Last updated: 2020-10-20

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Knit directory: scATACseq-topics/

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Rmd f8bea96 kevinlkx 2020-10-20 clustering with k = 13 topics

Here we explore the structure in the Cusanovich et al (2018) ATAC-seq data inferred from the multinomial topic model with \(k = 13\).

Load packages and some functions used in this analysis.

library(Matrix)
library(dplyr)
library(ggplot2)
library(cowplot)
library(fastTopics)
source("code/plots.R")

Load the data. The counts are no longer needed at this stage of the analysis.

data.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/data/Cusanovich_2018/processed_data/"
load(file.path(data.dir, "Cusanovich_2018.RData"))
rm(counts)

Plot PCs of the topic proportions

We first use PCA to explore the structure inferred from the multinomial topic model with \(k = 13\):

Load the \(k = 13\) Poisson NMF fit.

out.dir <- "/project2/mstephens/kevinluo/scATACseq-topics/output/Cusanovich_2018"
fit <- readRDS(file.path(out.dir, "/fit-Cusanovich2018-scd-ex-k=13.rds"))$fit

Plot PCs of the topic proportions.

p1 <- pca_plot(poisson2multinom(fit),pcs = 1:2,fill = "none")
p2 <- pca_plot(poisson2multinom(fit),pcs = 3:4,fill = "none")
p3 <- pca_plot(poisson2multinom(fit),pcs = 5:6,fill = "none")
p4 <- pca_plot(poisson2multinom(fit),pcs = 7:8,fill = "none")
p5 <- pca_plot(poisson2multinom(fit),pcs = 9:10,fill = "none")
p6 <- pca_plot(poisson2multinom(fit),pcs = 11:12,fill = "none")

plot_grid(p1,p2,p3,p4,p5,p6)

Version Author Date
a38788b kevinlkx 2020-10-20

Some of the structure is more evident from “hexbin” plots showing the density of the points.

breaks <- c(0,1,5,10,100,Inf)
p7 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 1:2, breaks = breaks) + guides(fill = "none")
p8 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 3:4, breaks = breaks) + guides(fill = "none")
p9 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 5:6, breaks = breaks) + guides(fill = "none")
p10 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 7:8, breaks = breaks) + guides(fill = "none")
p11 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 9:10, breaks = breaks) + guides(fill = "none")
p12 <- pca_hexbin_plot(poisson2multinom(fit), pcs = 11:12, breaks = breaks) + guides(fill = "none")

plot_grid(p7,p8,p9,p10,p11,p12)

Version Author Date
a38788b kevinlkx 2020-10-20

Layer the tissue and cell labels onto the PC plots

Next, we layer the tissue and cell labels onto the PC plots.

PCs 1 and 2:

p13 <- labeled_pca_plot(fit,1:2,samples$tissue,font_size = 7,
                       legend_label = "tissue")
p14 <- labeled_pca_plot(fit,1:2,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p13,p14,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

PCs 3 and 4:

p15 <- labeled_pca_plot(fit,3:4,samples$tissue,font_size = 7,
                        legend_label = "tissue")
p16 <- labeled_pca_plot(fit,3:4,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p15,p16,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

PCs 5 and 6:

p17 <- labeled_pca_plot(fit,5:6,samples$tissue,font_size = 7,
                       legend_label = "tissue")
p18 <- labeled_pca_plot(fit,5:6,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p17,p18,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

PCs 7 and 8:

p19 <- labeled_pca_plot(fit,7:8,samples$tissue,font_size = 7,
                       legend_label = "tissue")
p20 <- labeled_pca_plot(fit,7:8,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p19,p20,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

PCs 9 and 10:

p21 <- labeled_pca_plot(fit,9:10,samples$tissue,font_size = 7,
                       legend_label = "tissue")
p22 <- labeled_pca_plot(fit,9:10,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p21,p22,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

PCs 11 and 12:

p23 <- labeled_pca_plot(fit,11:12,samples$tissue,font_size = 7,
                       legend_label = "tissue")
p24 <- labeled_pca_plot(fit,11:12,samples$cell_label,font_size = 7,
                        legend_label = "cell_label")
plot_grid(p23,p24,rel_widths = c(8,11))

Version Author Date
a38788b kevinlkx 2020-10-20

k-means clustering on topic proportions

Define clusters using k-means, and then create structure plot based on the clusters from k-means.

