TreeCorTreat: Tree-based correlation screen for phenotype-associated transcriptomic features and cell types
15 November 2021
Package
TreeCorTreat 0.9.0
Contents
- 1 Overview
- 2 Input data and data preparation
- 3 Module 1: Data integration
- 4 Module 2: Define cell types at multiple resolutions
- 5 Module 3: Identify association between cell type proportion and sample phenotype
- 6 Module 4: Identify association between global gene expression and sample phenotype
- 7 Module 5: Identify differentially expressed genes
- 8 Module 6: Visualization
- 9 TreeCorTreat supports versatile multi-resolution analysis
- 10 Analysis of multivariate outcomes
- 11 Adjusting for covariates in TreeCorTreat analysis
1 Overview
Single-cell RNA-seq experiments with multiple samples are increasingly used to discover cell types and their molecular features that may influence samples’ phenotype (e.g. disease). However, analyzing and visualizing the complex cell type-phenotype association remains nontrivial. TreeCorTreat is an open source R package that tackles this problem by using a tree-based correlation screen to analyze and visualize the association between phenotype and transcriptomic features and cell types at multiple cell type resolution levels. With TreeCorTreat, one can conveniently explore and compare different feature types, phenotypic traits, analysis protocols and datasets, and evaluate the impacts of potential confounders.
TreeCorTreat takes a gene expression matrix (raw count), cell-level metadata and sample-level metadata as input. It provides a whole pipeline to integrate data across samples, identify cell clusters and their hierarchical structure, evaluate the association between sample phenotype and cell type at different resolution levels in terms of both cell type proportion and gene expression, and summarize and visualize the results in a tree structured TreeCorTreat plot. This pipeline consists of six functional modules:
- Module 1: Data integration
- Module 2: Define cell types at multiple resolutions
- Module 3: Identify association between cell type proportion and sample phenotype
- Module 4: Identify association between global gene expression and sample phenotype
- Module 5: Identify differentially expressed genes
- Module 6: Visualization via TreeCorTreat plot
The modular structure provides users with the flexibility to skip certain analysis steps and replace them by users’ own data or analysis functions.
For more details, please check our paper describing the TreeCorTreat package: - Tree-based Correlation Screen and Visualization for Exploring Phenotype-Cell Type Association in Multiple Sample Single-Cell RNA-Sequencing Experiments. bioRxiv 2021.10.27.466024; doi: https://doi.org/10.1101/2021.10.27.466024
2 Input data and data preparation
The input for TreeCorTreat consists of three components: a gene expression matrix E (G\(\times\)C, with rows representing G genes and columns representing C cells), cell-level metadata M (C\(\times\)K, where K \(\geq\) 2) and sample-level metadata L (S\(\times\)J, where J \(\geq\) 2). To avoid redundancy and save storage space, we split metadata into cell-level and sample-level metadata. Cell-level metadata primarily annotates cell-level information and must contain at least 2 columns for each cell: cell barcode and corresponding sample identifier (ID). An optional third column can be added to provide cell-type annotation. Cell barcode is used to couple cell-level metadata M and gene expression matrix E. Sample-level metadata documents a sample’s phenotype(s) of interests (e.g. clinical outcome) and other related covariates (e.g. age and sex). The first column contains unique sample IDs, and the remaining columns contain phenotypes and covariates. The cell-level metadata and sample-level metadata can be linked via unique sample IDs.
Here, we include 8 single-cell RNA-seq PBMC samples downloaded from ArrayExpress: E-MTAB-9357 as an example to illustrate TreeCorTreat pipeline. If you are interested in how to prepare input data from scratch, please refer to the Data Preparation tutorial here.
# load in TreeCorTreat
options(warn=-1)
suppressMessages(library(TreeCorTreat))
suppressMessages(library(ggplot2))
suppressMessages(library(dplyr))
suppressMessages(library(tidyr))
data(raw_data)
str(raw_data)## List of 3
## $ sample_meta:'data.frame': 8 obs. of 5 variables:
## ..$ sample : chr [1:8] "HD-17-Su" "HD-19-Su" "HD-23-Su" "HD-30-Su" ...
## ..$ study : chr [1:8] "Su" "Su" "Su" "Su" ...
## ..$ age : num [1:8] 52 79 26 35 33 63 88 84
## ..$ sex : chr [1:8] "F" "M" "F" "F" ...
## ..$ severity: chr [1:8] "HD" "HD" "HD" "HD" ...
## $ cell_meta :'data.frame': 16572 obs. of 2 variables:
## ..$ barcode: chr [1:16572] "HD-17-Su:345974" "HD-17-Su:345975" "HD-17-Su:345976" "HD-17-Su:345977" ...
## ..$ sample : chr [1:16572] "HD-17-Su" "HD-17-Su" "HD-17-Su" "HD-17-Su" ...
## $ count :Formal class 'dgCMatrix' [package "Matrix"] with 6 slots
## .. ..@ i : int [1:17088466] 10 77 81 419 683 873 1496 2208 2218 2229 ...
## .. ..@ p : int [1:16573] 0 796 1378 2418 3480 4540 5982 7295 8011 8927 ...
## .. ..@ Dim : int [1:2] 19668 16572
## .. ..@ Dimnames:List of 2
## .. .. ..$ : chr [1:19668] "A1BG" "A1BG-AS1" "A2M" "A2M-AS1" ...
## .. .. ..$ : chr [1:16572] "HD-17-Su:345974" "HD-17-Su:345975" "HD-17-Su:345976" "HD-17-Su:345977" ...
## .. ..@ x : num [1:17088466] 1 1 1 7 1 1 1 1 1 1 ...
## .. ..@ factors : list()raw_data is a list that contains three elements: sample-level metadata, cell-level metadata and gene expression matrix (raw count). There are 8 samples (4 healthy donors (HD) and 4 Severe (Se) COVID-19 samples), 16572 cells and 19668 genes.
