Visualize and interpret droplet and pulse-seq topics
Peter Carbonetto
Last updated: 2020-08-19
Checks: 7 0
Knit directory: single-cell-topics/analysis/
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| File | Version | Author | Date | Message |
|---|---|---|---|---|
| Rmd | d35cb03 | Peter Carbonetto | 2020-08-19 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | 368a74a | Peter Carbonetto | 2020-08-19 | Added some text to plots_tracheal_epithelium analysis. |
| Rmd | 223406b | Peter Carbonetto | 2020-08-19 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | aca46cc | Peter Carbonetto | 2020-08-19 | Added manual clustering of droplet samples based on PCs. |
| Rmd | 38f811b | Peter Carbonetto | 2020-08-19 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| Rmd | 343747e | Peter Carbonetto | 2020-08-19 | Small edit to figure dimensions. |
| html | 5a35bbd | Peter Carbonetto | 2020-08-19 | Added labeled PCA plot; adjusted plot dimensions in |
| Rmd | fb91075 | Peter Carbonetto | 2020-08-19 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | 8b9b528 | Peter Carbonetto | 2020-08-19 | Added more PCA plots to plots_tracheal_epithelium analysis. |
| Rmd | ee7cbf1 | Peter Carbonetto | 2020-08-19 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | c517ea2 | Peter Carbonetto | 2020-08-18 | Small fix to one of the PCA plots in plots_tracheal_epithelium. |
| Rmd | 8f5c210 | Peter Carbonetto | 2020-08-18 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | 01afbd2 | Peter Carbonetto | 2020-08-18 | Added some PC plots to the plots_tracheal_epithelium analysis. |
| Rmd | f1c7d02 | Peter Carbonetto | 2020-08-18 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| html | 0a04fc1 | Peter Carbonetto | 2020-08-18 | Added abundance plots to plots_tracheal_epithelium analysis. |
| Rmd | f914f7e | Peter Carbonetto | 2020-08-18 | wflow_publish(“plots_tracheal_epithelium.Rmd”) |
| Rmd | 61917ad | Peter Carbonetto | 2020-08-18 | Working on new analysis, plots_tracheal_epithelium.Rmd. |
TO DO: Add introductory text here.
Load the packages used in the analysis below.
# library(fastTopics)
devtools::load_all("~/git/fastTopics")
library(ggplot2)
library(cowplot)
source("../code/plots.R")Load the sample annotations. (The count data are no longer needed at this stage of the analysis.)
load("../data/droplet.RData")
samples_droplet <- samples
load("../data/pulseseq.RData")
samples_pulseseq <- samples
rm(samples,counts)Load the \(k = 7\) Poisson NMF model fit for the droplet data, and the \(k = 11\) Poisson NMF fit for the pulse-seq data.
fit_droplet <- readRDS("../output/droplet/rds/fit-droplet-scd-ex-k=7.rds")$fit
fit_pulseseq <- readRDS("../output/pulseseq/rds/fit-pulseseq-scd-ex-k=11.rds")$fitRare and abundant cell types in the droplet data:
p1 <- create_abundance_plot(fit_droplet)
print(p1)
| Version | Author | Date |
|---|---|---|
| 0a04fc1 | Peter Carbonetto | 2020-08-18 |
The first topic is indeed very rare; only 43 samples have a greater than 10% contribution from this topic.
sum(poisson2multinom(fit_droplet)$L[,1] > 0.1)
# [1] 43Rare and abundant cell types in the pulse-seq data:
p2 <- create_abundance_plot(fit_pulseseq)
print(p2)
| Version | Author | Date |
|---|---|---|
| 0a04fc1 | Peter Carbonetto | 2020-08-18 |
[Explain why we don’t use t-SNE.] Substructure is evident from principal components of droplet topic proportions:
p3 <- basic_pca_plot(fit_droplet,c("PC1","PC2"))
p4 <- basic_pca_plot(fit_droplet,c("PC4","PC5"))
p5 <- basic_pca_plot(fit_droplet,c("PC5","PC6"))
plot_grid(p3,p4,p5,nrow = 1)
TO DO: Add PC plots with topic proportions here.
p6 <- pca_plot(poisson2multinom(fit_droplet),pcs = 1:2,k = c(2,5,6),
plot_grid_call = function (plots)
do.call(plot_grid,c(lapply(plots,
function (x) x + guides(fill = "none")),list(nrow = 1))))
print(p6)
Principal components 4 and 5:
p7 <- pca_plot(poisson2multinom(fit_droplet),pcs = 4:5,k = c(3,4,5,7),
plot_grid_call = function (plots)
do.call(plot_grid,c(lapply(plots,
function (x) x + guides(fill = "none")),list(nrow = 1))))
print(p7)
Principal components 5 and 6:
p8 <- pca_plot(poisson2multinom(fit_droplet),pcs = 5:6,k = 1) +
guides(fill = "none")
print(p8)
| Version | Author | Date |
|---|---|---|
| 8b9b528 | Peter Carbonetto | 2020-08-19 |
[Note that PCs 3 and 7 does not reveal any additional substructure, so are not included in our plots.]
