# Load required packages
suppressPackageStartupMessages({
library(treekoR)
library(SingleCellExperiment)
library(ggtree)
})treekoR is a novel framework that aims to utilise the hierarchical nature of single cell cytometry data, to find robust and interpretable associations between cell subsets and patient clinical end points. This is achieved by deriving the tree structure of cell clusters, followed by measuring the %parent (proportions of each node in the tree relative to the number of cells belonging to the immediate parent node), in addition to the %total (proportion of cells in each node relative to all cells). These proportions are then used in significance testing and classification models to determine which cell subpopulation proportions most correlated with the patient clinical outcome of interest. treekoR then provides an interactive visualisation which helps to highlight these results.
SingleCellExperiment
containing samples of flow cytometry expression data from 39 patients.
This data represents a subset of a dataset that was originally used by
De Biasi et al. (2020) for the characterisation of CD8+ T cells,
comparing between COVID-19 patients and healthy controls.treekoR requires the following information in the variables:
exprs: Single cell expression data (n × p), where p is the number of markers, and
n is the number of cellsclusters: a vector of length n representing the cell type or
cluster of each cell (can be character or
numeric)classes: a vector of length n containing the patient
outcome/class each cell belongs tosamples: a vector of length n identifying the patient each cell
belongs toIn this example: the clusters contain 100 clusters
generated by FlowSOM; classes identify whether the cell
belongs to a COVID-19 or healthy patient; and samples
identify which cell the patient comes from.
The scaled median marker expression for each cluster is calculated which is used to construct a hierarchical tree.
In this step, the choice of hierarchical aggregation method (which
determines the structure of the tree) is determined. By default the
framework chooses HOPACH to construct the tree via the
hierarchy_method argument, however any of the methods in
hclust can be used (see @ref(hc-methods)).
Proportions of each cell cluster in the tree are calculated - both the proportion relative to all and proportion relative to the hierarchical parent. These proportions are used in a two sample t-test, testing for equal means between the patient clinical outcome using both types of proportions.
tested_tree <- testTree(phylo=clust_tree$clust_tree,
clusters=clusters,
samples=samples,
classes=classes,
pos_class_name=NULL)node: unique identifier for each node in the
hierarchical treeparent: the node of the parentisTip: whether the node is a leaf node in the treeclusters: the clusters belonging to the corresponding
nodestat_all: test statistic obtained from testing between
conditions using the proportion of the node relative to all cells
(%total) in each sample. pval_total is the corresponding
p-value (unadjusted)stat_parent: test statistic obtained from testing
between conditions using the proportion of the node relative to cells in
the parent node (%parent) in each sample. pval_parent is
the corresponding p-value (unadjusted)res_df <- getTreeResults(tested_tree)
head(res_df, 10)
#> parent node isTip clusters stat_total stat_parent pval_total
#> 51 139 51 TRUE 77 1.0823881 4.439944 0.290116635
#> 52 139 52 TRUE 66 -1.9247385 -4.439944 0.069760334
#> 63 147 63 TRUE 41 1.8107720 3.337019 0.080505219
#> 67 150 67 TRUE 32 1.7508315 3.229075 0.092603817
#> 136 135 136 FALSE 58, 67, 57 1.4290582 -3.198607 0.163963670
#> 71 150 71 TRUE 54 -2.3514750 -2.829766 0.033743471
#> 125 100 125 FALSE 85, 84, .... 2.7073249 2.707325 0.011833319
#> 20 115 20 TRUE 89 -0.7764417 -2.897438 0.446585533
#> 116 115 116 FALSE 59, 88, .... 3.0537776 2.897438 0.005462897
#> 140 100 140 FALSE 35, 43, .... -2.5102007 -2.510201 0.017809639
#> pval_parent
#> 51 0.0003193122
#> 52 0.0003193122
#> 63 0.0022734266
#> 67 0.0033277959
#> 136 0.0041906737
#> 71 0.0082310590
#> 125 0.0118333189
#> 20 0.0124821272
#> 116 0.0124821272
#> 140 0.0178096392The results of the previous steps are visualised by a coloured tree with a corresponding heatmap. The heatmap displays the median scaled marker expressions of each cluster to help understand what cell type each cluster may represent, and the tree not only reveals how clusters have been hierarchically aggregated, but is coloured on each node by the test statistic obtained when testing using the proportions relative to all of that node, with the branch connecting the child to the parent coloured by the test statistic obtained when testing using the proportions relative to parent of the child node.
plotInteractiveHeatmap(tested_tree,
clust_med_df = clust_tree$median_freq,
clusters=clusters)
#> Warning: Removed 1 row containing missing values or values outside the scale range
#> (`geom_interactive_point()`).Below we change the hierarchical aggregation technique to
average-linkage hierarchical clustering. The available options include
any of the available ethods in the hclust() function, ie,
one of "ward.D", "ward.D2",
"single", "complete", "average",
"mcquitty", "median" or
"centroid"
clust_tree <- getClusterTree(exprs,
clusters,
hierarchy_method="average")
tested_tree <- testTree(clust_tree$clust_tree,
clusters=clusters,
samples=samples,
classes=classes,
pos_class_name=NULL)
plotInteractiveHeatmap(tested_tree,
clust_med_df = clust_tree$median_freq,
clusters=clusters)
#> Warning: Removed 1 row containing missing values or values outside the scale range
#> (`geom_interactive_point()`).Below we change the significance test to use count models instead of
a t-test/Wilcoxon test. The available methods are "GLMM" or
"edgeR"
clust_tree <- getClusterTree(exprs,
clusters,
hierarchy_method="hopach")
tested_tree_edgeR <- testTree(clust_tree$clust_tree,
clusters=clusters,
samples=samples,
classes=classes,
sig_test="edgeR",
pos_class_name="COV")
#> Using classic mode.
