Introduction

To determine cell type heterogeneity between samples jointly, this code treats each individual (unstrained condition) as its own separate sample and performs integration across individuals. It then finds clusters within the integrated data and characterizes the clusters.

Load data and packages

The ANT1.2 seurat object was generated by running the preprocessing code in Pre-processing of raw 10x files into count matrices and demultiplexing.

library(Seurat)
library(gridExtra)
library(ggplot2)
library(cowplot)
library(tidyverse)

Registered S3 method overwritten by 'cli':
  method     from    
  print.boxx spatstat

── Attaching packages ────────────────────────────────── tidyverse 1.3.0 ──

✓ tibble  3.0.4     ✓ dplyr   1.0.2
✓ tidyr   1.1.2     ✓ stringr 1.4.0
✓ readr   1.3.1     ✓ forcats 0.4.0
✓ purrr   0.3.4     

── Conflicts ───────────────────────────────────── tidyverse_conflicts() ──
x dplyr::combine() masks gridExtra::combine()
x dplyr::filter()  masks stats::filter()
x dplyr::lag()     masks stats::lag()

library(ashr)
library(Matrix)

Attaching package: 'Matrix'

The following objects are masked from 'package:tidyr':

    expand, pack, unpack

ANT1.2 <- readRDS("data/ANT1_2.rds")

Split data into individuals

First, we split the merged seurat object into separate objects corresponding to the individual from which cells originated. Then integrate across the individuals using SCT transform

#split data into each individual/sample
NA18855_Unstrain <- subset(ANT1.2, labels == "NA18855_Unstrain")
NA18856_Unstrain <- subset(ANT1.2, labels == "NA18856_Unstrain")
NA19160_Unstrain <- subset(ANT1.2, labels == "NA19160_Unstrain")

# Create a seurat object merged for all the unstrain samples (because we are missing one individual for the strained samples)
seurat.list <- list(NA18855_Unstrain, NA18856_Unstrain, NA19160_Unstrain)
for (i in 1:length(seurat.list)) {
    seurat.list[[i]] <- SCTransform(seurat.list[[i]], verbose = FALSE)
}

SCT.features <- SelectIntegrationFeatures(object.list = seurat.list, nfeatures = 5000)
seurat.list <- PrepSCTIntegration(object.list = seurat.list, anchor.features = SCT.features, 
    verbose = FALSE)

#find anchors
seurat.anchors <- FindIntegrationAnchors(object.list = seurat.list, normalization.method = "SCT", 
    anchor.features = SCT.features, verbose = FALSE)
SCT.integrated <- IntegrateData(anchorset = seurat.anchors, normalization.method = "SCT",
    verbose = FALSE)

#visualized integrated
SCT.integrated <- RunPCA(SCT.integrated, verbose = FALSE, npcs = 100)
DimPlot(SCT.integrated, reduction = "pca", group.by = c("labels"))

Past versions of “split single cell data-1.png”

ElbowPlot(SCT.integrated, ndims = 100) #38 PCs?

Past versions of “split single cell data-2.png”

SCT.integrated <- FindNeighbors(SCT.integrated, dims = 1:38)

Computing nearest neighbor graph

Computing SNN

SCT.integrated <- FindClusters(SCT.integrated, resolution = 0.4)

Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck

Number of nodes: 1203
Number of edges: 67564

Running Louvain algorithm...
Maximum modularity in 10 random starts: 0.6657
Number of communities: 3
Elapsed time: 0 seconds

DimPlot(SCT.integrated, group.by = c("labels"), reduction = "pca")

Past versions of “split single cell data-3.png”

DimPlot(SCT.integrated, group.by = c("seurat_clusters"), reduction = "pca")

Past versions of “split single cell data-4.png”

SCT.integrated <- RunUMAP(SCT.integrated, dims = 1:38)

