RNASeq

# loading packages
library(tidyverse)
library(kableExtra)
library(ggtext)
library(DESeq2)
library(RColorBrewer)
library(viridis)
library(grid)
library(ggpubr)
library(patchwork)
library(plotly)
library(htmltools)
library(htmlwidgets)
library(tximport)
library(vegan)
library(fgsea)
library(ggh4x)
library(Cairo)
library(lme4)
library(lmerTest) 
library(emmeans)

Data Input

All data inputs and outputs in this repo are relative to FOLDER_PATH. Please, save the provided data frames in RDS format in the dataframes folder.

# Define directory paths based on R.environ
FOLDER_PATH <- Sys.getenv("BOX_PATH_RSVBacPublication") 
dataframes_folder <- file.path(FOLDER_PATH, "dataframes")

# Create subfolder for output files
outputs_folder <- file.path(FOLDER_PATH, "outputs/RNASeq")
if (!file.exists(outputs_folder)) {
  dir.create(outputs_folder, recursive = TRUE)
}

Bac.Vir_colors = c("#CC9564", "#a89de0", "#f54590", "#2e67f2", "#856141","#6855c9","#9f2d5e", "#1e439d")
Bac.Vir_fill <- c("white", "white", "white", "white", "#856141","#6855c9","#9f2d5e", "#1e439d")

RNASeq Upstream Analysis

This part of analysis was conducted on the HPC cluster managed by the Biostatistics and Informatics Shared Resource (BISR) and supported by an NCI P30-CA125123 and Institutional funds from the Dan L Duncan Comprehensive Cancer Center and Baylor College of Medicine. Data was transferred to the HPC using rsync and MD5 were verified for all samples to ensure data integrity. The folder scripts includes all the scripts used for this part of the analysis.

Genome Mapping and Expression Quantification

RNA-seq reads were aligned using STAR v2.7.10b (Dobin et al., 2012) and gene expression was quantified using RSEM v1.3.3 (Li and Dewey, 2011). The Homo sapiens GRCh38 reference genome and corresponding annotation files were downloaded from Ensembl (soft-masked version, release 113) and used to generate index files for STAR (with --sjdbOverhang 149) and RSEM. STAR was run with --quantMode TranscriptomeSAM to generate transcriptome-aligned BAM files. After confirming that the percentage of aligned reads was consistent across samples, BAM files were randomly subsampled to match the sample with the lowest read count (95 million reads per sample) using samtools v1.17 (Danecek et al., 2021). rsem-calculate-expression was run on the subsampled BAM files using --estimate-rspd to improve quantification accuracy.

Gene-level quantification results were imported into R using tximport (Soneson et al., 2015), which produces length-scaled counts and an offset matrix for DESeq2. Transcripts with zero length were corrected to a length of one to avoid computational errors during normalization. We include the resulting txi object in the dataframes folder, as well as metadata, gene annotation and msigdbr, as RDS files for reproducibility.

# Read the provided RDS files
dataframeID <- "RSVbac"
txi <- readRDS(file.path(dataframes_folder, paste0(dataframeID, "_RNASeq_human_txi.rds")))
meta <- readRDS(file.path(dataframes_folder, paste0(dataframeID, "_RNASeq_meta.rds")))
gene_anno <- readRDS(file.path(dataframes_folder, paste0("Homo_sapiens.GRCh38.113.rds")))
msigdbr_human <- readRDS(file.path(dataframes_folder, "Homo_sapiens.msigdbr.2025.1.Hs.rds"))

Saturation Curves

Rarefaction curves were generated using the vegan (Oksanen et al., 2024) package to confirm that sequencing depth was sufficient to capture the majority of expressed genes. Subsets of the reads in a sample are taken and the number of detected genes in each of these subsets is plotted.

make_rarefaction_plot <- function(txi) {

  # Prepare count matrix
  df_counts_matrix <- round(txi$counts)
  raremax <- round(min(rowSums(t(df_counts_matrix))), 0)
  
  # Generate rarefaction data
  rarefy_df <- rarecurve(
    t(df_counts_matrix),
    step = round(raremax / 10, 0),
    sample = raremax,
    tidy = TRUE
  )
  
  # Clean up metadata and separate identifiers
  rarefy_df <- rarefy_df %>%
    mutate(Site = gsub("\\s*\\|\\s*", "|", Site)) %>%
    separate(Site, into = c("species", "virus", "line", "exp"), sep = "\\|", remove = FALSE) %>%
    mutate(
      virus = factor(virus),
      line = factor(line),
      species = factor(species, levels = c("NB", "Spn", "Hin", "Dpi"))
    )
  
  # Make plot
  plot_rarefy <- ggplot(rarefy_df, aes(
    x = Sample, y = Species,
    color = interaction(species, virus, sep = " "),
    shape = interaction(line, exp, sep = " ", lex.order = TRUE)
  )) +
    geom_point(size = 1.5) +
    scale_shape_manual(values = c(1, 2, 15, 16, 17)) +
    scale_color_manual(values = Bac.Vir_colors) +
    labs(x = "reads", y = "genes", color = "Sample", shape = "Exp") +
    scale_y_continuous(expand = c(0, 0), limits = c(0, max(rarefy_df$Species) * 1.02)) +
    theme_bw(base_size = 14) +
    theme(
      axis.text.x = element_text(angle = 30, hjust = 1),
      panel.grid.major = element_blank(),
      panel.grid.minor = element_blank(),
      plot.margin = margin(t = 15, r = 0, b = -18, l = 0)
    )
  
  return(plot_rarefy)
}

make_rarefaction_plot(txi)

This analysis confirmed adequate sequencing depth. We therefore proceeded to quality assessment and normalization of the gene expression data.

Exploratory DESeq Analysis

An initial model was fit for normalization and quality assessment only. We use it to explore the data and identify possible batch effects due to sequencing the data in two independent runs, without considering any interaction between variables. The final model for differential expression analysis is described in the next section.