3 Module 1: Data integration
Harmony algorithm is applied to integrate cells from different samples and embed them into a common low-dimensional space. We adapt the RunHarmony and BuildClusterTree functions from Seurat v3 and wrap the following steps into a standalone treecor_harmony function with tunable parameters: library size normalization, integration feature selection, Principal Component Analysis (PCA), Harmony integration, unsupervised louvain clustering, Uniform Manifold Approximation and Projection (UMAP), hierarchical clustering of louvain clusters, and differentially expression analysis to identify cell type marker genes to facilitate annotation.
Specifically, for library size normalization, gene counts for a given cell are divided by total counts for that cell, multiplied by a scaling factor (\(10^4\)) and applied a natural log transformation. Features for integrating samples are obtained by choosing highly variable genes (HVGs) based on variance-mean relationship within each individual sample (using SelectIntegrationFeatures function) and ranking the features based on the number of samples where they are identified as HVGs. By default, top 2000 HVGs are chosen and fed into downstream PCA procedure. Harmony is carried out based on the top 20 PCs and the corrected harmony embedding is obtained. Top 20 harmony coordinates are then used for downstream louvain clustering and UMAP analyses (using FindClusters and RunUMAP functions in Seurat).
# data integration
set.seed(12345)
output_dir <- paste0(getwd(),'/')
integration <- treecor_harmony(count = raw_data[['count']], sample_meta = raw_data[['sample_meta']], output_dir = output_dir)This step is the most time-consuming and RAM intensive module in our evaluation. It may take up to days to run using a large dataset (>\(10^5\) cells). Instead of using this default integration pipeline, users can also use their own integration results in a Seurat object or provide cell cluster labels in an additional ‘celltype’ column in the cell-level metadata and skip the integration step.
The integrated Seurat object will be stored in a specified output directory and filtered gene expression matrix or cell-level metadata can be extracted by access_data_seurat command. The filtered gene expression matrix and cell-level metadata will be used for downstream analysis.
# list integration result and data extracted from Seurat object
integrated_data <- access_data_seurat(seurat_obj = integration,output_dir = output_dir)The integrated_data is a list object that contains three components: count (for gene expression matrix), cell_meta (filtered cell-level metadata) and phylo_tree (hierarchical clustering of cell types). In our example, no cell has been filtered out. For demonstration purpose, we extracted updated cell-level metadata (with additional columns including ‘celltype’ and UMAP coordinates) using integrated_data$cell_meta and stored as integrated_cellmeta data object.
data(integrated_cellmeta)
head(integrated_cellmeta)## barcode sample celltype UMAP_1 UMAP_2
## 1 HD-17-Su:345974 HD-17-Su 3 -3.393676 -7.0051720
## 2 HD-17-Su:345975 HD-17-Su 2 -5.090366 6.9038575
## 3 HD-17-Su:345976 HD-17-Su 2 -6.407369 6.9599665
## 4 HD-17-Su:345977 HD-17-Su 5 -6.225506 3.5054925
## 5 HD-17-Su:345978 HD-17-Su 1 8.189426 0.8951219
## 6 HD-17-Su:345979 HD-17-Su 9 9.156926 -4.1967079# data for downstream analysis
sample_meta <- raw_data[['sample_meta']]
dim(sample_meta)## [1] 8 5count <- raw_data[['count']]
dim(count)## [1] 19668 16572cell_meta <- integrated_cellmeta
dim(cell_meta)## [1] 16572 54 Module 2: Define cell types at multiple resolutions
The previous steps defined cell clusters either by louvain clustering or based on user-provided cell-level metadata (i.e. the optional column 3 in the cell metadata). Users can add text labels to annotate the cell type of each cell cluster based on top differentially expressed genes or known cell type marker genes. Here, we overlay several gene markers (e.g. CD3D, CD19 and CD68) on UMAP to roughly annotate cell clusters.
## plot UMAP and annotate cell type ID
# prepare cell type ID label
label_text <- integrated_cellmeta %>% group_by(celltype) %>% summarise(UMAP_1 = median(UMAP_1),UMAP_2 = median(UMAP_2))
# plot UMAP
ggplot(integrated_cellmeta,aes(x=UMAP_1,y=UMAP_2,color = celltype)) +
geom_point(size = 0.1) +
geom_text(data = label_text,aes(x=UMAP_1,y=UMAP_2,label = celltype),color = 'black',size = 5) +
theme_classic() +
guides(color = guide_legend(override.aes = list(size = 3))) 
## Overlay gene markers on top of UMAP
genes <- c('CD3D','CD14','CD19','NCAM1','CD4','CD8A',
'FCGR3A','CD1C','CD68','CD79A','CSF3R',
'CD33','CCR7','CD38','CD27','KLRD1')
# subset CPM normalized gene expression matrix
rc <- Matrix::colSums(count)
sub_count <- count[genes,] %>% as.matrix
sub_norm <- log2(t(t(sub_count)/rc*1e6 + 1)) %>% as.matrix
# prepare a long data format
df_marker <- t(sub_norm) %>% data.frame %>% mutate(barcode = rownames(.)) %>% gather(gene,expr,-barcode) %>% inner_join(cell_meta %>% select(barcode,UMAP_1,UMAP_2))
colnames(df_marker)## [1] "barcode" "gene" "expr" "UMAP_1" "UMAP_2"# ggplot2: each gene as a panel
ggplot(df_marker,aes(x = UMAP_1,y = UMAP_2,col = expr)) +
geom_point(size = 0.01, shape = ".") +
scale_colour_viridis_c(option = 'C',direction = 1) +
facet_wrap(~ gene) +
theme_classic(base_size = 12) +
theme(legend.position = 'bottom')
We roughly categorize 16 cell clusters into 3 large cell-types based on gene markers: B cells (CD19), T cells (CD3D) and Monocytes (CD14).
