testDS_limma: Test for differential states: method 'diffcyt-DS-limma'

In diffcyt: Differential discovery in high-dimensional cytometry via high-resolution clustering

Description

Calculate tests for differential states within cell populations using method 'diffcyt-DS-limma'

Usage

testDS_limma(
  d_counts,
  d_medians,
  design,
  contrast,
  block_id = NULL,
  trend = TRUE,
  weights = TRUE,
  markers_to_test = NULL,
  min_cells = 3,
  min_samples = NULL,
  plot = FALSE,
  path = "."
)

Arguments

d_counts	SummarizedExperiment object containing cluster cell counts, from calcCounts.
d_medians	SummarizedExperiment object containing cluster medians (median marker expression for each cluster-sample combination), from calcMedians. Assumed to contain a logical vector id_state_markers in the meta-data (accessed with metadata(d_medians)$id_state_markers), which identifies the set of 'cell state' markers in the list of assays.
design	Design matrix, created with createDesignMatrix. See createDesignMatrix for details.
contrast	Contrast matrix, created with createContrast. See createContrast for details.
block_id	(Optional) Vector or factor of block IDs (e.g. patient IDs) for paired experimental designs, to be included as random effects. If provided, the block IDs will be included as random effects using the limma duplicateCorrelation methodology. Alternatively, block IDs can be included as fixed effects in the design matrix (createDesignMatrix). See details.
trend	(Optional) Whether to fit a mean-variance trend when calculating moderated tests with function eBayes from limma package. When trend = TRUE, this is known as the limma-trend method (Law et al., 2014; Phipson et al., 2016). Default = TRUE.
weights	(Optional) Whether to use cluster cell counts as precision weights (across all samples and clusters); this allows the limma model fitting functions to account for uncertainty due to the total number of cells per sample (library sizes) and total number of cells per cluster. Default = TRUE.
markers_to_test	(Optional) Logical vector specifying which markers to test for differential expression (from the set of markers stored in the assays of d_medians). Default = all 'cell state' markers, which are identified by the logical vector id_state_markers stored in the meta-data of d_medians.
min_cells	Filtering parameter. Default = 3. Clusters are kept for differential testing if they have at least min_cells cells in at least min_samples samples.
min_samples	Filtering parameter. Default = number of samples / 2, which is appropriate for two-group comparisons (of equal size). Clusters are kept for differential testing if they have at least min_cells cells in at least min_samples samples.
plot	Whether to save diagnostic plot. Default = FALSE.
path	Path for diagnostic plot, if plot = TRUE. Default = current working directory.

Details

Calculates tests for differential states within cell populations (i.e. differential expression of cell state markers within clusters). Clusters are defined using cell type markers, and cell states are characterized by the median transformed expression of cell state markers.

This method uses the limma package (Ritchie et al. 2015, Nucleic Acids Research) to fit models and calculate moderated tests at the cluster level. Moderated tests improve statistical power by sharing information on variability (i.e. variance across samples for a single cluster) between clusters. By default, we provide option trend = TRUE to the limma eBayes function; this fits a mean-variance trend when calculating moderated tests, which is also known as the limma-trend method (Law et al., 2014; Phipson et al., 2016). Diagnostic plots are shown if plot = TRUE.

The experimental design must be specified using a design matrix, which can be created with createDesignMatrix. Flexible experimental designs are possible, including blocking (e.g. paired designs), batch effects, and continuous covariates. See createDesignMatrix for more details.

For paired designs, either fixed effects or random effects can be used. Fixed effects are simpler, but random effects may improve power in data sets with unbalanced designs or very large numbers of samples. To use fixed effects, provide the block IDs (e.g. patient IDs) to createDesignMatrix. To use random effects, provide the block_id argument here instead. This will make use of the limma duplicateCorrelation methodology. Note that >2 measures per sample are not possible in this case (fixed effects should be used instead). Block IDs should not be included in the design matrix if the limma duplicateCorrelation methodology is used.

