Contents

1 Introduction

RCSL is an R toolkit for single-cell clustering and trajectory analysis using single-cell RNA-seq data.

2 Installation

2.0.1 Install RCSL package and other requirements

RCSL can be installed directly from GitHub with ‘devtools’.

library(devtools)
devtools::install_github("QinglinMei/RCSL")

Now we can load RCSL. We also load the SingleCellExperiment, ggplot2 and igraph package.

library(RCSL)
library(SingleCellExperiment)
#> Loading required package: SummarizedExperiment
#> Loading required package: MatrixGenerics
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library(ggplot2)
library(igraph)
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library(umap)

3 Run RCSL

3.1 Load dataset (yan)

We illustrate the usage of RCSL on a human preimplantation embryos and embryonic stem cells(Yan et al., (2013)). The yan data is distributed together with the RCSL package, with 90 cells and 20,214 genes:

head(ann)
#>                 cell_type1
#> Oocyte..1.RPKM.     zygote
#> Oocyte..2.RPKM.     zygote
#> Oocyte..3.RPKM.     zygote
#> Zygote..1.RPKM.     zygote
#> Zygote..2.RPKM.     zygote
#> Zygote..3.RPKM.     zygote
yan[1:3, 1:3]
#>          Oocyte..1.RPKM. Oocyte..2.RPKM. Oocyte..3.RPKM.
#> C9orf152             0.0             0.0             0.0
#> RPS11             1219.9          1021.1           931.6
#> ELMO2                7.0            12.2             9.3
origData <- yan
label <- ann$cell_type1

3.2 1. Pre-processing

In practice, we find it always beneficial to pre-process single-cell RNA-seq datasets, including: 1. Log transformation. 2. Gene filter

data <- log2(as.matrix(origData) + 1)
gfData <- GenesFilter(data)

3.3 2. Calculate the initial similarity matrix S

resSimS <- SimS(gfData)
#> Calculate the Spearman correlation 
#> Calculate the Nerighbor Representation 
#> Find neighbors by KNN(Euclidean)

3.4 3. Estimate the number of clusters C

Estimated_C <- EstClusters(resSimS$drData,resSimS$S)
#> ======== Calculate maximal strongly connected components ======== 
#> ======== Calculate maximal strongly connected components ======== 
#> ======== Calculate maximal strongly connected components ========

3.5 4. Calculate the block diagonal matrix B

resBDSM <- BDSM(resSimS$S, Estimated_C)
#> ======== Calculate maximal strongly connected components ========

4 Calculate accuracy of the clustering

ARI_RCSL <- igraph::compare(resBDSM$y, label, method = "adjusted.rand")

5 Trajectory analysis to time-series datasets

DataName <- "Yan"
res_TrajecAnalysis <- TrajectoryAnalysis(gfData, resSimS$drData, resSimS$S,
                                         clustRes = resBDSM$y, TrueLabel = label, 
                                         startPoint = 1, dataName = DataName)

6 Display the constructed MST

res_TrajecAnalysis$MSTPlot

7 Display the plot of the pseudo-temporal ordering

res_TrajecAnalysis$PseudoTimePlot

8 Display the plot of the inferred developmental trajectory

res_TrajecAnalysis$TrajectoryPlot