# new celltype annotation (must contain `old` and `new` two columns)
new_label <- data.frame(old = sort(factor(unique(cell_meta$celltype,levels = 1:16)))) %>%
mutate(large_celltype = ifelse(old %in% c(1,9,11,12,13,16),'Mono',
ifelse(old %in% c(2:6,8,10,14,15),'T','B')),
new = paste0(large_celltype,'_c',old)) %>% select(-large_celltype)
new_label## old new
## 1 1 Mono_c1
## 2 10 T_c10
## 3 11 Mono_c11
## 4 12 Mono_c12
## 5 13 Mono_c13
## 6 14 T_c14
## 7 15 T_c15
## 8 16 Mono_c16
## 9 2 T_c2
## 10 3 T_c3
## 11 4 T_c4
## 12 5 T_c5
## 13 6 T_c6
## 14 7 B_c7
## 15 8 T_c8
## 16 9 Mono_c94.1 Modify cell type annotation
Users can modify the cell type label via modify_label function to update cell type annotations in cell_meta:
# modify cell type names in `cell_meta`
cell_meta <- modify_label(new_label,hierarchy_list = NULL,cell_meta)$cell_meta## Modify label in cell_metahead(cell_meta[,c('barcode','sample','celltype')])## barcode sample celltype
## 1 HD-17-Su:345974 HD-17-Su T_c3
## 2 HD-17-Su:345975 HD-17-Su T_c2
## 3 HD-17-Su:345976 HD-17-Su T_c2
## 4 HD-17-Su:345977 HD-17-Su T_c5
## 5 HD-17-Su:345978 HD-17-Su Mono_c1
## 6 HD-17-Su:345979 HD-17-Su Mono_c9Similarly, one can also update cell type annotation in hierarchical tree structure using this function
(i.e. modify_label(new_label,hierarchy_list = hierarchy_list,cell_meta = NULL)$hierarchy_list).
4.2 Construct hierarchical tree structure
To facilitate association analyses at multiple resolutions, cell clusters are further clustered hierarchically. The tree can be derived using either a data-driven approach or a knowledge-based approach.
The tree can be either provided by users based on prior knowledge (knowledge-based) or derived from the data using hierarchical clustering (data-driven). For data-driven approach, a phylogenetic tree is built on PC space by applying treecor_harmony function (or BuildClusterTree from Seurat R package).
4.2.1 Data-driven approach
In the data-driven approach, the tree is generated by the hierarchical clustering of the louvain clusters by applying treecor_harmony function (or BuildClusterTree from Seurat R package). Specifically, PC scores are first averaged across cells within each cell cluster and a hierarchical tree is constructed. The data-driven approach could provide an unbiased way to infer underlying tree structure, but it can be challenging to annotate every intermediate tree nodes.
4.2.2 Knowledge-based approach
In the knowledge-based approach, users can specify the tree based on their prior knowledge by providing a string to describe the parent-children relationship of clusters at different granularity levels. The leaf nodes in the tree correspond to cell clusters obtained from either the louvain clustering in the integration step or the ‘celltype’ column from cell metadata.
# specify input string with special character ('@') to distinguish non-leaf node from leaves
input_string <- '@All(@B(B_c7),@T(T_c15,T_c14,T_c10,T_c8,T_c6,T_c5,T_c4,T_c3,T_c2),@Monocyte(Mono_c16,Mono_c13,Mono_c12,Mono_c11,Mono_c9,Mono_c1))'
# extract hierarchy from string
hierarchy_structure <- extract_hrchy_string(input_string,special_character = '@', plot = T)
For demonstration purpose, we will use knowledge-based hierarchical structure in subsequent analysis.
5 Module 3: Identify association between cell type proportion and sample phenotype
Analyses of cell type proportions are wrapped into a standalone function treecor_ctprop. Here we first define the feature for each tree node and then evaluate the association between the feature and the sample phenotype. For a leaf node cell cluster, the feature is defined as the proportion of cells in a sample that fall into that node. For an intermediate parent node or root node, users can choose to define the feature in one of Aggregate (default setting), Concatenate leaf nodes or Concatenate immediate children. Once the feature for each node is defined, we will go through each tree node to examine the correlation (or other summary statistic) between its feature and the sample phenotype. The way to compute the summary statistic depends on whether the phenotype is univariate or multivariate:
- Univariate phenotype/Multivariate phenotype analyzed separately:
- Pearson correlation (default) with correlation sign
- Spearman correlation with correlation sign
- Canonical correlation
- Chi-squared statistic (via likelihood ratio test between a full model (with phenotype as explanatory variables) and a reduced model (intercept-only)) with coefficient sign
- Multivariate phenotype analyzed jointly:
- Canonical correlation
- Chi-squared statistic (via likelihood ratio test)
The permutation-based p-value can be obtained and adjusted p-value is computed using Benjamini & Yekutieli procedure.
We can first use a kernel density plot to visualize cell density stratified by disease severity.
# density plot on UMAP embeddings
treecor_celldensityplot(cell_meta,
sample_meta,
row_variable = 'study',
col_variable = 'severity',
row_combined = F)
Next, we can assess the association between cell type proportion and disease severity using treecor_ctprop() pipeline:
# cell type prop pipeline (default)
res_ctprop_full <- treecor_ctprop(hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity',
method = "aggregate",
analysis_type = "pearson",
num_permutations = 100)
names(res_ctprop_full)## [1] "canonical_corr" "pc_ls"The first element is a table of computed summary statistic (e.g. pearson correlation) along with p-value and adjusted p-value. The second element is a list of PC matrices for each cell type. However, in default setting (aggregate), proportion is aggregated as a vector (1-dimension) and thus we do not conduct PCA in this scenario.