The contrast matrix specifying the contrast of interest can be created with createContrast. See createContrast for more details.

By default, differential tests are performed for all cell state markers (which are identified with the vector id_state_markers stored in the meta-data of the cluster medians input object). The optional argument markers_to_test allows the user to specify a different set of markers to test (e.g. to investigate differences for cell type markers).

Filtering: Clusters are kept for differential testing if they have at least min_cells cells in at least min_samples samples. This removes clusters with very low cell counts across conditions, to improve power.

Weights: By default, cluster cell counts are used as precision weights (across all samples and clusters); allowing the limma model fitting functions to account for uncertainty due to the total number of cells per sample (library sizes) and total number of cells per cluster. This option can also be disabled with weights = FALSE, if required.

Value

Returns a new SummarizedExperiment object, where rows = cluster-marker combinations, and columns = samples. In the rows, clusters are repeated for each cell state marker (i.e. the sheets or assays from the previous d_medians object are stacked into a single matrix). Differential test results are stored in the rowData slot. Results include raw p-values (p_val) and adjusted p-values (p_adj) from the limma moderated tests, which can be used to rank cluster-marker combinations by evidence for differential states within cell populations. Additional output columns from the limma tests are also included. The results can be accessed with the rowData accessor function.

Examples

# For a complete workflow example demonstrating each step in the 'diffcyt' pipeline, 
# see the package vignette.

# Function to create random data (one sample)
d_random <- function(n = 20000, mean = 0, sd = 1, ncol = 20, cofactor = 5) {
  d <- sinh(matrix(rnorm(n, mean, sd), ncol = ncol)) * cofactor
  colnames(d) <- paste0("marker", sprintf("%02d", 1:ncol))
  d
}

# Create random data (without differential signal)
set.seed(123)
d_input <- list(
  sample1 = d_random(), 
  sample2 = d_random(), 
  sample3 = d_random(), 
  sample4 = d_random()
)

# Add differential states (DS) signal
ix_DS <- 901:1000
ix_cols_type <- 1:10
ix_cols_DS <- 19:20
d_input[[1]][ix_DS, ix_cols_type] <- d_random(n = 1000, mean = 3, ncol = 10)
d_input[[2]][ix_DS, ix_cols_type] <- d_random(n = 1000, mean = 3, ncol = 10)
d_input[[3]][ix_DS, c(ix_cols_type, ix_cols_DS)] <- d_random(n = 1200, mean = 3, ncol = 12)
d_input[[4]][ix_DS, c(ix_cols_type, ix_cols_DS)] <- d_random(n = 1200, mean = 3, ncol = 12)

experiment_info <- data.frame(
  sample_id = factor(paste0("sample", 1:4)), 
  group_id = factor(c("group1", "group1", "group2", "group2")), 
  stringsAsFactors = FALSE
)

marker_info <- data.frame(
  channel_name = paste0("channel", sprintf("%03d", 1:20)), 
  marker_name = paste0("marker", sprintf("%02d", 1:20)), 
  marker_class = factor(c(rep("type", 10), rep("state", 10)), 
                        levels = c("type", "state", "none")), 
  stringsAsFactors = FALSE
)

# Prepare data
d_se <- prepareData(d_input, experiment_info, marker_info)

# Transform data
d_se <- transformData(d_se)

# Generate clusters
d_se <- generateClusters(d_se)

# Calculate counts
d_counts <- calcCounts(d_se)

# Calculate medians
d_medians <- calcMedians(d_se)

# Create design matrix
design <- createDesignMatrix(experiment_info, cols_design = "group_id")

# Create contrast matrix
contrast <- createContrast(c(0, 1))

# Test for differential states (DS) within clusters
res_DS <- testDS_limma(d_counts, d_medians, design, contrast)

diffcyt documentation built on Nov. 8, 2020, 6:37 p.m.