# DESeq design 
dds_exp <- DESeqDataSetFromTximport(txi,
                                colData = meta,
                                design = ~ species + virus + line + seq_run)

# Run DESeq
dds_exp <- DESeq(dds_exp)

# See design
design(dds_exp)

~species + virus + line + seq_run

We performed an unbiased (blind = TRUE, independent of the dds design formula) transformation of the data for exploratory analysis, calculated based on variance stabilizing transformations (VST) (Anders and Huber, 2010; Huber et al., 2003; Tibshirani, 1988). Principal component analysis was performed on the VST data using the top 500 genes by variance to assess the primary sources of variation and evaluate potential batch effects.

vsd_exp <- vst(dds_exp, blind = TRUE)
pcaData <- plotPCA(vsd_exp, intgroup = c("species", "virus", "line", "seq_run"), returnData = TRUE) %>%
  mutate(line = paste0("HNO", line))
percentVar <- round(100 * attr(pcaData, "percentVar"))

pPCA <- ggplot(pcaData, aes(PC1, PC2)) +
  geom_point(aes(fill = Bac.Vir_label, color = Bac.Vir_label, shape = line), 
             size = 4, stroke = 1) +  
  scale_shape_manual(values = c(21, 24)) + 
  scale_color_manual(values = Bac.Vir_colors) + 
  scale_fill_manual(values = Bac.Vir_fill) +
  ggtitle("Principal Component Analysis (blind = TRUE)") +
  xlab(paste0("PC1: ", percentVar[1], "% variance")) +
  ylab(paste0("PC2: ", percentVar[2], "% variance")) +
  labs(fill = "Condition", color = "Condition", shape = "HNO Line") +
  theme_bw(base_size = 14) +
  theme(
    panel.grid.major = element_blank(),
    panel.grid.minor = element_blank(),
    legend.text = element_markdown(size = 14),
  ) +
    guides(
    color = guide_legend(title = "Condition", override.aes = list(color = Bac.Vir_colors, shape = 22))
  ) + coord_fixed()

pPCA

# Save PCA plot
saveRDS(pPCA, file = file.path(outputs_folder, "RSVbac_RNASeq_human_PCA.rds"))

Samples from different sequencing runs and experimental dates were interspersed rather than clustered, indicating negligible batch effects.

DGE virus * species

Based on the exploratory analysis, we decided to exclude seq_run and exp from the final DESeq2 model, as they did not contribute significantly to the observed variation.

When using DESeqDataSetFromTximport(), tximport helps generate an offset to correct for differences in average transcript length across samples. This adjustment accounts for potential bias introduced by differential transcript usage (DTU), ensuring that gene-level differential expression analysis remains accurate.

# DESeq design 
dds <- DESeqDataSetFromTximport(txi,
                                colData = meta,
                                design = ~ virus + species + virus:species + line)

The Ensembl annotation gets added to the dds for follow up plots and tables.

# Convert rowData(dds) to a data frame for merging
rowdata_df <- as.data.frame(rowData(dds))

# Add rownames as a new column in the dataframe
rowdata_df$ensembl_gene_id <- rownames(rowdata_df)

# Merge with rowData(dds)
rowdata_df <- left_join(rowdata_df, gene_anno, by = "ensembl_gene_id")

# Assign back to dds
rowData(dds) <- DataFrame(rowdata_df, row.names = rowdata_df$ensembl_gene_id)

# Check if the row names of dds and rowData are still correctly aligned
#all(rownames(dds) == rownames(rowData(dds))) # Should return TRUE if correct

This design captures all the biological variables of interest:

# Run DESeq
dds <- DESeq(dds)

# See results names design
design(dds)

~virus + species + virus:species + line

#resultsNames(dds)

• virus + species + virus:species Includes main effects of virus and species, as well as their interaction. This allows us to test whether the effect of virus depends on the species. This is equivalent to virus * species.
• line Models differences between the two organoid lines.

We apply a variance stabilizing transformation using vst() from the DESeq2 package. This transformation makes the variance roughly independent of the mean, which is especially useful for PCA and clustering. It produces log2-like values that are more homoscedastic than raw or normalized counts.

# Run VST
vst_matrix <- vst(dds)

# Save as assay in dds
assays(dds)[["vst"]] <- assay(vst_matrix) 

vst_matrix <- assays(dds)[["vst"]]

saveRDS(vst_matrix, file = file.path(outputs_folder, paste0(dataframeID, "_RNASeq_vst_matrix.rds")))

Contrasts Results

We implemented the adaptive shrinkage method (ashr(Stephens, 2016)) to obtain more accurate and stable estimates of log₂ fold changes (LFC) for each gene across contrasts. This approach mitigates the impact of low counts and high variability, which can otherwise lead to exaggerated LFC estimates. Shrinkage was performed using the lfcShrink() function from the DESeq2 package.

# Function to run a contrast analysis and generate plots
run_contrast_analysis <- function(dds, FC_cutoff, contrast_name, 
                                 contrast = NULL, coef = NULL,
                                 ylim = NULL, show_outlier = TRUE) {
  # Check inputs
  if (!is.null(contrast)) {
    res <- results(dds, contrast = contrast, alpha = 0.05)
    resLFC <- lfcShrink(dds, contrast = contrast, type = "ashr")
  } else if (!is.null(coef)) {
    res <- results(dds, name = coef, alpha = 0.05)
    resLFC <- lfcShrink(dds, coef = coef, type = "ashr")
  } else {
    stop("Please provide either a 'contrast' or a 'coef'.")
  }

  # Results table
  res_df <- as.data.frame(res)
  res_df$Geneid <- rownames(res_df)
  res_df$contrast <- contrast_name
  res_df$log2FoldChange_shrunk <- resLFC$log2FoldChange

  # Merge with gene annotation (adds hgnc_symbol, etc.)
  res_df <- left_join(res_df, gene_anno, by = c("Geneid" = "ensembl_gene_id"))
  rownames(res_df) <- NULL

  # Make label: Symbol (Geneid) + stats
  res_df <- res_df %>%
    mutate(symbol = ifelse(!is.na(hgnc_symbol) & hgnc_symbol != "",
                           hgnc_symbol, Geneid))

  # Determine ylim if not provided
  if (is.null(ylim)) {
    ylim_val <- quantile(abs(res_df$log2FoldChange_shrunk), 0.99, na.rm = TRUE)
    ylim <- c(-ylim_val, ylim_val)
  }

  # Identify significant genes and outliers for plotting
  res_df <- res_df %>%
    filter(!is.na(baseMean), !is.na(log2FoldChange_shrunk)) %>%
    mutate(
      sig_category = case_when(
        is.na(padj) ~ "No-pvalue",
        padj > 0.05 ~ "p-value >0.05",
        padj <= 0.05 & abs(log2FoldChange_shrunk) <= 0 ~ "p-value <0.05",
        padj <= 0.05 & log2FoldChange_shrunk > 0 ~ "Up",
        padj <= 0.05 & log2FoldChange_shrunk < -0 ~ "Down"
      ),
      sig_category = factor(sig_category, levels = c("No-pvalue","p-value >0.05","p-value <0.05","Up","Down")),
      is_outlier = log2FoldChange_shrunk < ylim[1] | log2FoldChange_shrunk > ylim[2],
      log2FoldChange_shrunk_clipped = case_when(
        log2FoldChange_shrunk < ylim[1] ~ ylim[1] * 0.98,  # Slightly inside boundary
        log2FoldChange_shrunk > ylim[2] ~ ylim[2] * 0.98,
        TRUE ~ log2FoldChange_shrunk
      ),
      point_shape = if_else(is_outlier & show_outlier, 18, 16)
    ) %>%
    select(Geneid, contrast, everything())
  