# extract Pearson correlation
res_ctprop <- res_ctprop_full[[1]] %>% mutate(severity.absolute_cor = abs(severity.pearson))
head(res_ctprop)## id severity.method severity.analysis_type severity.pearson severity.p
## 1 1 aggregate pearson NA NA
## 2 2 aggregate pearson 0.1147058 0.82828283
## 3 3 aggregate pearson -0.7614817 0.08080808
## 4 4 aggregate pearson 0.7603030 0.08080808
## 5 5 aggregate pearson 0.1147058 0.82828283
## 6 6 aggregate pearson 0.5039696 0.11111111
## severity.adjp severity.direction severity.p.sign severity.adjp.sign x y
## 1 NA <NA> <NA> <NA> 6.25 2
## 2 1.0000000 + ns ns 0.00 1
## 3 0.7781478 - ns ns 5.00 1
## 4 0.7781478 + ns ns 12.50 1
## 5 1.0000000 + ns ns 0.00 0
## 6 0.8321858 + ns ns 1.00 0
## label leaf severity.absolute_cor
## 1 All FALSE NA
## 2 B FALSE 0.1147058
## 3 T FALSE 0.7614817
## 4 Monocyte FALSE 0.7603030
## 5 B_c7 TRUE 0.1147058
## 6 T_c15 TRUE 0.5039696colnames(res_ctprop)## [1] "id" "severity.method" "severity.analysis_type"
## [4] "severity.pearson" "severity.p" "severity.adjp"
## [7] "severity.direction" "severity.p.sign" "severity.adjp.sign"
## [10] "x" "y" "label"
## [13] "leaf" "severity.absolute_cor"Then we can use TreeCorTreat plot to visualize the result. Users can specify variables for different aesthetics (e.g. color, size, alpha). We will discuss TreeCorTreat plot in details later.
# TreeCorTreat plot visualization
treecortreatplot(hierarchy_structure,
annotated_df = res_ctprop,
response_variable = 'severity',
color_variable = 'direction',
size_variable = 'absolute_cor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2)
6 Module 4: Identify association between global gene expression and sample phenotype
Analyses of global gene expression are wrapped into a standalone function treecor_expr. Here we also first define the feature for each tree node and then evaluate the global gene expression-sample phenotype association. For a leaf node, we first pool all cells in the node to obtain a pseudobulk profile of the node. We then use these pseudobulk profiles from all samples to select highly variable genes using locally weighted scatterplot smoothing (LOWESS) procedure in R. The pseudobulk profile of the selected highly variable genes in each sample is used as the feature vector of the node. This feature vector is multivariate. For non-leaf nodes, users can choose to define the feature in one of Aggregate (default setting), Concatenate leaf nodes or Concatenate immediate children. Once the feature for each node is defined, we will go through each tree node to examine the correlation (or other summary statistic) between its feature and the sample phenotype. Users can choose either canonical correlation or F-statistic (Pillai-Bartlett, comparing a full model (with phenotype as explanatory variables) with a reduced model (intercept-only)).
The following code illustrates an example of association between gene expression and disease severity, using default setting (aggregate).
# gene expression pipeline
res_expr_full <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity',
method = 'aggregate',
analysis_type = 'cancor',
num_permutations = 100)
names(res_expr_full)## [1] "canonical_corr" "pc_ls"By running treecor_expr, it results in two elements: the first element is a summary table of computed summary statistic (e.g. canonical correlation) along with p-value and adjusted p-value for each cell type; and the second element is a list of PC matrices for each cell type.
# extract canonical correlation
res_expr <- res_expr_full[[1]]
head(res_expr)## id severity.cancor severity.p severity.adjp severity.direction
## 1 1 0.8942939 0.03030303 0.4360897 +
## 2 2 0.6428883 0.15151515 0.9911129 +
## 3 3 0.9297493 0.03030303 0.4360897 +
## 4 4 0.8324347 0.03030303 0.4360897 +
## 5 5 0.6428883 0.15151515 0.9911129 +
## 6 6 0.8807827 0.04040404 0.4845441 +
## severity.p.sign severity.adjp.sign x y label leaf
## 1 sig ns 6.25 2 All FALSE
## 2 ns ns 0.00 1 B FALSE
## 3 sig ns 5.00 1 T FALSE
## 4 sig ns 12.50 1 Monocyte FALSE
## 5 ns ns 0.00 0 B_c7 TRUE
## 6 sig ns 1.00 0 T_c15 TRUEThe summary table is used to generate TreeCorTreat plot:
# visualize
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2)
In addition to summary table, one can also assess each cell type separately by using PC matrices from gene expression analysis.
# extract PCA list
pc_expr <- res_expr_full[[2]]
names(pc_expr)## [1] "All" "B" "T" "Monocyte" "B_c7" "T_c15"
## [7] "T_c14" "T_c10" "T_c8" "T_c6" "T_c5" "T_c4"
## [13] "T_c3" "T_c2" "Mono_c16" "Mono_c13" "Mono_c12" "Mono_c11"
## [19] "Mono_c9" "Mono_c1"# extract PCA matrix corresponding to 'T' tree node
pc_expr[['T']]## PC1 PC2
## HD-17-Su -44.684639 2.823131
## HD-19-Su -51.774059 4.264000
## HD-23-Su -51.352355 -23.472410
## HD-30-Su -39.098933 -37.230307
## Se-137.1-Su 73.681369 47.520847
## Se-178.1-Su 11.966558 20.281313
## Se-180.1-Su 6.457736 53.349695
## Se-181.1-Su 94.804324 -67.536270The list of PC matrices are named by cell type names specified in celltype column of cell-level metadata (either user-specified or inferred from data-driven approach). Thus, users can extract PC coordinates of each sample of a given cell type (e.g. T cell category) and visualize sample-level PCA plot using treecor_samplepcaplot.