21:05:46 UMAP embedding parameters a = 0.9922 b = 1.112

21:05:46 Read 1203 rows and found 38 numeric columns

21:05:46 Using Annoy for neighbor search, n_neighbors = 30

21:05:46 Building Annoy index with metric = cosine, n_trees = 50

0%   10   20   30   40   50   60   70   80   90   100%

[----|----|----|----|----|----|----|----|----|----|

**************************************************|
21:05:46 Writing NN index file to temp file /tmp/RtmpVstoIG/file2f0197c684e6a
21:05:46 Searching Annoy index using 1 thread, search_k = 3000
21:05:46 Annoy recall = 100%
21:05:47 Commencing smooth kNN distance calibration using 1 thread
21:05:47 Initializing from normalized Laplacian + noise
21:05:48 Commencing optimization for 500 epochs, with 45058 positive edges
21:05:51 Optimization finished

p1_SCT <- DimPlot(SCT.integrated, group.by = c("orig.ident"))
p2_SCT <- DimPlot(SCT.integrated, group.by = c("labels"))
p3_SCT <- DimPlot(SCT.integrated, group.by = c("seurat_clusters"))
grid.arrange(p1_SCT, p2_SCT, p3_SCT, nrow = 2)

Past versions of “split single cell data-5.png”

#for figures: remove legend and add in later
p1_figure <- p2_SCT + theme(legend.position = "none")
p2_figure <- p3_SCT + theme(legend.position = "none") + scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
grid.arrange(p1_figure, p2_figure)

Past versions of “split single cell data-6.png”

p2_SCT 

Past versions of “split single cell data-7.png”

p3_SCT + scale_color_manual(values=c("plum3", "#E69F00", "#654321"))

Past versions of “split single cell data-8.png”

SCT.integrated@active.ident <- SCT.integrated$integrated_snn_res.0.4

Examine clusters in integrated data

We identified 3 clusters in the integrated data. We now characterize them by gene expression for a chondrogenic marker gene to get a sense of which cluster(s) represent more mature chondrocytes.

cluster_memberships <- as.matrix(table(SCT.integrated@meta.data$integrated_snn_res.0.4, SCT.integrated@meta.data$labels))
cluster_memberships

   
    NA18855_Unstrain NA18856_Unstrain NA19160_Unstrain
  0              429              280              156
  1              129               81               51
  2               28               34               15

cluster_memberships_proportions <- prop.table(cluster_memberships, margin = 2)
cluster_memberships_proportions

   
    NA18855_Unstrain NA18856_Unstrain NA19160_Unstrain
  0       0.73208191       0.70886076       0.70270270
  1       0.22013652       0.20506329       0.22972973
  2       0.04778157       0.08607595       0.06756757

#make a nice proportions bar plot for a figure
long_cluster_memberships_proportions <- as_tibble(as.data.frame(cluster_memberships_proportions))
ggplot(long_cluster_memberships_proportions, aes(x = Var2, y = Freq, fill = Var1)) +
     geom_bar(position = "dodge", stat = "identity") + 
     scale_fill_manual(values=c("plum3", "#E69F00", "#654321")) + 
     theme_cowplot() +
     labs(title = "Proportion of cells from each individual \n assigned to each unsupervised cluster", x = "", y = "Proportion", fill = "Cluster")

Past versions of “examine clusters-1.png”

Explore some cluster marker genes

Here, we fit a poisson ash model to raw counts from each of the three clusters for chondrocyte marker genes and plot their distributions broken down by cluster and by individual.