# Create custom MA plot (ggplot version)
  ma_plot <- ggplot(res_df, aes(x = log2(baseMean), 
                         y = if_else(is_outlier & show_outlier, 
                                     log2FoldChange_shrunk_clipped, 
                                     log2FoldChange_shrunk))) +
    geom_point(aes(color = sig_category, shape = point_shape), 
               size = 1, alpha = 0.6) +
    scale_shape_identity() + 
    geom_hline(yintercept = 0, linetype = "solid", color = "black", linewidth = 0.3) +
    geom_hline(yintercept = c(-FC_cutoff, FC_cutoff), 
               linetype = "dashed", linewidth = 0.5) +
    scale_color_manual(
      values = c("Up" = "salmon3", "Down" = "salmon3", 
                 "No-pvalue" = "gray60", "p-value >0.05" = "gray40", "p-value <0.05" = "salmon3"),
      name = ""
    ) +
    coord_cartesian(ylim = ylim) +
    labs(
      title = contrast_name,
      x = "log2(mean expression)",
      y = "Shrunken log2FC"
    ) +
    theme_bw(base_size = 10) +
    theme(
      panel.grid.major = element_blank(),
      panel.grid.minor = element_blank(),
      plot.title = element_text(hjust = 0.5, size = 11, face = "bold"),
      legend.position = "none"
    )
  
  # Volcano plot (ggplot)
  volcano_plot <- ggplot(res_df, aes(x = log2FoldChange_shrunk, y = -log10(padj))) +
    geom_point(aes(color = sig_category), size = 1, alpha = 0.6) +
    scale_color_manual(
      values = c("Up" = "salmon3", "Down" = "salmon3", 
                 "No-pvalue" = "gray60", "p-value >0.05" = "gray40", "p-value <0.05" = "salmon3"),
      name = ""
    ) +
    geom_vline(xintercept = c(-FC_cutoff, FC_cutoff), linetype = "dashed") +
    geom_hline(yintercept = -log10(0.05), linetype = "dashed") +
    labs(title = contrast_name,
         x = "Shrunken log2FC",
         y = "-log10(adjusted p-value)") +
    theme_bw(base_size = 10) +
    theme(
      panel.grid.major = element_blank(),
      panel.grid.minor = element_blank(),
      plot.title = element_text(hjust = 0.5, size = 11, face = "bold"),
      legend.position = "none"
    )
  
  return(list(
    results = res_df,
    ma_plot = ma_plot,
    volcano_plot = volcano_plot
  ))
}

# Create output folder for individual contrasts results
contrasts_folder <- file.path(outputs_folder, "Contrasts_individual_results")

if (!dir.exists(contrasts_folder)) {
  dir.create(contrasts_folder, recursive = TRUE)
}

Virus TRUE vs FALSE in each Species

This section tests the effect of virus infection (TRUE vs FALSE) within each bacterial species specifically:

• For the reference species NB, the virus effect is captured by the main coefficient virus_TRUE_vs_FALSE.
• For other species (Dpi, Spn, Hin), the virus effect is a combination of the main virus effect plus the interaction term (e.g., virusTRUE.speciesDpi).

# Define contrast list with full virus effect per species:
contrast_Virus_TvsF <- list(
  VirusTvsF_NB  = "virus_TRUE_vs_FALSE",                                   # virus effect in NB, does not need interaction
  VirusTvsF_Spn = list(c("virus_TRUE_vs_FALSE", "virusTRUE.speciesSpn")),  # virus effect in Spn
  VirusTvsF_Hin = list(c("virus_TRUE_vs_FALSE", "virusTRUE.speciesHin")),   # virus effect in Hin
  VirusTvsF_Dpi = list(c("virus_TRUE_vs_FALSE", "virusTRUE.speciesDpi"))  # virus effect in Dpi
)

# Run contrasts
res_Virus_TvsF <- imap(contrast_Virus_TvsF, function(contrast_val, name) {
  if (is.character(contrast_val)) {
    run_contrast_analysis(dds, FC_cutoff = 2, contrast_name = name, coef = contrast_val, ylim = c(-5, 5))
  } else {
    run_contrast_analysis(dds, FC_cutoff = 2, contrast_name = name, contrast = contrast_val, ylim = c(-5, 5))
  }
})

# Save individual contrast results R file
for (contrast_name in names(res_Virus_TvsF)) {
  saveRDS(res_Virus_TvsF[[contrast_name]], 
          file = file.path(contrasts_folder, paste0("DESeq2_contrast_", contrast_name, ".rds")))
}

# Summarize table based on sig_category for each contrast
summary_Virus_TvsF <- map_dfr(res_Virus_TvsF, 
                                           ~ .x$results %>%
                                             group_by(sig_category) %>%
                                             summarise(count = n()) %>%
                                             pivot_wider(names_from = sig_category, values_from = count) %>%
                                             mutate_all(~ replace(., is.na(.), 0)),
                                           .id = "contrast")
                                        
kable(summary_Virus_TvsF)

contrast	No-pvalue	p-value >0.05	Up	Down
VirusTvsF_NB	46313	19780	5841	6998
VirusTvsF_Spn	48363	22313	3931	4325
VirusTvsF_Hin	45288	20352	5941	7351
VirusTvsF_Dpi	46313	20083	5612	6924

wrap_plots(map(res_Virus_TvsF, "ma_plot"), nrow = 1)

wrap_plots(map(res_Virus_TvsF, "volcano_plot"), nrow = 1)

Difference in Virus Effect Compared to NB

Here we focus on whether the virus effect differs between each species and the reference species NB. This highlights species-specific modulation of virus response by comparing the virus effect in each species against the baseline virus effect in NB.

• We use the interaction terms alone (e.g., virusTRUE.speciesDpi), which quantify how much the virus effect in each species deviates from that in NB.