# PCA plot (using `T` node)
treecor_samplepcaplot(sample_meta,
pca_matrix = pc_expr[['T']],
response_variable = 'severity',
font_size = 10,
point_size = 3)
We can observe that samples’ severity progressively changes from healthy to severe along the direction indicated by the dashed line, which corresponds to an optimal axis inferred from CCA that maximizes the correlation between the severity and samples’ coordinates in the embedded space.
7 Module 5: Identify differentially expressed genes
The analysis of differentially expressed genes (DEGs) is wrapped into a function treecor_deg(). Here we first compute the pseudobulk gene expression profile for each tree node using the ‘Aggregate’ approach as before. In other words, for each node all cells from its descendants are pooled and aggregated into a pseudobulk profile in each sample. Pseudobulk profiles are normalized by library size. Limma was then used to conduct differential expression analysis:
Univariate phenotype/Multivariate phenotype analyzed separately: Fit a limma model by regressing gene expression against phenotype with covariate adjustment. DEGs passing a user-specified false discovery rate (FDR) cutoff (default cutoff = 0.05) will be reported for each node and saved into csv files along with log fold change, t-statistics, p-value and FDR.
Multivariate phenotype analyzed jointly: Multiple traits are combined into a univariate variable using either a data-driven approach (PC1) or user-specified weights (linear combination of multiple traits using weights). Then the aggregated trait will be analyzed as a univariate phnenotype.
# DEG pipeline
res_deg <- treecor_deg(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity')
names(res_deg)## [1] "dge.summary" "dge.ls" "pseudobulk.ls"# 1st: summary of number of DEGs
head(res_deg$dge.summary) # or head(res_deg[[1]])## label severity.num_deg x y id leaf
## 1 All 0 6.25 2 1 FALSE
## 2 B 0 0.00 1 2 FALSE
## 3 T 993 5.00 1 3 FALSE
## 4 Monocyte 2 12.50 1 4 FALSE
## 5 B_c7 0 0.00 0 5 TRUE
## 6 T_c15 456 1.00 0 6 TRUE# 2nd: extract DEGs for a specific tree node using cell type name
# (1) check phenotypes
names(res_deg$dge.ls)## [1] "severity"# (2) choose a phenotype
severity.dge.ls <- res_deg$dge.ls[['severity']]
# (3) extract DEGs of T cell category
head(severity.dge.ls[['T']]) ## severitySe.logFC severitySe.t severitySe.p severitySe.fdr
## FADS1 2.221299 13.884431 2.351480e-07 0.004165177
## JCHAIN 3.036823 12.451043 5.964215e-07 0.005282207
## INPPL1 1.755148 10.250902 3.058248e-06 0.017470015
## APOBEC3H 2.250345 9.908317 4.052855e-06 0.017470015
## GPR68 1.693644 9.515158 5.657245e-06 0.017470015
## CORO1C 1.813891 9.220954 7.316652e-06 0.017470015# 3rd: extract sample-level pseudobulk for a specific tree node using cell type name
names(res_deg$pseudobulk.ls) # or names(res_deg[[3]])## [1] "All" "B" "T" "Monocyte" "B_c7" "T_c15"
## [7] "T_c14" "T_c10" "T_c8" "T_c6" "T_c5" "T_c4"
## [13] "T_c3" "T_c2" "Mono_c16" "Mono_c13" "Mono_c12" "Mono_c11"
## [19] "Mono_c9" "Mono_c1"head(res_deg$pseudobulk.ls[['T']]) ## HD-17-Su HD-19-Su HD-23-Su HD-30-Su Se-137.1-Su Se-178.1-Su
## A1BG 5.493462 5.506674 5.6802740 5.876471 4.7791551 5.3461643
## A1BG-AS1 2.652148 1.854222 2.3063366 2.420313 2.9822341 2.2278498
## A2M 0.000000 0.000000 0.1968054 0.000000 0.6555113 0.3599841
## A2M-AS1 1.814409 1.413839 1.2995822 1.852688 3.5050187 2.9638223
## A4GALT 0.000000 0.000000 0.3699580 0.000000 0.0000000 0.0000000
## AAAS 4.653329 4.235223 4.1063058 4.665398 5.1026623 4.7283047
## Se-180.1-Su Se-181.1-Su
## A1BG 5.516765 4.809994
## A1BG-AS1 0.000000 2.002053
## A2M 0.000000 1.001369
## A2M-AS1 3.578594 3.809896
## A4GALT 0.000000 0.000000
## AAAS 4.315328 4.250520treecor_deg returns a list of three elements: first element summarizes number of DEGs for each cell type, and the second element corresponds to specific DEGs identified within each cell cluster for each phenotype (response_variable) and third one stores sample-level pseudobulk gene expression matrix (each row is a gene and each column is a sample) for each cell type.
Depending on whether multivariate phenotype is analyzed separately or jointly, there will be additional columns in the summary table (i.e. 1st element - dge.summary) that documents number of DEGs with respect to different phenotypes and additional elements in the DEG list (i.e. 2nd element - dge.ls).