#run poisson ash to data per cluster and per indivdiual, then show the estimated prior distribution
#plot curves for genes separated by indivdual (information for indivdual contained in labels)
plot_by_labels <- function(gene, counts, labels){
     #create dataframe to store the cdf axes and the groups
     dataframe_cdf <- data.frame(x = c(), y = c(), label = c())
     for(individual in unique(labels)){ #in the case that the labels supplied contain cluster, it will do this by cluster instead
          x <- counts[gene, labels == individual]
          s <- colSums(counts[,labels == individual]) #sum of molecule counts per sample
          lam <- x/s
          fit <- ashr::ash_pois(x, s, mixcompdist = "halfuniform")
          cdf <- ashr::cdf.ash(fit, seq(from = 0, to = max(lam), length.out = 1000))
          temp_df <- cbind(cdf$x, t(cdf$y), individual)
          names(temp_df) <- c("x", "y", "label")
          dataframe_cdf <- rbind(dataframe_cdf, temp_df)
     }
     names(dataframe_cdf) <- c("x", "y", "label")
     dataframe_cdf$x <- as.character(dataframe_cdf$x)
     dataframe_cdf$y <- as.character(dataframe_cdf$y)
     dataframe_cdf$x <- as.numeric(dataframe_cdf$x)
     dataframe_cdf$y <- as.numeric(dataframe_cdf$y)
     #use df to make our plot
     plot <- ggplot(dataframe_cdf, aes(x = x, y = y, color = as.factor(label), group=as.factor(label))) +
          geom_point() + 
          labs(title = paste0(gene), x = "Latent gene expression", y = "CDF", color = "Label") +
          theme_cowplot()
     return(plot)
}


#plots for figures
#COL11A1
a <- plot_by_labels("COL11A1", SCT.integrated@assays$RNA@counts, SCT.integrated$integrated_snn_res.0.4) + 
     theme(legend.position = "none") + 
     scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
b <- plot_by_labels("COL11A1", SCT.integrated@assays$RNA@counts, SCT.integrated$labels) + 
     theme(legend.position = "none") 
grid.arrange(a, b, nrow = 1)

Past versions of “poisson ash-1.png”

#SOX5
a <- plot_by_labels("SOX5", SCT.integrated@assays$RNA@counts, SCT.integrated$integrated_snn_res.0.4) + 
     theme(legend.position = "none") + 
     scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
b <- plot_by_labels("SOX5", SCT.integrated@assays$RNA@counts, SCT.integrated$labels) + 
     theme(legend.position = "none") 
grid.arrange(a, b, nrow = 1)

Past versions of “poisson ash-2.png”

#SOX6
a <- plot_by_labels("SOX6", SCT.integrated@assays$RNA@counts, SCT.integrated$integrated_snn_res.0.4) + 
     theme(legend.position = "none") + 
     scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
b <- plot_by_labels("SOX6", SCT.integrated@assays$RNA@counts, SCT.integrated$labels) + 
     theme(legend.position = "none") 
grid.arrange(a, b, nrow = 1)

Past versions of “poisson ash-3.png”

#TIMP2
a <- plot_by_labels("TIMP2", SCT.integrated@assays$RNA@counts, SCT.integrated$integrated_snn_res.0.4) + 
     theme(legend.position = "none") + 
     scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
b <- plot_by_labels("TIMP2", SCT.integrated@assays$RNA@counts, SCT.integrated$labels) + 
     theme(legend.position = "none") 
grid.arrange(a, b, nrow = 1)

Past versions of “poisson ash-4.png”

#TIMP3
a <- plot_by_labels("TIMP3", SCT.integrated@assays$RNA@counts, SCT.integrated$integrated_snn_res.0.4) + 
     theme(legend.position = "none") + 
     scale_color_manual(values=c("plum3", "#E69F00", "#654321"))
b <- plot_by_labels("TIMP3", SCT.integrated@assays$RNA@counts, SCT.integrated$labels) + 
     theme(legend.position = "none") 
grid.arrange(a, b, nrow = 1)

Past versions of “poisson ash-5.png”

Session information

sessionInfo()