# Define contrast list of interaction terms (difference in virus effect compared to NB, interaction terms only)
contrast_VirusEffect_diff_vsNB <- list(
  VirusEffectDiff_Spn = "virusTRUE.speciesSpn",
  VirusEffectDiff_Hin = "virusTRUE.speciesHin",
  VirusEffectDiff_Dpi = "virusTRUE.speciesDpi"
)

# Run contrasts using coef names (interaction terms)
res_VirusEffect_diff_vsNB <- imap(contrast_VirusEffect_diff_vsNB, 
                                  ~ run_contrast_analysis(dds, FC_cutoff = 2, contrast_name = .y, coef = .x, ylim = c(-3, 3)))

# Save individual contrast results R file
for (contrast_name in names(res_VirusEffect_diff_vsNB)) {
  saveRDS(res_VirusEffect_diff_vsNB[[contrast_name]], 
          file = file.path(contrasts_folder, paste0("DESeq2_contrast_", contrast_name, ".rds")))
}

# Summarize table based on sig_category for each contrast
summary_VirusEffect_diff_vsNB <- map_dfr(res_VirusEffect_diff_vsNB, 
                                           ~ .x$results %>%
                                             group_by(sig_category) %>%
                                             summarise(count = n()) %>%
                                             pivot_wider(names_from = sig_category, values_from = count) %>%
                                             mutate_all(~ replace(., is.na(.), 0)),
                                           .id = "contrast")
                                        
kable(summary_VirusEffect_diff_vsNB)

contrast	No-pvalue	p-value >0.05	Up	Down
VirusEffectDiff_Spn	58602	18705	597	1028
VirusEffectDiff_Hin	54504	24399	6	23
VirusEffectDiff_Dpi	61676	17059	36	161

wrap_plots(map(res_VirusEffect_diff_vsNB, "ma_plot"), nrow = 1)

wrap_plots(map(res_VirusEffect_diff_vsNB, "volcano_plot"), nrow = 1)

Bacteria vs NB in Virus FALSE

This comparison evaluates how each bacterial species differs from the reference species NB when virus is absent (virus = FALSE). This reveals baseline species-specific gene expression differences without virus influence.

• Because virus FALSE is the baseline in the model, these contrasts use only the main species effects (e.g., species_Dpi_vs_NB).

# These don't need the interaction term because virus FALSE is the baseline
contrast_Species_vsNB_VF <- list(
  SpnvsNB_VF = "species_Spn_vs_NB",
  HinvsNB_VF = "species_Hin_vs_NB",
  DpivsNB_VF = "species_Dpi_vs_NB"
)

# Run all contrasts and generate plots
res_Species_vsNB_VF <- imap(contrast_Species_vsNB_VF,
                            ~ run_contrast_analysis(dds, FC_cutoff = 2, contrast_name = .y, coef = .x, ylim = c(-3, 3)))

# Save individual contrast results R file
for (contrast_name in names(res_Species_vsNB_VF)) {
  saveRDS(res_Species_vsNB_VF[[contrast_name]], 
          file = file.path(contrasts_folder, paste0("DESeq2_contrast_", contrast_name, ".rds")))
}

# Summarize table based on sig_category for each contrast
summary_Species_vsNB_VF <- map_dfr(res_Species_vsNB_VF, 
                                   ~ .x$results %>%
                                     group_by(sig_category) %>%
                                     summarise(count = n()) %>%
                                     pivot_wider(names_from = sig_category, values_from = count) %>%
                                     mutate_all(~ replace(., is.na(.), 0)),
                                   .id = "contrast")
kable(summary_Species_vsNB_VF)

contrast	No-pvalue	p-value >0.05	Up	Down
SpnvsNB_VF	54504	24030	328	70
HinvsNB_VF	47338	31513	80	1
DpivsNB_VF	49386	29443	97	6

wrap_plots(map(res_Species_vsNB_VF, "ma_plot"), nrow = 1)

wrap_plots(map(res_Species_vsNB_VF, "volcano_plot"), nrow = 1)

Bacteria vs NB in Virus TRUE

Here we test the species differences under virus infection (virus = TRUE). This reveals species-specific gene expression differences that happens under virus influence.

• These contrasts combine the main species effect plus the interaction with virus (e.g., species_Dpi_vs_NB + virusTRUE.speciesDpi).

# These include the interaction terms to reflect virus-specific responses
contrast_Species_vsNB_VT <- list(
  SpnvsNB_VT = list(c("species_Spn_vs_NB", "virusTRUE.speciesSpn")),
  HinvsNB_VT = list(c("species_Hin_vs_NB", "virusTRUE.speciesHin")),
  DpivsNB_VT = list(c("species_Dpi_vs_NB", "virusTRUE.speciesDpi"))
)

# Run all contrasts and generate plots
res_Species_vsNB_VT <- imap(contrast_Species_vsNB_VT,
                            ~ run_contrast_analysis(dds, FC_cutoff = 2, contrast_name = .y, contrast = .x, ylim = c(-3, 3)))

# Save individual contrast results R file
for (contrast_name in names(res_Species_vsNB_VT)) {
  saveRDS(res_Species_vsNB_VT[[contrast_name]], 
          file = file.path(contrasts_folder, paste0("DESeq2_contrast_", contrast_name, ".rds")))
}

# Summarize table based on sig_category for each contrast
summary_Species_vsNB_VT <- map_dfr(res_Species_vsNB_VT, 
                                   ~ .x$results %>%
                                     group_by(sig_category) %>%
                                     summarise(count = n()) %>%
                                     pivot_wider(names_from = sig_category, values_from = count) %>%
                                     mutate_all(~ replace(., is.na(.), 0)),
                                   .id = "contrast")
kable(summary_Species_vsNB_VT)

contrast	No-pvalue	p-value >0.05	Up	Down
SpnvsNB_VT	58602	17814	1089	1427
HinvsNB_VT	60651	17855	121	305
DpivsNB_VT	57578	20495	244	615

wrap_plots(map(res_Species_vsNB_VT, "ma_plot"), nrow = 1)

wrap_plots(map(res_Species_vsNB_VT, "volcano_plot"), nrow = 1)

Line (9009 vs 9007)

This section examines the effect of HNO line on gene expression, independent of species and virus status. This contrast helps identify baseline donor-specific transcriptional differences, which may reflect biological variation.

• In this setup, 9007 is the reference, and the coefficient line_9009_vs_9007 captures the log2 fold change in gene expression for line 9009 relative to line 9007, averaged across all species and virus conditions.

res_Line_9009vs9007 <- run_contrast_analysis(dds, FC_cutoff = 2, ylim = c(-5, 5),
  contrast_name = "Line_9009_vs_9007",
  coef = "line_9009_vs_9007")

# Save individual contrast results R file
saveRDS(res_Line_9009vs9007, 
        file = file.path(contrasts_folder, "DESeq2_contrast_Line_9009_vs_9007.rds"))

# Summarize table based on sig_category for the contrast
summary_Line_9009vs9007 <- res_Line_9009vs9007$results %>%
  group_by(sig_category) %>%
  summarise(count = n()) %>%
  pivot_wider(names_from = sig_category, values_from = count) %>%
  mutate_all(~ replace(., is.na(.), 0))
kable(summary_Line_9009vs9007)

No-pvalue	p-value >0.05	Up	Down
45288	24853	4221	4570

#figures side by side
wrap_plots(
  res_Line_9009vs9007$ma_plot,
  res_Line_9009vs9007$volcano_plot,
  nrow = 1
)

Combine all contrast results

# Flatten and combine all contrast result objects into one table
all_contrast_results <- list(
  res_Virus_TvsF,
  res_VirusEffect_diff_vsNB,
  res_Species_vsNB_VF,
  res_Species_vsNB_VT,
  list(Line_9009vs9007 = res_Line_9009vs9007)) %>% flatten()

# Build a single DE table
DESeqData <- map_dfr(all_contrast_results, "results") %>%
  select(Geneid, contrast, everything()) %>%
  select(1:match("sig_category", names(.)))

