Awesome

redPATH

redPATH reconstructs the pseudo development time of cell lineages in single-cell RNA-seq data. It formulates the problem of pseudo temporal ordering into a Hamiltonian path problem and attempts to recover the pseudo development time in single-cell RNA-seq datasets. We provide a comprehensive analysis software tool with robust performance.

Overview

Table of content

• Installation
  • Required Packages
  • Install
• Example Usage
  • Preprocessing
  • redPATH pseudotime
  • Biological Analysis
• Citation
• Maintenance

Installation

- Required Packages

car, combinat, doParallel, dplyr, energy, ggplot2, GOsummaries, gplots, MASS, mclust, minerva, plotly, Rcpp, RcppArmadillo, scater

- Install

After downloading the package, please extract and rename the folder to "redPATH"

require(devtools)
setwd("where redPATH folder is located")
install("redPATH", args = c("--no-multiarch")

Example Usage:

- Preprocessing

Assuming your input data is a m genes (rows) by n cells (cols) matrix. Here, a neural stem cell (145 cells) dataset from 2015 (1) is used as an example analysis and a diseased example (MGH107 - 252 cells) dataset is also provided from 2017 (2).

library(redPATH)
input_data <- llorens_exprs # A sample single cell dataset

# Reduce genes to ones which are related to Cell Development, Cell Morphology, Cell Differentiation, Cell Fate and Cell Maturation (from Gene Ontology)
subset_GO_data <- get_GO_exp(input_data, gene_type = "gene_names", organism = "mouse", takelog = F)

# Filter genes using a simple dispersion threshold
filtered_GO_data <- filter_exp(subset_GO_data, dispersion_threshold = 10, threads = 2)

An optional function is also available to identify the G0-like cells:

sample_mean_score <- llorens_reCAT_scores
# Get kmeans clusters
kmeans_clusters <- kmeans_clustering(sample_mean_score$mean_scores, k = 5)

test_for_g0 <- statistical_test(sample_mean_score$mean_scores, kmeans_clusters, threshold = 0.001)

# Example output:
# [1] "Possible group of G0-like cells is: 2"

new_input_data <- input_data[,-which(kmeans_clusters == 2)]

# Evaluation plots:
d1 <- distribution_plot(sample_mean_score$mean_scores, kmeans_clusters)
d2 <- mean_score_plot(sample_mean_score$mean_scores, kmeans_clusters, reCAT_ordIndex)

- redPATH pseudotime

redpath_pseudotime <- redpath(filtered_GO_data, threadnum = 4, base_path_range = c(4:8))
# base_path_range is set to the estimated number of cell types, k0, and k0 +4. 

# Plot specific gene expression along the pseudotime
cdk_gene_exprs <- get_specific_gene_exprs(full_gene_exprs_data = input_data, gene_name = "cdk11", type = "match", organism = "mm")

plot_cdk <- plot_specific_gene_exprs(cdk_gene_exprs, redpath_pseudotime, color_label = cell_type_label, order = T)
require(scater)
multiplot(plotlist = plot_cdk, layout = matrix(c(1:2), 2, 1)) # For a total number of 2 plots

- Biological Analysis

1. Discovery of potential marker genes

# Distance Correlation and Maximal Information Coefficient calculation
dcor_result <- calc_correlation(full_exprs_matrix = input_data, redpath_pseudotime, type = "dcor") 
mic_result <- calc_correlation(full_exprs_matrix = input_data, redpath_pseudotime, type = "mic")

# Union Selection
selected_result <- gene_selection(full_exprs_matrix = input_data, redpath_pseudotime, dcor_result, mic_result, threshold = 0.5)

# Intersection Selection
selected_result <- gene_selection_intersection(full_exprs_matrix = input_data, redpath_pseudotime, dcor_result, mic_result, threshold = 0.5)

# selected_result returns a m genes by n cells matrix with the selected significant genes

2. Heatmap & Gene Ontology Analysis

- Producing heatmaps

# Calculating the ON/HIGH or OFF/LOW state of each cell for each gene.
gene_state_result <- calc_all_hmm_diff_parallel(selected_result, cls_num = 2, threadnum = 4) 

# Optional: plots the top 10 significant genes
hmm_plots <- plot_hmm_full(selected_result, redpath_pseudotime, gene_state_result, cell_type_label = llorens_labels, num_plots = 10)
require(scater)
multiplot(plotlist = hmm_plots, layout = matrix(c(1:10), 5, 2)) # For a total number of 10 plots

# Infer gene clusters using hierarchical clustering
inferred_gene_cluster <- get_gene_cluster(sorted_matrix = gene_state_result, cls_num = 8)

# Heatmap plot
cell_labels <- llorens_labels
heatmap_plot <- plot_heatmap_analysis(sorted_matrix = inferred_gene_cluster, redpath_pseudotime, cell_labels = llorens_labels, gene_cluster = inferred_gene_cluster, input_type = "hmm")

- Gene Ontology plots

GO_summary_result <- infer_GO_summaries(inferred_gene_cluster, organism = "mmusculus")
plot(GO_summary_result, fontsize = 8)

3. 3D Cell proliferation and differentiation plots:

- 3D plot with cell type labels and cell cycle stages

# normalizedResultLst can be loaded from the output .RData file from reCAT.
p1 <- plot_cycle_diff_3d(normalizedResultLst, redpath_pseudotime, cell_cycle_labels, cell_type_labels)
p1

- Plot with marker gene

# normalizedResultLst can be loaded from the output .RData file from reCAT.

# get specific genes of interest:
cdk_gene_exprs <- get_specific_gene_exprs(input_data, gene_name = "cdk11", type = "match", organism = "mm")
p1 <- plot_diff_3d(normalizedResultLst, redpath_pseudotime, cell_cycle_labels, cdk_gene_exprs)
p1

Citation & References

This work is currently under submission. Reconstructing the pseudo development time of cell lineages in single-cell RNA-seq data and applications in cancer.

References:

1. Llorens-Bobadilla, E., Zhao, S., Baser, A., Saiz-Castro, G., Zwadlo, K. and Martin-Villalba, A. (2015) Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury. Cell Stem Cell, 17, 329-340.

2. Venteicher, A.S., Tirosh, I., Hebert, C., Yizhak, K., Neftel, C., Filbin, M.G., Hovestadt, V., Escalante, L.E., Shaw, M.L., Rodman, C. et al. (2017) Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq. Science, 355.

Maintenance

If there's any questions / problems regarding redPATH, please feel free to contact Ken Xie - xkk17@mails.tsinghua.edu.cn. Thank you!