R version 3.6.1 (2019-07-05)
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] Matrix_1.2-18   ashr_2.2-47     forcats_0.4.0   stringr_1.4.0  
 [5] dplyr_1.0.2     purrr_0.3.4     readr_1.3.1     tidyr_1.1.2    
 [9] tibble_3.0.4    tidyverse_1.3.0 cowplot_1.1.0   ggplot2_3.3.3  
[13] gridExtra_2.3   Seurat_3.2.3   

loaded via a namespace (and not attached):
  [1] readxl_1.3.1          backports_1.1.10      workflowr_1.6.2      
  [4] plyr_1.8.6            igraph_1.2.4.1        lazyeval_0.2.2       
  [7] splines_3.6.1         listenv_0.8.0         scattermore_0.7      
 [10] digest_0.6.27         invgamma_1.1          htmltools_0.5.0      
 [13] SQUAREM_2020.4        gdata_2.18.0          fansi_0.4.1          
 [16] magrittr_2.0.1        tensor_1.5            cluster_2.1.0        
 [19] ROCR_1.0-7            globals_0.12.5        modelr_0.1.8         
 [22] matrixStats_0.57.0    colorspace_2.0-0      rvest_0.3.6          
 [25] rappdirs_0.3.1        ggrepel_0.9.0         haven_2.3.1          
 [28] xfun_0.8              crayon_1.3.4          jsonlite_1.7.2       
 [31] spatstat_1.64-1       spatstat.data_1.7-0   survival_2.44-1.1    
 [34] zoo_1.8-8             glue_1.4.2            polyclip_1.10-0      
 [37] gtable_0.3.0          leiden_0.3.1          future.apply_1.3.0   
 [40] abind_1.4-5           scales_1.1.1          DBI_1.1.0            
 [43] miniUI_0.1.1.1        Rcpp_1.0.5            viridisLite_0.3.0    
 [46] xtable_1.8-4          reticulate_1.16       rsvd_1.0.1           
 [49] truncnorm_1.0-8       htmlwidgets_1.5.2     httr_1.4.2           
 [52] gplots_3.0.1.1        RColorBrewer_1.1-2    ellipsis_0.3.1       
 [55] ica_1.0-2             farver_2.0.3          pkgconfig_2.0.3      
 [58] uwot_0.1.10           dbplyr_1.4.2          deldir_0.1-23        
 [61] labeling_0.4.2        tidyselect_1.1.0      rlang_0.4.10         
 [64] reshape2_1.4.3        later_1.1.0.1         munsell_0.5.0        
 [67] cellranger_1.1.0      tools_3.6.1           cli_2.2.0            
 [70] generics_0.0.2        broom_0.7.0           ggridges_0.5.1       
 [73] evaluate_0.14         yaml_2.2.1            goftest_1.2-2        
 [76] npsurv_0.4-0          knitr_1.23            fs_1.3.1             
 [79] fitdistrplus_1.0-14   caTools_1.17.1.2      RANN_2.6.1           
 [82] pbapply_1.4-0         future_1.18.0         nlme_3.1-140         
 [85] whisker_0.3-2         mime_0.9              xml2_1.3.2           
 [88] compiler_3.6.1        rstudioapi_0.13       plotly_4.9.2.1       
 [91] png_0.1-7             lsei_1.2-0            spatstat.utils_1.17-0
 [94] reprex_0.3.0          stringi_1.4.6         RSpectra_0.15-0      
 [97] lattice_0.20-41       vctrs_0.3.6           pillar_1.4.7         
[100] lifecycle_0.2.0       lmtest_0.9-37         RcppAnnoy_0.0.18     
[103] data.table_1.13.0     bitops_1.0-6          irlba_2.3.3          
[106] httpuv_1.5.1          patchwork_1.1.0       R6_2.5.0             
[109] promises_1.1.1        KernSmooth_2.23-15    codetools_0.2-16     
[112] MASS_7.3-52           gtools_3.8.1          assertthat_0.2.1     
[115] rprojroot_2.0.2       withr_2.3.0           sctransform_0.3.2    
[118] mgcv_1.8-28           parallel_3.6.1        hms_0.5.3            
[121] grid_3.6.1            rpart_4.1-15          rmarkdown_1.13       
[124] Rtsne_0.15            git2r_0.26.1          mixsqp_0.3-43        
[127] shiny_1.3.2           lubridate_1.7.9