# Save the results
write.csv(DESeqData, file = file.path(outputs_folder, "RSVbac_DESeq_human_Contrasts.csv"), row.names = FALSE)
saveRDS(DESeqData, file = file.path(outputs_folder, "RSVbac_DESeq_human_Contrasts.rds"))

saveRDS(dds, file = file.path(outputs_folder, "RSVbac_DESeq_human_dds.rds"))

GSEA

The original Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005) method ranks genes by expression changes and tests for enrichment of predefined gene sets using a weighted Kolmogorov–Smirnov-like statistic. Here we use Fast Gene Set Enrichment Analysis (fgsea) (Korotkevich et al., 2016), specifically the fgseaMultilevel() function, based on an adaptive multilevel Monte Carlo algorithm that drastically reduces runtime. This allows us to estimate arbitrarily low p-values (e.g., as small as 10⁻¹⁰⁰), enabling standard multiple testing corrections like Benjamini-Hochberg.

# Function to Run GSEA on a single contrast using Ensembl gene IDs
run_fgsea <- function(contrasts_df, each_contrast, msigdb, collection, subcollection = NULL) {
  # Build Ensembl-based rank vector from the selected contrast table
  ranks <- contrasts_df %>%
    filter(contrast == each_contrast) %>%
    filter(!is.na(stat))
  ranks_vec <- setNames(ranks$stat, ranks$Geneid)

  # Filter the msigdb collection and convert to pathways list
  msig_sub <- msigdb %>%
    { if (!is.null(subcollection)) {
        filter(., gs_collection == collection, gs_subcollection == subcollection)
      } else {
        filter(., gs_collection == collection)
      }
    } %>%
    dplyr::select(gs_name, ensembl_gene) %>%
    filter(!is.na(ensembl_gene), ensembl_gene != "") %>%
    distinct()

  if (nrow(msig_sub) == 0L) {
    stop("No gene sets found for collection = ", collection,
         if (!is.null(subcollection)) paste0(", subcollection = ", subcollection) else "")
  }
  
  pathways <- split(msig_sub$ensembl_gene, msig_sub$gs_name)
  
  # Run fgsea (multilevel for accurate small p-values)
  fgseaRes <- fgseaMultilevel(pathways = pathways, stats = ranks_vec) %>%
    arrange(padj, pval)

  return(list(
    pathways = pathways,
    fgseaRes = fgseaRes)
  )
}

# Function to run GSEA on multiple contrasts and extract filtered NES matrix
run_multi_contrast_gsea <- function(contrasts_df, contrasts_list, msigdb, collection, subcollection = NULL,
                                    padj_threshold = 0.05, nes_threshold = NULL) {
  
  # Run GSEA on each contrast using the standalone run_fgsea function
  fgsea_results <- map(
    contrasts_list %>% set_names(),
    ~ {
      run_fgsea(
        contrasts_df = contrasts_df,
        each_contrast = .x,
        msigdb = msigdb,
        collection = collection,
        subcollection = subcollection
      )
    }
  )
  
  # Extract results from each contrast into a single dataframe
  nes_df <- map_dfr(
    names(fgsea_results),
    ~ {
      fgsea_results[[.x]]$fgseaRes %>%
        as_tibble() %>%
        dplyr::select(pathway, NES, padj) %>%
        mutate(contrast = .x)
    }
  ) %>%
    mutate(pathway = str_remove(pathway, "^[^_]+_"))
    
  # Find pathways that meet significance threshold in at least one contrast
  sig_pathways <- nes_df %>%
    filter(padj < padj_threshold) %>%
    pull(pathway) %>%
    unique()
  
  # Additionally filter by NES threshold if provided
  # Keep pathways where absolute NES exceeds threshold in at least one contrast
  if (!is.null(nes_threshold)) {
    nes_filtered_pathways <- nes_df %>%
      filter(abs(NES) >= nes_threshold) %>%
      pull(pathway) %>%
      unique()
    
    # Take intersection: must meet BOTH padj AND NES criteria
    sig_pathways <- intersect(sig_pathways, nes_filtered_pathways)
  }
  
  # Create NES matrix for selected pathways
  nes_matrix <- nes_df %>%
    filter(pathway %in% sig_pathways) %>%
    dplyr::select(pathway, contrast, NES) %>%
    pivot_wider(names_from = contrast, values_from = NES, values_fill = 0) %>%
    column_to_rownames("pathway") %>%
    as.matrix()
  
  # Create padj matrix for significance annotation
  padj_matrix <- nes_df %>%
    filter(pathway %in% sig_pathways) %>%
    dplyr::select(pathway, contrast, padj) %>%
    pivot_wider(names_from = contrast, values_from = padj, values_fill = 1) %>%
    column_to_rownames("pathway") %>%
    as.matrix()
  
  # Sort pathways by their range (max variation across contrasts)
  # This puts the most dynamically regulated pathways at the top
  nes_range <- apply(nes_matrix, 1, function(x) max(x) - min(x))
  sort_order <- order(nes_range, decreasing = TRUE)
  
  nes_matrix <- nes_matrix[sort_order, , drop = FALSE]
  padj_matrix <- padj_matrix[sort_order, , drop = FALSE]
  
  # Return both raw results and filtered matrices
  return(list(
    raw_fgsea = fgsea_results,      
    nes_matrix = nes_matrix,
    padj_matrix = padj_matrix,
    combined_df = nes_df,
    sig_pathways = sig_pathways
  ))
}

# Function to create grouped heatmaps of NES values with significance annotations
plot_fgsea_grouped_ggplot <- function(multi_contrast_gsea_res, pathway_groups, category_colors,
                                      padj_threshold = 0.05, title = "GSEA NES Heatmap (Grouped)",
                                      nicknames = NULL) {
  
  # Extract matrices
  nes_matrix <- multi_contrast_gsea_res$nes_matrix
  padj_matrix <- multi_contrast_gsea_res$padj_matrix
  
  # Apply nicknames if provided
  if (!is.null(nicknames)) {
    colnames(nes_matrix) <- nicknames[colnames(nes_matrix)]
    colnames(padj_matrix) <- nicknames[colnames(padj_matrix)]
  }
  
  # Convert to long format
  nes_df <- as.data.frame(nes_matrix) %>%
    rownames_to_column("pathway") %>%
    pivot_longer(-pathway, names_to = "contrast", values_to = "NES")
  
  padj_df <- as.data.frame(padj_matrix) %>%
    rownames_to_column("pathway") %>%
    pivot_longer(-pathway, names_to = "contrast", values_to = "padj")
  