plotInteractiveHeatmap(tested_tree_edgeR,
clust_med_df = clust_tree$median_freq,
clusters=clusters)You can also extract edgeR FDR values as so:
head(tested_tree_edgeR$data[,c("parent", "node", "isTip", "clusters", "FDR_parent", "FDR_total")])
#> # A tibble: 6 × 6
#> parent node isTip clusters FDR_parent FDR_total
#> <int> <dbl> <lgl> <list> <dbl> <dbl>
#> 1 102 1 TRUE <chr [1]> 0.890 0.158
#> 2 102 2 TRUE <chr [1]> 0.808 0.157
#> 3 104 3 TRUE <chr [1]> 0.794 0.920
#> 4 104 4 TRUE <chr [1]> 0.278 0.107
#> 5 105 5 TRUE <chr [1]> 0.794 0.331
#> 6 105 6 TRUE <chr [1]> 0.943 0.636Extract cell type proportions (both %total and %parent), as well as the geometric mean of each marker per cell type. These can then be used for further investigation via visualisation or machine learning.
The function below returns a dataframe containing the absolute proportions and proportions to parent for each cell type for each sample.
perc_parent_n_celltype denotes the proportion of the
cell type relative to its parent (%parent) n generations away, ie.
perc_parent_1_2 is the proportion of cluster 2 relative to
its direct parent in the hierarchical tree.
perc_total_celltype denotes the proportion of the cell
type relative all cells (%total)
prop_df <- getCellProp(phylo=clust_tree$clust_tree,
clusters=clusters,
samples=samples,
classes=classes)
head(prop_df[,1:8])
#> sample_id class perc_total_92 perc_total_91 perc_total_7 perc_total_50
#> 1 COV1 COV 0.029411765 0.005882353 0.01176471 0.000000000
#> 2 COV10 COV 0.066666667 0.044444444 0.00000000 0.005555556
#> 3 COV12 COV 0.051428571 0.000000000 0.03428571 0.000000000
#> 4 COV13 COV 0.006410256 0.000000000 0.01923077 0.012820513
#> 5 COV14 COV 0.019867550 0.019867550 0.00000000 0.006622517
#> 6 COV15 COV 0.000000000 0.000000000 0.00000000 0.000000000
#> perc_total_5 perc_total_39
#> 1 0.005882353 0.000000000
#> 2 0.016666667 0.005555556
#> 3 0.005714286 0.000000000
#> 4 0.038461538 0.000000000
#> 5 0.006622517 0.000000000
#> 6 0.006993007 0.000000000The function below returns a dataframe containing the geometric mean for each marker for each cell type for each sample.
m_gmean_celltype denotes the geometric mean of marker m
in the cell type per sample
means_df <- getCellGMeans(clust_tree$clust_tree,
exprs=exprs,
clusters=clusters,
samples=samples,
classes=classes)
head(means_df[,1:8])
#> sample_id class CD45RA B525-FITC-H_gmean_92 CD45RA B525-FITC-H_gmean_91
#> 1 COV1 COV 8.026009 10.271191
#> 2 COV10 COV 8.804902 8.432155
#> 3 COV12 COV 8.268734 NaN
#> 4 COV13 COV 10.221825 NaN
#> 5 COV14 COV 9.768303 10.470112
#> 6 COV15 COV NaN NaN
#> CD45RA B525-FITC-H_gmean_7 CD45RA B525-FITC-H_gmean_50
#> 1 7.551528 NaN
#> 2 NaN 6.360800
#> 3 6.611482 NaN
#> 4 8.369937 10.629523
#> 5 NaN 8.597188
#> 6 NaN NaN
#> CD45RA B525-FITC-H_gmean_5 CD45RA B525-FITC-H_gmean_39
#> 1 7.126569 NaN
#> 2 8.496825 7.683541
#> 3 9.265149 NaN
#> 4 8.178744 NaN
#> 5 8.543981 NaN
#> 6 11.036683 NaNsessionInfo()
#> R version 4.4.2 (2024-10-31)
#> Platform: x86_64-pc-linux-gnu
#> Running under: Ubuntu 24.04.1 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=C
#> [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
#>
#> time zone: Etc/UTC
#> tzcode source: system (glibc)
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods
#> [8] base
#>
#> other attached packages:
#> [1] ggtree_3.15.0 SingleCellExperiment_1.29.1
#> [3] SummarizedExperiment_1.37.0 Biobase_2.67.0
#> [5] GenomicRanges_1.59.1 GenomeInfoDb_1.43.1
#> [7] IRanges_2.41.1 S4Vectors_0.45.2
#> [9] BiocGenerics_0.53.3 generics_0.1.3
#> [11] MatrixGenerics_1.19.0 matrixStats_1.4.1
#> [13] treekoR_1.15.0 BiocStyle_2.35.0
#>
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#> [11] MASS_7.3-61 backports_1.5.0
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#> [21] buildtools_1.0.0 minqa_1.2.8
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#> [99] clue_0.3-66 scales_1.3.0
#> [101] png_0.1-8 ggfun_0.1.7
#> [103] colorRamps_2.3.4 knitr_1.49
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#> [123] beachmat_2.23.1 cluster_2.1.6
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#> [131] rlang_1.1.4 crayon_1.5.3
#> [133] ggsignif_0.6.4 labeling_0.4.3
#> [135] FlowSOM_2.15.0 ggbeeswarm_0.7.2
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