Four suggested by PCA analysis, plus a “background” cluster (\(c_0\)):
droplet_cluster_colors <- c("gray","darkblue","gold","yellowgreen","firebrick")
pca_droplet <- prcomp(poisson2multinom(fit_droplet)$L)$x
n <- nrow(pca_droplet)
x <- rep("c0",n)
pc1 <- pca_droplet[,"PC1"]
pc2 <- pca_droplet[,"PC2"]
pc6 <- pca_droplet[,"PC6"]
x[pc2 > -0.1] <- "c1"
x[pc6 < -0.04] <- "c2"
x[(pc1 - 0)^2 + (pc2 + 0.75)^2 < 0.09] <- "c3"
x[(pc1 - 0.5)^2 + (pc2 + 0.9)^2 < 0.04] <- "c4"
samples_droplet$cluster <- factor(x)
p9 <- pca_plot_with_labels(fit_droplet,c("PC1","PC2"),samples_droplet$cluster,
droplet_cluster_colors) + labs(fill = "cluster")
p10 <- pca_plot_with_labels(fit_droplet,c("PC4","PC5"),samples_droplet$cluster,
droplet_cluster_colors) + labs(fill = "cluster")
p11 <- pca_plot_with_labels(fit_droplet,c("PC5","PC6"),samples_droplet$cluster,
droplet_cluster_colors) + labs(fill = "cluster")
plot_grid(p9,p10,p11)
The vast majority of the droplet samples are in the \(c_1\) cluster:
table(samples_droplet$cluster)
#
# c0 c1 c2 c3 c4
# 73 6533 50 162 375This suggests separate treatment of the \(c_1\) cluster.
TO DO: Add more text here.
set.seed(1)
droplet_topic_colors <- c("gold","royalblue","turquoise","lightgreen",
"forestgreen","firebrick","olivedrab")
topic_ordering <- c(1,5,6,2,3,4,7)
fit_droplet_rare <- select(poisson2multinom(fit_droplet),
loadings = which(samples_droplet$cluster != "c1" &
samples_droplet$cluster != "c4"))
tsne_droplet_rare <- tsne_from_topics(fit_droplet_rare,dims = 1,perplexity = 30,
verbose = FALSE)
p12 <- structure_plot(fit_droplet_rare,rows = order(tsne_droplet_rare$Y),
topics = topic_ordering,
colors = droplet_topic_colors[topic_ordering])
print(p12)
set.seed(1)
fit_droplet_abundant <-
select(poisson2multinom(fit_droplet),
loadings = which(samples_droplet$cluster == "c1" |
samples_droplet$cluster == "c4"))
tsne_droplet_abundant <- tsne_from_topics(fit_droplet_abundant,dims = 1,n = 1000,
perplexity = 100,verbose = FALSE)
Y <- tsne_droplet_abundant$Y
p13 <- structure_plot(fit_droplet_abundant,
rows = rownames(Y)[order(Y)],
topics = topic_ordering,
colors = droplet_topic_colors[topic_ordering])
print(p13)
It is helpful to compare these results with clustering reported in the Montoro et al (2018) paper. To make this comparison, we layer the 7 clusters on top of these PCs:
droplet_celltype_colors <-
c("royalblue", # Basal
"firebrick", # Ciliated
"forestgreen", # Club
"gold", # Goblet
"darkmagenta", # Ionocyte
"darkorange", # Neuroendocrine
"lightsteelblue") # Tuft
p9 <- pca_plot_with_labels(fit_droplet,c("PC1","PC2"),samples_droplet$tissue,
droplet_celltype_colors) + labs(fill = "celltype")
p10 <- pca_plot_with_labels(fit_droplet,c("PC4","PC5"),samples_droplet$tissue,
droplet_celltype_colors) + labs(fill = "celltype")
p11 <- pca_plot_with_labels(fit_droplet,c("PC5","PC6"),samples_droplet$tissue,
droplet_celltype_colors) + labs(fill = "celltype")
plot_grid(p9,p10,p11)
And likewise in the pulse-seq data:
p5 <- basic_pca_plot(fit_pulseseq,c("PC3","PC4"))
p6 <- basic_pca_plot(fit_pulseseq,c("PC5","PC6"))
plot_grid(p5,p6)ggplot(cbind(pca$x,data.frame(k2 = fit$L[,2])),
aes(x = PC1,y = PC2,fill = k2)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)
ggplot(cbind(pca$x,data.frame(k5 = fit$L[,5])),
aes(x = PC1,y = PC2,fill = k5)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)
ggplot(cbind(pca$x,data.frame(k6 = fit$L[,6])),