  # Merge NES and padj
  plot_df <- nes_df %>%
    left_join(padj_df, by = c("pathway", "contrast")) %>%
    left_join(pathway_groups, by = "pathway")
  
  # Set factor levels for consistent ordering
  plot_df$category <- factor(plot_df$category, levels = unique(pathway_groups$category))
  plot_df$contrast <- factor(plot_df$contrast, levels = colnames(nes_matrix))
  
  # Significance stars
  plot_df$star <- ifelse(plot_df$padj < padj_threshold, "*", "")
  
  # Plot
  ggplot(plot_df, aes(x = contrast, y = pathway, fill = NES)) +
    geom_tile(color = "white") +
    geom_text(aes(label = star), color = "black", size = 6, vjust = 0.75) +
    facet_grid2(category ~ ., scales = "free_y", space = "free_y", switch = "y",
                strip = strip_themed(background_y = elem_list_rect(fill = category_colors))) +
    scale_y_discrete(expand = c(0,0), position = "right") +
    scale_x_discrete(expand = c(0,0)) +
    scale_fill_gradientn(colours = c("#009AD1", "#d9f1fa", "#fefbea", "#fae1e4", "#AD1457"),
                         rescaler = ~ scales::rescale_mid(.x, mid = 0)) +
    labs(x = "", y = "") +
    theme_bw(base_size = 12) +
    theme(
      panel.grid.major = element_blank(),
      panel.grid.minor = element_blank(),
      strip.text.y.left = element_text(size = 12, face = "bold"),
      axis.text.x = element_text(size = 14, color = "black", face = "bold"),
      axis.text.y = element_text(size = 11, color = "black"),
      panel.spacing = unit(0, 'pt'),
      legend.position = c(2.2, 0.92), 
      legend.direction = "vertical"
    ) 
}

We focus our GSEA analysis on the MSigDB Hallmark gene sets(Liberzon et al., 2015), which represent well-defined biological states and processes derived from multiple curated sources. These 50 gene sets provide interpretable signatures of key biological pathways without the redundancy present in larger collections. We organize the Hallmark pathways into five functional categories for clearer biological interpretation.

category_colors_HALLMARK <- c("#FF0000", "#2ca02c", "#9467bd", "#ff7f0e", "grey70")
pathway_groups_HALLMARK <- bind_rows(
  tibble(
    pathway = c("INTERFERON_ALPHA_RESPONSE", "INTERFERON_GAMMA_RESPONSE",
                "TNFA_SIGNALING_VIA_NFKB", "INFLAMMATORY_RESPONSE",
                "IL6_JAK_STAT3_SIGNALING", "IL2_STAT5_SIGNALING",
                "COMPLEMENT", "ALLOGRAFT_REJECTION"),
    category = "Immune Response"
  ),
  
  tibble(
    pathway = c("TGF_BETA_SIGNALING", "E2F_TARGETS", "G2M_CHECKPOINT", 
                "MYC_TARGETS_V1", "MYC_TARGETS_V2", "MITOTIC_SPINDLE"),
    category = "Cell Cycle"
  ),
  
  tibble(
    pathway = c("APOPTOSIS", "P53_PATHWAY", "HYPOXIA", 
                "UV_RESPONSE_UP", "UV_RESPONSE_DN",
                "UNFOLDED_PROTEIN_RESPONSE"),
    category = "Stress"
  ),
  
  tibble(
    pathway = c("MTORC1_SIGNALING", "PI3K_AKT_MTOR_SIGNALING", 
                "KRAS_SIGNALING_UP", "EPITHELIAL_MESENCHYMAL_TRANSITION",
                "MYOGENESIS", "ANGIOGENESIS"),
    category = "Growth Signaling"
  ),

  tibble(
    pathway = c("OXIDATIVE_PHOSPHORYLATION", "GLYCOLYSIS", 
                "FATTY_ACID_METABOLISM", "CHOLESTEROL_HOMEOSTASIS",
                "APICAL_JUNCTION", "COAGULATION",
                "PROTEIN_SECRETION", "ANDROGEN_RESPONSE", 
                "XENOBIOTIC_METABOLISM", "ADIPOGENESIS",
                "DNA_REPAIR", "APICAL_SURFACE",
                "BILE_ACID_METABOLISM", "PANCREAS_BETA_CELLS",
                "REACTIVE_OXYGEN_SPECIES_PATHWAY", "WNT_BETA_CATENIN_SIGNALING",
                "ESTROGEN_RESPONSE_EARLY", "ESTROGEN_RESPONSE_LATE"),
    category = "Other"
  )
)

Multi-Contrast GSEA: Virus Effect Differences

Here we perform GSEA on the interaction contrasts (VirusEffectDiff_Spn, VirusEffectDiff_Hin, VirusEffectDiff_Dpi) to identify pathways that are differentially regulated by virus infection across bacterial species.

Gene Sets are included in the heatmap if they meet both criteria:

1. Significance: padj < 0.05 in at least one contrast
2. Effect size: |NES| ≥ 1.5 in at least one contrast

This dual filtering ensures we focus on pathways with both statistical significance and substantial biological effect sizes. The resulting NES matrix is sorted by pathway variance across contrasts, placing the most dynamically regulated pathways at the top. The heatmap uses a diverging color scale (blue → white → red) centered at NES = 0, with asterisks (*) indicating padj < 0.05.

# Define contrasts and nicknames for cleaner labels
multi_list_VirusEffect_diff_vsNB <- c("VirusEffectDiff_Spn", "VirusEffectDiff_Hin", "VirusEffectDiff_Dpi")
contrast_nicknames <- c(VirusEffectDiff_Spn = "Spn", VirusEffectDiff_Hin = "Hin", VirusEffectDiff_Dpi = "Dpi")

# Run multi-contrast GSEA
multi_gsea_VirusEffect_diff_vsNB <- run_multi_contrast_gsea(DESeqData, multi_list_VirusEffect_diff_vsNB, msigdbr_human, 
                                                            collection = "H", padj_threshold = 0.05, nes_threshold = 1.5)

# Generate grouped heatmap
multi_gsea_VirusEffect_diff_vsNB_heatmap <- plot_fgsea_grouped_ggplot(multi_gsea_VirusEffect_diff_vsNB, 
                                                                      pathway_groups_HALLMARK, category_colors_HALLMARK,
                                                                      padj_threshold = 0.05,
                                                                      title = "GSEA NES: Virus Effect Difference",
                                                                      contrast_nicknames)
multi_gsea_VirusEffect_diff_vsNB_heatmap