Define clusters using k-means with \(k = 14\):

set.seed(10)

clusters.14 <- factor(kmeans(poisson2multinom(fit)$L,centers = 14)$cluster)
print(sort(table(clusters.14),decreasing = TRUE))
# clusters.14
#    13     7     3     2     4     1    12    10     8    14     5     6    11 
# 18096 10936  9331  7693  6077  5817  4184  3994  3627  3306  2964  2821  2073 
#     9 
#   254

Structure plot based on k-means clusters

colors <- c("#a6cee3","#1f78b4","#b2df8a","#33a02c","#fb9a99","#e31a1c",
            "#fdbf6f","#ff7f00","#cab2d6","#6a3d9a","#ffff99","#b15928",
            "gray")
rows <- sample(nrow(fit$L),4000)

p25 <- structure_plot(select(poisson2multinom(fit),loadings = rows),
                     grouping = clusters.14[rows],n = Inf,gap = 20,
                     perplexity = 50,topics = 1:13,colors = colors,
                     num_threads = 4,verbose = FALSE)
# Perplexity automatically changed to 42 because original setting of 50 was too large for the number of samples (131)
# Perplexity automatically changed to 1 because original setting of 50 was too large for the number of samples (7)
# Perplexity automatically changed to 26 because original setting of 50 was too large for the number of samples (82)
# Perplexity automatically changed to 44 because original setting of 50 was too large for the number of samples (137)
print(p25)

Version Author Date
a38788b kevinlkx 2020-10-20

Define clusters using k-means with \(k = 15\):

set.seed(10)

clusters.15 <- factor(kmeans(poisson2multinom(fit)$L,centers = 15)$cluster)
print(sort(table(clusters.15),decreasing = TRUE))
# clusters.15
#    13     7     3     9     8     4     1    14    12     2    15     5     6 
# 14936  8426  8043  6796  6757  5951  5784  4314  4168  3657  3649  2960  2738 
#    10    11 
#  1697  1297

Structure plot based on k-means clusters

colors <- c("#a6cee3","#1f78b4","#b2df8a","#33a02c","#fb9a99","#e31a1c",
            "#fdbf6f","#ff7f00","#cab2d6","#6a3d9a","#ffff99","#b15928",
            "gray")
rows <- sample(nrow(fit$L),4000)

p26 <- structure_plot(select(poisson2multinom(fit),loadings = rows),
                     grouping = clusters.15[rows],n = Inf,gap = 20,
                     perplexity = 50,topics = 1:13,colors = colors,
                     num_threads = 4,verbose = FALSE)
# Perplexity automatically changed to 42 because original setting of 50 was too large for the number of samples (131)
# Perplexity automatically changed to 48 because original setting of 50 was too large for the number of samples (149)
# Perplexity automatically changed to 23 because original setting of 50 was too large for the number of samples (75)
# Perplexity automatically changed to 13 because original setting of 50 was too large for the number of samples (44)
print(p26)

Version Author Date
a38788b kevinlkx 2020-10-20

Define clusters using k-means with \(k = 20\):

set.seed(10)

clusters.20 <- factor(kmeans(poisson2multinom(fit)$L,centers = 20)$cluster)
print(sort(table(clusters.20),decreasing = TRUE))
# clusters.20
#    17    13     4     1     3    20     8    12     7     2    15    14    18 
# 13635  6703  5963  5809  5557  5057  5045  4012  3783  3492  3490  3147  2989 
#     5     6    11    16    10    19     9 
#  2958  2738  2158  1839  1756   721   321

Structure plot based on k-means clusters

colors <- c("#a6cee3","#1f78b4","#b2df8a","#33a02c","#fb9a99","#e31a1c",
            "#fdbf6f","#ff7f00","#cab2d6","#6a3d9a","#ffff99","#b15928",
            "gray")
rows <- sample(nrow(fit$L),4000)
p27 <- structure_plot(select(poisson2multinom(fit),loadings = rows),
                     grouping = clusters.20[rows],n = Inf,gap = 20,
                     perplexity = 50,topics = 1:13,colors = colors,
                     num_threads = 4,verbose = FALSE)
# Perplexity automatically changed to 43 because original setting of 50 was too large for the number of samples (133)
# Perplexity automatically changed to 1 because original setting of 50 was too large for the number of samples (9)
# Perplexity automatically changed to 26 because original setting of 50 was too large for the number of samples (82)
# Perplexity automatically changed to 25 because original setting of 50 was too large for the number of samples (81)
# Perplexity automatically changed to 43 because original setting of 50 was too large for the number of samples (134)
# Perplexity automatically changed to 27 because original setting of 50 was too large for the number of samples (86)
# Perplexity automatically changed to 48 because original setting of 50 was too large for the number of samples (150)
# Perplexity automatically changed to 10 because original setting of 50 was too large for the number of samples (35)
print(p27)

