this:

# Construct Conos object, where pl is a list of pagoda2 objectscon<-Conos$new(pl)# Build joint graphcon$buildGraph()# Find communitiescon$findCommunities()# Generate embeddingcon$embedGraph()# Plot joint graphcon$plotGraph()# Plot panel with joint clustering resultscon$plotPanel()

To see more documentation on the classConos, run?Conos.

Please see the following tutorials for detailed examples of how to use conos:

• HTML version
• Markdown version

• HTML version
• Markdown version

Note that for integration withScanpy, users need to save conos files to disk from an R session, and then load these files into Python.

Save conos for Scanpy:

• HTML version
• Markdown version

Load conos files into Scanpy:

• Jupyter Notebook

• HTML version
• Markdown version

First of all, in order to obtain an RNA velocity plot from aConos object you have to use thedropEst pipeline to align and annotate your single-cell RNA-seq measurements. You can seethis tutorial andthis shell script to see how it can be done. In this example we specifically assume that when running dropEst you have used the-V option to get estimates of unspliced/spliced counts from the dropEst directly. Secondly, you need thevelocyto.R package for the actual velocity estimation and visualisation.

After running dropEst you should have 2 files for each of the samples:

• sample.rds (matrix of counts)
• sample.matrices.rds (3 matrices of exons, introns and spanning reads)

The.matrices.rds files are the velocity files. Load them into R in a list (same order as you give to conos). Load, preprocess and integrate with conos the count matrices (.rds) as you normally would. Before running the velocity, you must at least create an embedding and run the leiden clustering. Finally, you can estimate the velocity as follows:

### Assuming con is your Conos object and cms.list is the list of your velocity files ###library(velocyto.R)# Preprocess the velocity files to match the Conos objectvi<- velocityInfoConos(cms.list=cms.list,con=con,n.odgenes=2e3,verbose=TRUE)# Estimate RNA velocityvel.info<-vi %$%  gene.relative.velocity.estimates(emat,nmat,cell.dist=cell.dist,deltaT=1,kCells=25,fit.quantile=0.05,n.cores=4)# Visualise the velocity on your Conos embedding# Takes a very long time!# Assign to a variable to speed up subsequent recalculationscc.velo<- show.velocity.on.embedding.cor(vi$emb,vel.info,n=200,scale='sqrt',cell.colors= ac(vi$cell.colors,alpha=0.5),cex=0.8,grid.n=50,cell.border.alpha=0,arrow.scale=3,arrow.lwd=0.6,n.cores=4,xlab="UMAP1",ylab="UMAP2")# Use cc=cc.velo$cc when running again (skips the most time consuming delta projections step)show.velocity.on.embedding.cor(vi$emb,vel.info,cc=cc.velo$cc,n=200,scale='sqrt',cell.colors= ac(vi$cell.colors,alpha=0.5),cex=0.8,arrow.scale=15,show.grid.flow=TRUE,min.grid.cell.mass=0.5,grid.n=40,arrow.lwd=2,do.par=F,cell.border.alpha=0.1,n.cores=4,xlab="UMAP1",ylab="UMAP2")

To install the stable version fromCRAN, use:

install.packages('conos')

To install the latest version ofconos, use:

install.packages('devtools')devtools::install_github('kharchenkolab/conos')

The dependencies are inherited frompagoda2. Note that this package also has the dependencyigraph, which requires various libraries to install correctly. Please see the installation instructions at that page for more details, along with the github READMEhere.

To install system dependencies usingapt-get, use the following:

sudo apt-get updatesudo apt-get -y install libcurl4-openssl-dev libssl-dev libxml2-dev libgmp-dev libglpk-dev

For Red Hat distributions usingyum, use the following command:

sudo yum updatesudo yum install openssl-devel libcurl-devel libxml2-devel gmp-devel glpk-devel

Using the Mac OS package managerHomebrew, try the following command:

brew updatebrew install openssl curl-openssl libxml2 glpk gmp

(You may need to runbrew uninstall curl in order forbrew install curl-openssl to be successful.)

As of version 1.3.1,conos should successfully install on Mac OS. However, if there are issues, please refer to the following wiki page for further instructions on installingconos with Mac OS:Installing conos for Mac OS

If your system configuration is making it difficult to installconos natively, an alternative way to getconos running is through a docker container.

Note: On Mac OS X, Docker Machine has Memory and CPU limits. To control it, please check instructions either forCLI or forDocker Desktop.

The docker distribution has the latest version and also includes thepagoda2 package. To start a docker container, firstinstall docker on your platform and then start thepagoda2 container with the following command in the shell:

docker run -p 8787:8787 -e PASSWORD=pass pkharchenkolab/conos:latest

The first time you run this command, it will download several large images so make sure that you have fast internet access setup. You can then point your browser tohttp://localhost:8787/ to get an Rstudio environment withpagoda2 andconos installed (please log in using credentials username=rstudio, password=pass). Explore thedocker --mount option to allow access of the docker image to your local files.

Note: If you already downloaded the docker image and want to update it, please pull the latest image with:

docker pull pkharchenkolab/conos:latest

If you want to build image by your own, download theDockerfile (available in this repo under/docker) and run to following command to build it:

docker build -t conos .

This will create a "conos" docker image on your system (please be patient, as the build could take approximately 30-50 minutes to finish).You can then run it using the following command:

docker run -d -p 8787:8787 -e PASSWORD=pass --name conos -it conos

If you find this software useful for your research, please cite the correspondingpaper:

Barkas N., Petukhov V., Nikolaeva D., Lozinsky Y., Demharter S., Khodosevich K., & Kharchenko P.V. Joint analysis of heterogeneous single-cell RNA-seq dataset collections. Nature Methods, (2019). doi:10.1038/s41592-019-0466-z

The R package can be cited as:

Viktor Petukhov, Nikolas Barkas, Peter Kharchenko, and EvanBiederstedt (2021). conos: Clustering on Network of Samples. Rpackage version 1.5.2.