# Save heatmap and NES values
ggsave(filename = file.path(outputs_folder, "GSEA_VirusEffectDiff_NES_Heatmap.png"), 
       plot = multi_gsea_VirusEffect_diff_vsNB_heatmap, width = 6, height = 10, dpi = 300)
saveRDS(multi_gsea_VirusEffect_diff_vsNB_heatmap, file = file.path(outputs_folder, "GSEA_VirusEffectDiff_NES_Heatmap.rds"))

saveRDS(multi_gsea_VirusEffect_diff_vsNB, file = file.path(outputs_folder, "GSEA_VirusEffectDiff_Results.rds"))
write_csv(multi_gsea_VirusEffect_diff_vsNB$combined_df, file.path(outputs_folder, "GSEA_VirusEffectDiff_NES_Values.csv"))

Leading-Edge Gene Extraction

Leading-edge genes are the subset of genes in each pathway that contribute most to the enrichment signal. We extract these genes to:

1. Understand which specific genes drive pathway enrichment
2. Identify overlapping genes between pathways
3. Visualize expression patterns of key pathway members

Since genes can appear in multiple pathways, we provide two approaches for visualization:

1. Grouped assignment (used in the categorical heatmaps): Each gene is assigned to a single pathway based on the highest absolute NES value across all contrasts. This prevents double-counting when summarizing pathway-level statistics.

2. All leading-edge genes (used the custom pathway plot): Genes can appear in multiple pathways if they contribute to enrichment in each. This preserves the full biological complexity but may count the same gene multiple times.

# Function to extract leading-edge genes from multi-contrast GSEA results
extract_le_genes_from_gsea <- function(multi_contrast_gsea_res, msigdb, collection, subcollection = NULL) {
  
  # Extract the components we need from the results object
  raw_fgsea <- multi_contrast_gsea_res$raw_fgsea
  sig_pathways <- multi_contrast_gsea_res$sig_pathways
  contrasts_analyzed <- names(raw_fgsea)
  
  # Collect leading-edge genes ONLY from pathways that passed filtering
  all_leading_edge <- map_dfr(
    contrasts_analyzed,
    ~ {
      contrast_name <- .x
      
      # Get the full GSEA results for this contrast
      fgsea_res <- raw_fgsea[[contrast_name]]$fgseaRes %>%
        as_tibble() %>%
        filter(!is.na(leadingEdge)) %>%
        mutate(pathway = str_remove(pathway, "^[^_]+_")) %>%
        filter(pathway %in% sig_pathways)
      
      if (nrow(fgsea_res) == 0) return(tibble())
      
      # Unnest the leading edge genes for these significant pathways
      fgsea_res %>%
        dplyr::select(pathway, pval, padj, ES, NES, leadingEdge) %>%
        unnest(leadingEdge) %>%
        dplyr::rename(gene = leadingEdge) %>%
        mutate(
          contrast = contrast_name,
          abs_NES = abs(NES)
        )
    }
  )
  
  # Assign ONE pathway per gene based on highest absolute NES
  gene_to_pathway <- all_leading_edge %>%
    group_by(gene) %>%
    slice_max(abs_NES, n = 1, with_ties = FALSE) %>%
    ungroup() %>%
    dplyr::select(gene, pathway) %>%
    dplyr::rename(gsea_pathway = pathway) 
  
  # Filter all_leading_edge to keep only the assigned gene-pathway combinations
  grouped_leading_edge <- all_leading_edge %>%
    inner_join(gene_to_pathway, by = c("gene" = "gene", "pathway" = "gsea_pathway"))
  
  # Get the list of unique pathways represented
  pathways_list <- unique(gene_to_pathway$gsea_pathway)
  
  # Summary table of leading edge genes per pathway
  le_summary <- all_leading_edge %>%
    group_by(pathway) %>%
    summarise(
      Total_LE_Genes_All_Contrasts = n(),
      LE_Genes = n_distinct(gene),
      .groups = "drop"
    ) %>%
    left_join(
      grouped_leading_edge %>%
        group_by(pathway) %>%
        summarise(
          Grouped_LE_Genes = n_distinct(gene),
          .groups = "drop"
        ),
      by = "pathway"
    ) %>%
    mutate(
      Grouped_LE_Genes = replace_na(Grouped_LE_Genes, 0),
      Genes_Removed = LE_Genes - Grouped_LE_Genes,
      Pct_Retained = round((Grouped_LE_Genes / LE_Genes) * 100, 1)
    ) %>%
    arrange(desc(LE_Genes))
  
  # Get basal pathway sizes from msigdb
  msig_sub <- msigdb %>%
    { if (!is.null(subcollection)) {
        filter(., gs_collection == collection, gs_subcollection == subcollection)
      } else {
        filter(., gs_collection == collection)
      }
    } %>%
    mutate(gs_name = str_remove(gs_name, "^[^_]+_")) %>%
    filter(gs_name %in% sig_pathways) %>%
    dplyr::select(gs_name, ensembl_gene) %>%
    filter(!is.na(ensembl_gene), ensembl_gene != "") %>%
    distinct()
  
  # Get pathway sizes
  pathway_sizes <- msig_sub %>%
    group_by(gs_name) %>%
    summarise(Genes_In_Pathway = n(), .groups = "drop") %>%
    dplyr::rename(pathway = gs_name)
  
  # Add to summary
  le_summary <- le_summary %>%
    left_join(pathway_sizes, by = "pathway") %>%
    mutate(
      Pct_LE_of_Total = round((Grouped_LE_Genes / Genes_In_Pathway) * 100, 1)
    ) %>%
    dplyr::select(pathway, Genes_In_Pathway, LE_Genes, 
                  Grouped_LE_Genes, Genes_Removed, Pct_Retained, Pct_LE_of_Total) %>%
      arrange(desc(Pct_Retained))
  
  # Pairwise overlap table
  # Convert to gene lists for overlap calculation
  all_genes_list <- split(msig_sub$ensembl_gene, msig_sub$gs_name)
  
  # Get all LE genes per pathway (before grouping)
  le_genes_by_pathway <- all_leading_edge %>%
    distinct(pathway, gene)
  le_genes_list <- split(le_genes_by_pathway$gene, le_genes_by_pathway$pathway)
  
  # Make sure we only compare pathways that are in both lists
  pathway_names <- intersect(names(all_genes_list), names(le_genes_list))
  n_pathways <- length(pathway_names)
  
  if (n_pathways < 2) {
    overlap_table <- tibble()
  } else {
    overlap_table <- map_dfr(1:(n_pathways-1), function(i) {
      map_dfr((i+1):n_pathways, function(j) {
        p1 <- pathway_names[i]
        p2 <- pathway_names[j]
        
        # Basal overlap (all genes in pathways)
        basal_set1 <- all_genes_list[[p1]]
        basal_set2 <- all_genes_list[[p2]]
        basal_intersection <- intersect(basal_set1, basal_set2)
        