Version Author Date
a38788b kevinlkx 2020-10-20

Refine clusters

We then further refine the clusters based on k-means result with \(k = 20\): merge “orange” clusters 9, 11, 14; merge “brown” clusters 3 and 10, 16, 19; merge “yellow” clusters 8 and 18. (maybe also merge the “red” clusters 2 and 4)

clusters.merged <- clusters.20
clusters.merged[clusters.20 %in% c(9,11,14)] <- 9
clusters.merged[clusters.20 %in% c(3,10,16,19)] <- 3
clusters.merged[clusters.20 %in% c(8,18)] <- 8
clusters.merged <- factor(clusters.merged, labels = paste0("c", c(1:length(unique(clusters.merged)))))
print(sort(table(clusters.merged),decreasing = TRUE))
# clusters.merged
#   c13    c3    c8   c11    c4    c1    c9   c14   c10    c7    c2   c12    c5 
# 13635  9873  8034  6703  5963  5809  5626  5057  4012  3783  3492  3490  2958 
#    c6 
#  2738
colors <- c("#a6cee3","#1f78b4","#b2df8a","#33a02c","#fb9a99","#e31a1c",
            "#fdbf6f","#ff7f00","#cab2d6","#6a3d9a","#ffff99","#b15928",
            "gray")
rows <- sample(nrow(fit$L),4000)
p28 <- structure_plot(select(poisson2multinom(fit),loadings = rows),
                     grouping = clusters.merged[rows],n = Inf,gap = 20,
                     perplexity = 50,topics = 1:13,colors = colors,
                     num_threads = 4,verbose = FALSE)
# Perplexity automatically changed to 43 because original setting of 50 was too large for the number of samples (134)
# Perplexity automatically changed to 47 because original setting of 50 was too large for the number of samples (147)
print(p28)

Version Author Date
a38788b kevinlkx 2020-10-20
samples$cluster_kmeans <- clusters.merged

freq_cluster_tissue <- with(samples,table(tissue,cluster_kmeans))
print(freq_cluster_tissue)
#                   cluster_kmeans
# tissue               c1   c2   c3   c4   c5   c6   c7   c8   c9  c10  c11  c12
#   BoneMarrow         33 1342  174  259    0 2453    0    1    0    0 3085    0
#   Cerebellum         80    8    0   29    0    0    0  387    0 1140   47  510
#   Heart            2084  128   15   64    5    0 3776   14    0    0  442    4
#   Kidney           1047   45   27   48 2953    0    0    4    3    0  347    1
#   LargeIntestine    328  107  109   70    0    0    1   32    0    0  310    5
#   Liver             334   96   41   36    0    0    0    0 5577    1   47    0
#   Lung             1468 1143 1017 2647    0    2    5   28   28    2  552    2
#   PreFrontalCortex   97   84    1  127    0    0    0 4453    0    2   39 1115
#   SmallIntestine    112   71   49    9    0    0    0    3   15    0 1601    0
#   Spleen              8  302 1033 2498    0   43    0    0    0    0  114    0
#   Testes             34    1    0    2    0  240    0   30    3    0   43    0
#   Thymus              6   56 7406   88    0    0    1    0    0    0   25    0
#   WholeBrain        178  109    1   86    0    0    0 3082    0 2867   51 1853
#                   cluster_kmeans
# tissue              c13  c14
#   BoneMarrow       1056    0
#   Cerebellum         73    4
#   Heart            1113    5
#   Kidney            158 1798
#   LargeIntestine   4617 1507
#   Liver              31    4
#   Lung             1378 1724
#   PreFrontalCortex   38    3
#   SmallIntestine   2212    5
#   Spleen             22    0
#   Testes           2367    3
#   Thymus             31    4
#   WholeBrain        539    0

The clusters defined by k-means on topic proportions reasonably identify the clusters shown in the PCA hexbin plots (below).

p29 <- labeled_pca_plot(fit,1:2,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
p30 <- labeled_pca_plot(fit,3:4,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
p31 <- labeled_pca_plot(fit,5:6,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
p32 <- labeled_pca_plot(fit,7:8,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
p33 <- labeled_pca_plot(fit,9:10,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
p34 <- labeled_pca_plot(fit,11:12,samples$cluster_kmeans,font_size = 7,
                       legend_label = "cluster_kmeans")
plot_grid(p29,p30,p31,p32,p33,p34)

Version Author Date
a38788b kevinlkx 2020-10-20
plot_grid(p7,p8,p9,p10,p11,p12)

Version Author Date
a38788b kevinlkx 2020-10-20

We then label the cells in each cluster with the known tissue labels.

