Install packages

Install package:

if(!require(devtools)){
  install.packages("devtools") # If not already installed
}
#> Loading required package: devtools
devtools::install_github("TomKellyGenetics/DoubletDetection", ref = "r-implementation")
#> Skipping install of 'DoubletDetection' from a github remote, the SHA1 (2f420ba5) has not changed since last install.
#>   Use `force = TRUE` to force installation

Install dependancies:

#devtools::install_github("JinmiaoChenLab/Rphenograph")
devtools::install_github("TomKellyGenetics/Rphenograph")
install.packages("hdf5r")
install.packages("Matrix")

Install hdf5 (if needed)

OS X (using Homebrew) brew install hdf5

Debian-based systems (including Ubuntu) sudo apt-get install libhdf5-dev

Systems supporting yum and RPMs sudo yum install hdf5-devel

HDF5 1.8.14 has been pre-compiled for Windows and is available at https://github.com/mannau/h5-libwin

load package (and dependancies)

library("DoubletDetection")
#> Loading required package: Matrix
#> Loading required package: Rphenograph
#> Loading required package: gplots
#> 
#> Attaching package: 'gplots'
#> The following object is masked from 'package:stats':
#> 
#>     lowess
#> Loading required package: Rtsne
#> Warning: replacing previous import 'Matrix::cov2cor' by 'stats::cov2cor'
#> when loading 'DoubletDetection'
#> Warning: replacing previous import 'Matrix::toeplitz' by 'stats::toeplitz'
#> when loading 'DoubletDetection'
#> Warning: replacing previous import 'gplots::lowess' by 'stats::lowess' when
#> loading 'DoubletDetection'
#> Warning: replacing previous import 'Matrix::update' by 'stats::update' when
#> loading 'DoubletDetection'

Download Data from 10X Genomics

These commands will be called from R in Linux and Mac systems. The files will need to be downloaded from the given url (into the working directory) in Windows.

#download files
if(!(file.exists("pbmc8k_filtered_gene_bc_matrices.tar.gz"))){
  system("wget http://cf.10xgenomics.com/samples/cell-exp/2.1.0/pbmc8k/pbmc8k_filtered_gene_bc_matrices.tar.gz")
  #extract compressed files 
  #system("tar -xvzf pbmc8k_filtered_gene_bc_matrices.tar.gz")
}

Load Counts Matrix

matrix_path <- 'filtered_gene_bc_matrices/GRCh38/matrix.mtx'
raw_counts <- DoubletDetection::load_mtx(matrix_path)
# Remove columns with all 0s
non_zero_genes <- apply(raw_counts, 1, function(x) sum(x) != 0)
raw_counts <- raw_counts[non_zero_genes, ]

Run Doublet Detection

Right now, phenograph is a bit talkative, so we suppress the output to avoid lots of text:

#> Warning in .Object$initialize(...): numeric input for n_iters taken as an
#> integer
#> [1] "function sum accepted as new_lib_as"
#> [1] "Reference Class BoostClassifier has been initialized"
#> Warning in clf$fit(raw_counts): Sparse raw_counts is automatically
#> densified
#> [1] "Iteration 1/50"
#> [1] "Creating downsampled doublets..."
#> [1] "running downsamplePair for synthetic cell 1"
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#> [1] "Normalizing..."
#> [1] "Running PCA..."
#> [1] "Clustering augmented data set with Phenograph..."
#> [1] "Setting directed to FALSE because prune is TRUE"
#> Run Rphenograph starts:
#>   -Input data of 10476 rows and 30 columns
#>   -k is set to 30
#>   Finding nearest neighbors...DONE ~ 4.387 s
#>   Compute jaccard coefficient between nearest-neighbor sets...DONE ~ 3.963 s
#>   Build undirected graph from the weighted links...Removing non-symmetric links...DONE ~ 1626.84 s
#>   Run louvain clustering on the graph ...DONE ~ 23.863 s
#> Run Rphenograph DONE, totally takes 1659.05300000001s.
#>   Return a community class
#>   -Modularity value: 0.8274768 
#>   -Number of clusters: 16
#> Error in sizes(fullcommunities[[2]]): could not find function "sizes"
print(paste("Time elapsed", end-start,"seconds,", (end-start) / clf.n_iters, clf.n_iters), "sec/iteration, for", n_inters, "iterations")
#> Error in paste("Time elapsed", end - start, "seconds,", (end - start)/clf.n_iters, : object 'clf.n_iters' not found

Visualize Results

Convergence of Doublet Calls

DoubletDetection::convergence(clf, save='convergence_test.pdf', show=TRUE)
#> [1] "Saving tSNE plot as file: convergence_test.pdf"
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Doublets on tSNE map

tsne_coords <- DoubletDetection::tsne_plot(raw_counts, doublets, save='tsne_test.pdf', show=True)
#> Error in prcomp(aug_counts, rank = n_components, center = TRUE, scale. = TRUE): object 'aug_counts' not found