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Pseudobulk DESeq2: within-group contrasts

pseudobulk_within.Rd

Splits the pseudobulks by split_var and, within each split level, fits a DESeq2 model whose contrast variable is the second term of dge_formula. Used to test for an effect (e.g. case vs. control) restricted to one cluster at a time, where pooling across clusters would mix biology and batch.

Usage

pseudobulk_within(
  dge_formula,
  counts_df,
  meta_data,
  split_var,
  vals_test,
  verbose,
  min_counts_per_sample,
  present_in_min_samples
)

Arguments

dge_formula

One-sided formula such as ~cluster + donor. The first term is treated as the contrast variable in "one_vs_all" and "pairwise" modes, or as the split variable in "within" mode (in which case the second term becomes the contrast variable). Additional terms are kept as covariates in the DESeq2 design.

counts_df

Feature-by-pseudobulk integer count matrix. Rows are features; columns must align with rows of meta_data.

meta_data

data.frame of pseudobulk metadata. One row per pseudobulk; should contain only the variables used in dge_formula.

split_var

Name of the column in meta_data to split on (the first term of dge_formula). Each unique level of split_var produces an independent DESeq2 fit.

vals_test

Character vector of contrast levels to test. If NULL (default), every level of the contrast variable is tested.

verbose

Logical. Print progress messages. Default TRUE.

min_counts_per_sample

Minimum count per pseudobulk for a gene to be considered expressed in that pseudobulk. Default 10.

present_in_min_samples

Minimum number of pseudobulks in which a gene must reach min_counts_per_sample to be retained. Default 5.

Value

data.frame of DESeq2 results with columns group (the value of split_var), feature, baseMean, log2FoldChange, lfcSE, stat, pvalue, padj.

Details

Two-level contrasts (factor or character) yield the standard <var>_<level2>_vs_<level1> Wald coefficient. Three or more levels are integer-encoded and the fit returns an ordinal trend. Most users should call pseudobulk_deseq2() with mode = "within" rather than this function directly. See the pseudobulk vignette for a worked example using case-vs-control DGE within each cell cluster.

See also

pseudobulk_deseq2(), pseudobulk_one_vs_all(), pseudobulk_pairwise()