We can visualize the number of DEGs using TreeCorTreat plot:
# visualize
treecortreatplot(hierarchy_structure,
annotated_df = res_deg$dge.summary,
response_variable = 'severity',
color_variable = 'num_deg',
size_variable = 'num_deg',
alpha_variable = NULL,
annotate_number = T,
annotate_number_column = 'num_deg',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2)
Additionally, we can use treecor_degheatmap to explore gene expression pattern for top \(n\) DEGs, by specifying \(n\) and direction/sign of log fold change:
# heatmap for top 10 DEGs with positive log fold change (enriched in Severe)
treecor_degheatmap(sample_meta,
pseudobulk = res_deg$pseudobulk.ls[['T']],
deg_result = res_deg$dge.ls$severity[['T']],
top_n = 10,
deg_logFC = "positive",
annotation_col = c('severity','sex','age'),
annotation_colors = list(severity = c('HD' = 'green','Se' = 'red'),
sex = c('F' = 'purple','M' = 'blue')))
# heatmap for top 10 DEGs with negative log fold change (enriched in Healthy)
treecor_degheatmap(sample_meta,
pseudobulk = res_deg$pseudobulk.ls[['T']],
deg_result = res_deg$dge.ls$severity[['T']],
top_n = 10,
deg_logFC = "negative",
annotation_col = c('severity','sex','age'),
annotation_colors = list(severity = c('HD' = 'green','Se' = 'red'),
sex = c('F' = 'purple','M' = 'blue')))
# Combined two plots above
treecor_degheatmap(sample_meta,
pseudobulk = res_deg$pseudobulk.ls[['T']],
deg_result = res_deg$dge.ls$severity[['T']],
top_n = 10,
deg_logFC = "both",
annotation_col = c('severity','sex','age'),
annotation_colors = list(severity = c('HD' = 'green','Se' = 'red'),
sex = c('F' = 'purple','M' = 'blue')))
8 Module 6: Visualization
TreeCorTreat provides a variety of functions to visualize both the intermediate and final results. For example, we can use treecor_celldensityplot to show cell type proportions for different sample groups (Module 3). treecor_samplepcaplot projects samples to a shared low-dimensional embedding and find optimal axis (Module 4).treecor_degheatmap shows top differentially expressed genes of a given cell type (Module 5). In particular, in order to summarize the final results, we developed a versatile function treecortreatplot to display the multi-resolution cell type-phenotype association in a format we call ‘TreeCorTreat plot’. The plot consists of a dendrogram showing the cell types’ hierarchical structure and information layers showing analysis results about each tree node.
8.1 Tree skeleton representation
For displaying the tree, the nodes can be connected using straight lines, a classical angle bend representation, or a quadratic bezier curve representation. Users can choose line aesthetics such as linetype (e.g. solid or dashed) and line color according to their preference.
8.1.1 Edge type
- Straight line
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: edge_path_type
edge_path_type = "link")
- Classical angle bend
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.6,
nonleaf_point_gap = 0.3,
## parameter: edge_path_type
edge_path_type = "elbow")
Based on our experience, we recommend to use classical angle bend in a data-driven approach to avoid intertwined lines.
- Quadratic Bezier curves
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: edge_path_type
edge_path_type = "diagonal")
8.1.2 Line aesthetics: line type and color
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: line_type
line_type = 'dashed',
## parameter: line_color
line_color = 'darkgreen')
8.1.3 Modify label or circle position
In some cases where label length (cell type name) is long or circle size is quite large, default label or circle position on the non-leaf part (left) may not work well. Therefore, we introduce nonleaf_label_pos and nonleaf_point_gap parameters which allow users to manually fix the label position or gap between multivariate phenotype. A tip is that if one have \(k\) phenotypes to be analyzed separately, one can first choose a proper gap between points (i.e. nonleaf_point_gap = 0.1) and let nonleaf_label_pos = $k*nonleaf_point_gap + small_number$ (i.e. let \(k=3\), nonleaf_label_pos = 0.1*3+0.2 = 0.5).In the above examples, we specify gap between points at 0.2 and label position at 0.4.
8.2 Leaf representation
For external node configuration, TreeCorTreat plot aligns cell types in the rows and phenotypes in the columns. There are three different ways to visualize the results: balloon plot, heatmap and bar plot. Users can use color (color_variable), size (size_variable) and transparency (alpha_variable) to encode different information.
8.2.1 Balloon plot
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: plot_type
plot_type = 'balloon')
In the above balloon plot, the size of the balloon reflects the magnitude of canonical correlation between global gene expression profile and disease severity and color as well as transparency correspond to p-value significance.
Alternatively, we can use color to represent canonical correlation and size/transparency to represent p-value significance using the following code:
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'p.sign',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: plot_type
plot_type = 'balloon')
8.2.2 Heatmap
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: plot_type
plot_type = 'heatmap')
8.2.3 Barplot
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'p.sign',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
## parameter: plot_type
plot_type = 'bar')
8.3 More advanced plotting figures
8.3.1 Annotate numbers
Users can specify which variable/color to be used in annotating TreeCorTreat plot via
annotate_number, annotate_number_column and annotate_number_color arguments.The following code demonstrates an example of overlaying canonical correlation on top of TreeCorTreat:
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
annotate_number = T,
annotate_number_column = 'cancor',
annotate_number_color = 'black')
8.3.2 Modify legend title
If we want to modify legend title, one way is to modify column names of annotated_df directly before running treecortreatplot. The column names for summary statistic and statistical significance are usually defined in response_variable.colname format (e.g. severity.cancor, severity.direction (only exists in cell type proportion analysis), severity.p, severity.p.sign, etc). Thus, it’s crucial to check the column names are in the correct format before generating TreeCorTreat plot.
Suppose we hope to modify names for both response variables (‘severity’) and legends (i.e. ‘cancor’ and ‘p.sign’) by capitalizing the first letter in ‘severity’ and ‘cancor’ and replace ‘p.sign’ by ‘P-value significance’.