Tissues:

samples$tissue <- as.factor(samples$tissue)
cat(length(levels(samples$tissue)), "tissues. \n")
table(samples$tissue)
# 13 tissues. 
# 
#       BoneMarrow       Cerebellum            Heart           Kidney 
#             8403             2278             7650             6431 
#   LargeIntestine            Liver             Lung PreFrontalCortex 
#             7086             6167             9996             5959 
#   SmallIntestine           Spleen           Testes           Thymus 
#             4077             4020             2723             7617 
#       WholeBrain 
#             8766

Plot the distribution of tissues by cluster.

library(plyr);library(dplyr)
# ------------------------------------------------------------------------------
# You have loaded plyr after dplyr - this is likely to cause problems.
# If you need functions from both plyr and dplyr, please load plyr first, then dplyr:
# library(plyr); library(dplyr)
# ------------------------------------------------------------------------------
# 
# Attaching package: 'plyr'
# The following objects are masked from 'package:dplyr':
# 
#     arrange, count, desc, failwith, id, mutate, rename, summarise,
#     summarize

freq_cluster_tissue <- count(samples, vars=c("cluster_kmeans","tissue")) 

# stacked barplot for the counts of tissues by clusters
p35 <- ggplot(freq_cluster_tissue, aes(fill=tissue, y=freq, x=cluster_kmeans)) + 
  geom_bar(position="stack", stat="identity") +
  theme_classic() + xlab("Cluster") + ylab("Number of cells") +
  guides(fill=guide_legend(ncol=2)) +
  theme(
  legend.title = element_text(size = 10),
  legend.text = element_text(size = 8)
  )
print(p35)

Version Author Date
a38788b kevinlkx 2020-10-20

sessionInfo()
# R version 3.5.1 (2018-07-02)
# Platform: x86_64-pc-linux-gnu (64-bit)
# Running under: Scientific Linux 7.4 (Nitrogen)
# 
# Matrix products: default
# BLAS/LAPACK: /software/openblas-0.2.19-el7-x86_64/lib/libopenblas_haswellp-r0.2.19.so
# 
# locale:
#  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
#  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
#  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
#  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
#  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
# [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
# 
# attached base packages:
# [1] stats     graphics  grDevices utils     datasets  methods   base     
# 
# other attached packages:
# [1] plyr_1.8.6         fastTopics_0.3-180 cowplot_1.0.0      ggplot2_3.3.0     
# [5] dplyr_0.8.5        Matrix_1.2-15      workflowr_1.6.2   
# 
# loaded via a namespace (and not attached):
#  [1] ggrepel_0.8.2      Rcpp_1.0.4.6       lattice_0.20-38    tidyr_0.8.3       
#  [5] prettyunits_1.1.1  assertthat_0.2.1   rprojroot_1.3-2    digest_0.6.25     
#  [9] R6_2.4.1           backports_1.1.7    MatrixModels_0.4-1 evaluate_0.14     
# [13] coda_0.19-2        httr_1.4.1         pillar_1.4.4       rlang_0.4.6       
# [17] progress_1.2.2     lazyeval_0.2.2     data.table_1.12.8  irlba_2.3.3       
# [21] SparseM_1.77       whisker_0.4        hexbin_1.28.1      rmarkdown_2.1     
# [25] labeling_0.3       Rtsne_0.15         stringr_1.4.0      htmlwidgets_1.5.1 
# [29] munsell_0.5.0      compiler_3.5.1     httpuv_1.5.3.1     xfun_0.14         
# [33] pkgconfig_2.0.3    mcmc_0.9-7         htmltools_0.4.0    tidyselect_0.2.5  
# [37] tibble_3.0.1       quadprog_1.5-5     viridisLite_0.3.0  crayon_1.3.4      
# [41] withr_2.1.2        later_1.0.0        MASS_7.3-51.6      grid_3.5.1        
# [45] jsonlite_1.6       gtable_0.3.0       lifecycle_0.2.0    git2r_0.27.1      
# [49] magrittr_1.5       scales_1.1.1       RcppParallel_4.4.3 stringi_1.4.6     
# [53] farver_2.0.3       fs_1.3.1           promises_1.1.0     ellipsis_0.3.1    
# [57] vctrs_0.3.0        tools_3.5.1        glue_1.4.1         purrr_0.3.4       
# [61] hms_0.4.2          yaml_2.2.0         colorspace_1.4-1   plotly_4.8.0      
# [65] knitr_1.28         quantreg_5.36      MCMCpack_1.4-4