aes(x = PC1,y = PC2,fill = k6)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)
ggplot(cbind(pca$x,data.frame(k1 = fit$L[,1])),
aes(x = PC5,y = PC6,fill = k1)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)ggplot(cbind(pca$x,data.frame(k3 = fit$L[,3])),
aes(x = PC1,y = PC2,fill = k3)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)
ggplot(cbind(pca$x,data.frame(k4 = fit$L[,4])),
aes(x = PC1,y = PC2,fill = k4)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_gradient2(low = "deepskyblue",mid = "gold",high = "orangered",
midpoint = 0.5) +
theme_cowplot(font_size = 12)ggplot(cbind(samples_droplet,pca$x),aes(x = PC1,y = PC2,fill = tissue)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_manual(values = clrs) +
theme_cowplot(font_size = 12)
ggplot(cbind(samples_droplet,pca$x),aes(x = PC5,y = PC6,fill = tissue)) +
geom_point(shape = 21,color = "white",,size = 2) +
scale_fill_manual(values = clrs) +
theme_cowplot(font_size = 12)clrs <- c("royalblue", # basal
"firebrick", # ciliated
"forestgreen", # club
"gold", # goblet
"darkmagenta", # ionocyte
"darkorange", # neuroendocrine
"tomato", # proliferating
"darkgray") # tuft
ggplot(cbind(samples_droplet,pca$x),aes(x = PC1,y = PC2,fill = tissue)) +
geom_point(shape = 21,color = "white",size = 2) +
scale_fill_manual(values = clrs) +
theme_cowplot(font_size = 12)
ggplot(cbind(samples_droplet,pca$x),aes(x = PC5,y = PC6,fill = tissue)) +
geom_point(shape = 21,color = "white",,size = 2) +
scale_fill_manual(values = clrs) +
theme_cowplot(font_size = 12)
sessionInfo()
# R version 3.6.2 (2019-12-12)
# Platform: x86_64-apple-darwin15.6.0 (64-bit)
# Running under: macOS Catalina 10.15.5
#
# Matrix products: default
# BLAS: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRblas.0.dylib
# LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib
#
# locale:
# [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
#
# attached base packages:
# [1] stats graphics grDevices utils datasets methods base
#
# other attached packages:
# [1] cowplot_1.0.0 ggplot2_3.3.0 fastTopics_0.3-161 testthat_2.3.1
#
# loaded via a namespace (and not attached):
# [1] httr_1.4.1 tidyr_1.0.0 pkgload_1.0.2
# [4] viridisLite_0.3.0 jsonlite_1.6 RcppParallel_4.4.4
# [7] assertthat_0.2.1 yaml_2.2.0 remotes_2.1.0
# [10] progress_1.2.2 ggrepel_0.9.0 sessioninfo_1.1.1
# [13] pillar_1.4.3 backports_1.1.5 lattice_0.20-38
# [16] quantreg_5.54 glue_1.3.1 quadprog_1.5-8
# [19] digest_0.6.23 promises_1.1.0 colorspace_1.4-1
# [22] htmltools_0.4.0 httpuv_1.5.2 Matrix_1.2-18
# [25] pkgconfig_2.0.3 devtools_2.2.1 SparseM_1.78
# [28] purrr_0.3.3 scales_1.1.0 processx_3.4.1
# [31] whisker_0.4 later_1.0.0 Rtsne_0.15
# [34] MatrixModels_0.4-1 git2r_0.26.1 tibble_2.1.3
# [37] farver_2.0.1 usethis_1.6.0 ellipsis_0.3.0
# [40] withr_2.1.2 lazyeval_0.2.2 cli_2.0.0
# [43] magrittr_1.5 crayon_1.3.4 memoise_1.1.0
# [46] mcmc_0.9-6 evaluate_0.14 ps_1.3.0
# [49] fs_1.3.1 fansi_0.4.0 MASS_7.3-51.4
# [52] pkgbuild_1.0.6 tools_3.6.2 data.table_1.12.8
# [55] prettyunits_1.1.1 hms_0.5.2 lifecycle_0.1.0
# [58] stringr_1.4.0 MCMCpack_1.4-5 plotly_4.9.2
# [61] munsell_0.5.0 irlba_2.3.3 callr_3.4.0
# [64] compiler_3.6.2 rlang_0.4.5 grid_3.6.2
# [67] rstudioapi_0.10 htmlwidgets_1.5.1 labeling_0.3
# [70] rmarkdown_2.3 gtable_0.3.0 R6_2.4.1
# [73] knitr_1.26 dplyr_0.8.3 zeallot_0.1.0
# [76] workflowr_1.6.2.9000 rprojroot_1.3-2 desc_1.2.0
# [79] stringi_1.4.3 Rcpp_1.0.3 vctrs_0.2.1
# [82] tidyselect_0.2.5 xfun_0.11 coda_0.19-3