## modify column names
colnames(res_expr)## [1] "id" "severity.cancor" "severity.p"
## [4] "severity.adjp" "severity.direction" "severity.p.sign"
## [7] "severity.adjp.sign" "x" "y"
## [10] "label" "leaf"res_expr_new <- res_expr
colnames(res_expr_new) <- gsub('severity','Severity',colnames(res_expr_new))
colnames(res_expr_new) <- gsub('cancor','Cancor',colnames(res_expr_new))
colnames(res_expr_new) <- gsub('\\.p.sign','\\.P-value significance',colnames(res_expr_new))
## check modified column names
colnames(res_expr_new)## [1] "id" "Severity.Cancor"
## [3] "Severity.p" "Severity.adjp"
## [5] "Severity.direction" "Severity.P-value significance"
## [7] "Severity.adjp.sign" "x"
## [9] "y" "label"
## [11] "leaf"## visualize
treecortreatplot(hierarchy_structure,
annotated_df = res_expr_new,
response_variable = 'Severity',
color_variable = 'P-value significance',
size_variable = 'Cancor',
alpha_variable = 'P-value significance',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon')
8.3.3 Modify label colors
Users can also modify label colors to highlight cell types of interest. The advanced setting can be configured by advanced_list argument by providing label_info with fixed column names: ‘label’ (cell type label) and ‘label.color’ (a color vector). For example, we can highlight cell types that have significant global gene expression-disease severity correlation in red:
label_info <- data.frame(label = res_expr$label,
label.color = ifelse(res_expr$severity.p.sign == 'ns','black','red'))
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
advanced_list = list(label_info = label_info))
8.3.4 Create your own mapping from levels to aesthetic values
Users can customize mapping from levels in the data (e.g. categorical variables) to certain aesthetic values (e.g. color/size/transparency) via breaks and values (alike scale_manual() in ggplot2). This can be also achieved by providing color_info or size_info or alpha_info in advanced_list argument. The following code illustrates an example of changing color for statistical significance as blue (non-significant) and red (significant) and modifying transparency:
advanced_list <- list(color_info = data.frame(breaks = c('ns','sig'), values = c('blue','red')),
alpha_info = data.frame(breaks = c('ns','sig'), values = c(0.5,1)))
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
advanced_list = advanced_list)
Alternatively, one can modify balloon size using the following code:
advanced_list <- list(size_info = data.frame(breaks = c('ns','sig'), values = c(4,8)))
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'p.sign',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
advanced_list = advanced_list)
8.3.5 Choose a different color palette
For categorical variable, color palette can be specified by color_info in advanced options; For continuous variable, we provide different palettes from RColorBrewer package (default is ‘Spectral’) and can be modified via palette as a element in the advanced_list.
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
advanced_list = list(palette = 'PiYG'))
8.3.6 Configure layout
The layout of TreeCorTreat plot can be modified via layout_widths in the advanced_list parameter, which specifies the relative left-to-right ratio (or non-leaf: leaf ratio).
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon',
advanced_list = list(layout_widths = c(2,1)))
8.4 Save TreeCorTreat plots
TreecorTreat plots can be saved into pdf, png or other graphical formats.
png('TreeCorTreat_plot.png', height = 8, width = 15, res = 300, units = 'in')
treecortreatplot(hierarchy_structure,
annotated_df = res_expr,
response_variable = 'severity',
color_variable = 'cancor',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.4,
nonleaf_point_gap = 0.2,
plot_type = 'balloon')
dev.off()9 TreeCorTreat supports versatile multi-resolution analysis
To analyze cell type-phenotype association at multiple resolutions, fine-grained cell clusters (children) are progressively merged into bigger clusters (parents). When a parent node is created by merging two or more child nodes, the information from the children can be combined in multiple different ways with different implications. The two basic methods to combine child nodes are ‘aggregation’ and ‘concatenation’. The ‘aggregation’ method pools all cells from the child nodes into a single pseudobulk sample which is used to represent the parent node. In the ‘concatenation’ method, one first pools cells within each child node to form a pseudobulk sample for that node. One represents each child node using a feature or a feature vector extracted from its pseudobulk sample (e.g., highly variable genes). The parent node is then represented by concatenating the feature vectors from all child nodes into a longer vector.
To support different needs of users, TreeCorTreat provides three different ways to combine child nodes: ‘aggregate’ (default), ‘concatenate leaf nodes’, and ‘concatenate immediate children’.
9.1 Aggregate
The default setting for both treecor_expr and treecor_ctprop is aggregation (see previous examples), where we aggregate raw read counts for gene expression or number of cells for cell composition for non-leaf nodes.
9.2 Concatenate leaf nodes
In the ‘concatenate leaf nodes’ approach, feature vectors of all terminal leaf nodes derived from a node are concatenated into a long vector to serve as the feature vector of the node. For global gene expression analysis, this will result in a concatenated vector consisting of pseudobulk expression of highly variable genes obtained from each leaf node. For cell type proportion analysis, this will result in a vector of cell type proportions of the leaf nodes.
# gene expression pipeline; concat_leaf
res_expr_concatLeaf <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity',
method = 'concat_leaf',
num_permutations = 100)$canonical_corr
head(res_expr_concatLeaf)## id severity.cancor severity.p severity.adjp severity.direction
## 1 1 0.7803956 0.03030303 0.4360897 +
## 2 2 0.6428883 0.15151515 0.9911129 +
## 3 3 0.8556124 0.03030303 0.4360897 +
## 4 4 0.6743079 0.03030303 0.4360897 +
## 5 5 0.6428883 0.15151515 0.9911129 +
## 6 6 0.8807827 0.04040404 0.4845441 +
## severity.p.sign severity.adjp.sign x y label leaf
## 1 sig ns 6.25 2 All FALSE
## 2 ns ns 0.00 1 B FALSE
## 3 sig ns 5.00 1 T FALSE
## 4 sig ns 12.50 1 Monocyte FALSE
## 5 ns ns 0.00 0 B_c7 TRUE
## 6 sig ns 1.00 0 T_c15 TRUE9.3 Concatenate immediate children
In the ‘concatenate immediate children’ approach, a node’s immediate children are first identified. The feature vector of each immediate child is obtained using the ‘aggregate’ approach. Then feature vector of target node is obtained by concatenating the feature vectors of its immediate children.
# gene expression pipeline; concat_immediate_children
res_expr_concatImmChild <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity',
method = 'concat_immediate_children',
num_permutations = 100)$canonical_corr
head(res_expr_concatImmChild)## id severity.cancor severity.p severity.adjp severity.direction
## 1 1 0.8504976 0.03030303 0.4360897 +
## 2 2 0.6428883 0.15151515 0.9911129 +
## 3 3 0.8556124 0.03030303 0.4360897 +
## 4 4 0.6743079 0.03030303 0.4360897 +
## 5 5 0.6428883 0.15151515 0.9911129 +
## 6 6 0.8807827 0.04040404 0.4845441 +
## severity.p.sign severity.adjp.sign x y label leaf
## 1 sig ns 6.25 2 All FALSE
## 2 ns ns 0.00 1 B FALSE
## 3 sig ns 5.00 1 T FALSE
## 4 sig ns 12.50 1 Monocyte FALSE
## 5 ns ns 0.00 0 B_c7 TRUE
## 6 sig ns 1.00 0 T_c15 TRUE# compare 3 methods (non-leaf nodes)
summary_ls <- list(res_expr %>% filter(!leaf) %>% select(id,label,severity.cancor) %>% rename(aggregate = severity.cancor),
res_expr_concatLeaf %>% filter(!leaf) %>% select(id,label,severity.cancor) %>% rename(concatLeaf = severity.cancor),
res_expr_concatImmChild %>% filter(!leaf) %>% select(id,label,severity.cancor) %>% rename(concatImmChild = severity.cancor))
summary <- Reduce(inner_join,summary_ls)
summary## id label aggregate concatLeaf concatImmChild
## 1 1 All 0.8942939 0.7803956 0.8504976
## 2 2 B 0.6428883 0.6428883 0.6428883
## 3 3 T 0.9297493 0.8556124 0.8556124
## 4 4 Monocyte 0.8324347 0.6743079 0.6743079For some broad cell categories like B, T and Monocytes, the canonical correlation of leaf concatenation and immediate children concatenation are the same because the immediate children set is same as its corresponding leaf nodes. Depending on different tree hierarchies, three approaches may have different results and implications. This can also be visualized using TreeCorTreat plot:
# aggregate
colnames(res_expr) <- gsub('severity','Severity\n(Agg)',colnames(res_expr))
# concat_leaf
colnames(res_expr_concatLeaf) <- gsub('severity','Severity\n(Leaf)',colnames(res_expr_concatLeaf))
# concat_immediate_children
colnames(res_expr_concatImmChild) <- gsub('severity','Severity\n(ImmChild)',colnames(res_expr_concatImmChild))
# combine three approaches
res_combined <- Reduce(inner_join,list(res_expr,res_expr_concatLeaf,res_expr_concatImmChild))
# plot
treecortreatplot(hierarchy_structure,
annotated_df = res_combined,
response_variable = c('Severity\n(Agg)','Severity\n(ImmChild)','Severity\n(Leaf)'),
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.8,
nonleaf_point_gap = 0.2,
plot_type = 'balloon')
10 Analysis of multivariate outcomes
When there are multiple phenotypic traits, one can either analyze each trait separately as a univariate phenotype or analyze them jointly as a multivariate phenotype. For analyzing association between a multivariate phenotype and cell type proportion or global gene expression, CCA is used to compute the canonical correlation between the phenotype and cell type features (i.e. cell type proportion or gene expression).
10.1 Separate evaluation
# individually
multi_expr_sep <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = c('severity','age'),
num_permutations = 100)$canonical_corr
# visualize
treecortreatplot(hierarchy_structure,
annotated_df = multi_expr_sep,
response_variable = c('severity','age'),
separate = T,
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.5,
nonleaf_point_gap = 0.2)
10.2 Joint evaluation
Alternatively, we can analyze both severity and age jointly.
# jointly
multi_expr_joint <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = c('severity','age'),
separate = F,
num_permutations = 100)$canonical_corr
# visualize
treecortreatplot(hierarchy_structure,
annotated_df = multi_expr_joint,
response_variable = c('severity','age'),
separate = F,
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.2,
nonleaf_point_gap = 0.1)
For analyzing differential gene expression, a multivariate phenotype is first transformed into a univariate phenotype either using the principal component analysis or using a linear combination of traits with weights specified by users. The transformed univariate phenotype, which combines information from multiple traits, is then used to run differential expression analysis.
# jointly
multi_deg_joint <- treecor_deg(count,
hierarchy_structure,
cell_meta,
sample_meta %>% mutate(severity = ifelse(severity == "HD", 0,1)),
response_variable = c('severity','age'),
separate = F,
save_as_csv = F)$dge.summary
head(multi_deg_joint)## label combined_phenotype.num_deg x y id leaf
## 1 All 1 6.25 2 1 FALSE
## 2 B 0 0.00 1 2 FALSE
## 3 T 700 5.00 1 3 FALSE
## 4 Monocyte 0 12.50 1 4 FALSE
## 5 B_c7 0 0.00 0 5 TRUE
## 6 T_c15 0 1.00 0 6 TRUE11 Adjusting for covariates in TreeCorTreat analysis
TreeCorTreat is capable of handling covariates in the analysis, allowing users to adjust for potential confounders or unwanted technical variation. For example, instead of viewing age as a phenotype, one can also view it as a covariate and ask which cell type features are associated with disease severity after accounting for age.
# adjust for age
expr_adjusted <- treecor_expr(count,
hierarchy_structure,
cell_meta,
sample_meta,
response_variable = 'severity',
formula = '~age',
num_permutations = 100)$canonical_corr
# visualize
treecortreatplot(hierarchy_structure,
annotated_df = expr_adjusted,
response_variable = 'severity',
color_variable = 'p.sign',
size_variable = 'cancor',
alpha_variable = 'p.sign',
font_size = 12,
nonleaf_label_pos = 0.2,
nonleaf_point_gap = 0.1)