        # Leading edge overlap (only grouped LE genes)
        le_set1 <- le_genes_list[[p1]]
        le_set2 <- le_genes_list[[p2]]
        le_intersection <- intersect(le_set1, le_set2)
        
        tibble(
          Pathway1 = p1,
          Pathway2 = p2,
          Basal_Size1 = length(basal_set1),
          Basal_Size2 = length(basal_set2),
          Basal_Shared = length(basal_intersection),
          Basal_Pct1 = round((length(basal_intersection) / length(basal_set1)) * 100, 1),
          Basal_Pct2 = round((length(basal_intersection) / length(basal_set2)) * 100, 1),
          LE_Size1 = length(le_set1),
          LE_Size2 = length(le_set2),
          LE_Shared = length(le_intersection),
          LE_Pct1 = round((length(le_intersection) / length(le_set1)) * 100, 1),
          LE_Pct2 = round((length(le_intersection) / length(le_set2)) * 100, 1)
        )
      })
    }) %>%
      arrange(desc(LE_Shared))
  }
  
  return(list(
    all_leading_edge = all_leading_edge,  
    grouped_leading_edge = grouped_leading_edge,
    pathways_list = pathways_list,       
    gene_to_pathway = gene_to_pathway,
    le_summary = le_summary,              
    overlap_table = overlap_table         
  ))
}

# Function to plot all leading edge genes in a pathway across contrasts 
plot_expression_by_pathway <- function(pathway_name, leading_edges,
                                       vst_matrix, meta) {
  
  # Get all genes that appear in leading edge for this pathway in ANY contrast
  pathway_genes <- leading_edges %>%
    filter(pathway == pathway_name) %>%
    pull(gene) %>%
    unique()
  
  # Check if any genes found
  if (length(pathway_genes) == 0) {
    warning(paste("No leading edge genes found for pathway: ", pathway_name))
    return(NULL)
  }
  
  # Get expression values from vst_matrix for these genes
  expression_df <- vst_matrix[pathway_genes, ] %>%
    as.data.frame() %>%
    rownames_to_column("Gene") %>%
    pivot_longer(-Gene, names_to = "Sample", values_to = "Expression") %>%
    left_join(meta, by = c("Sample" = "label"))
  
  # Compute mean expression by condition
  expression_summary <- expression_df %>%
    group_by(Gene, condition) %>%
    summarise(MeanExpression = mean(Expression), .groups = "drop")
  
  # Create matrix for heatmap
  expr_matrix <- expression_summary %>%
    pivot_wider(names_from = condition, values_from = MeanExpression) %>%
    column_to_rownames("Gene") %>%
    as.matrix()
  
  # Create condition to Bac.Vir_label mapping for later use
  condition_labels <- expression_df %>%
    select(condition, Bac.Vir_label) %>%
    distinct()

  # Map Ensembl IDs to symbols
  gene_info <- msigdbr_human %>%
    select(ensembl_gene, gene_symbol) %>%
    distinct() %>%
    filter(ensembl_gene %in% rownames(expr_matrix)) %>%
    distinct(ensembl_gene, gene_symbol)
  
  # Create new row names with symbols (and keep Ensembl ID if symbol missing or duplicate)
  new_rownames <- sapply(rownames(expr_matrix), function(ens_id) {
    symbol <- gene_info$gene_symbol[gene_info$ensembl_gene == ens_id]
    if (length(symbol) > 0 && !is.na(symbol) && symbol != "") {
      return(symbol)
    } else {
      return(ens_id)
    }
  })
  
  # Handle duplicate symbols by appending Ensembl ID
  if (any(duplicated(new_rownames))) {
    dup_names <- new_rownames[duplicated(new_rownames) | duplicated(new_rownames, fromLast = TRUE)]
    for (name in unique(dup_names)) {
      dup_indices <- which(new_rownames == name)
      if (length(dup_indices) > 1) {
        # Append Ensembl ID to duplicates
        new_rownames[dup_indices] <- paste0(name, " (", rownames(expr_matrix)[dup_indices], ")")
      }
    }
  }
  
  rownames(expr_matrix) <- new_rownames
  
  # Z-score scale the matrix by row
  expr_matrix_scaled <- t(scale(t(expr_matrix)))
  
  # Prepare data for ggplot
  heatmap_data <- expr_matrix_scaled %>%
    as.data.frame() %>%
    rownames_to_column("Gene") %>%
    pivot_longer(-Gene, names_to = "condition", values_to = "Z_score") %>%
    left_join(condition_labels, by = "condition")
  
  # Hierarchical clustering of genes
  gene_dist <- dist(expr_matrix_scaled)
  gene_hclust <- hclust(gene_dist)
  gene_order <- rownames(expr_matrix_scaled)[gene_hclust$order]
  
  # Set factor levels for ordering
  heatmap_data$Gene <- factor(heatmap_data$Gene, levels = gene_order)
  
  # Create ggplot heatmap
  heatmap_Z <- ggplot(heatmap_data, aes(x = Bac.Vir_label, y = Gene, fill = Z_score)) +
    geom_tile(color = "white", linewidth = 0.5) +
    scale_fill_gradientn(
      colours = c("#009AD1", "#d9f1fa", "#fefbea", "#fae1e4", "#AD1457"),
      name = "Z-score"
    ) +
    scale_x_discrete(expand = c(0, 0)) +
    scale_y_discrete(expand = c(0, 0)) +
    labs(
      title = paste0(pathway_name, " (", length(pathway_genes), " leading edge genes)"),
      x = "",
      y = ""
    ) +
    theme_bw(base_size = 10) +
    theme(
      panel.grid = element_blank(),
      axis.text.x = element_markdown(size = 9),
      axis.text.y = element_text(size = 7),
      plot.title = element_text(hjust = 0.5, face = "bold", size = 11),
      legend.position = "right"
    )
  
  return(list(
    gene_info = gene_info,
    heatmap_data = heatmap_data,
    heatmap_Z = heatmap_Z
  ))
}

# Function to plot all leading edge genes in a pathway category across contrasts 
plot_expression_by_category <- function(pathway_groups, pathway_colors = RColorBrewer::brewer.pal(n = 12, name = "Paired"),
                                        category_name, leading_edges, vst_matrix, meta) {
  
  # Get all pathways in this category
  pathways_in_category <- pathway_groups %>%
    filter(category == category_name) %>%
    pull(pathway)
  
  # Check if any pathways found
  if (length(pathways_in_category) == 0) {
    warning(paste("No pathways found for category:", category_name))
    return(NULL)  
  }
  
  # Get all genes in this category from grouped_leading_edge
  category_data <- leading_edges %>%
    filter(pathway %in% pathways_in_category)
  
  if (nrow(category_data) == 0) {
    warning(paste("No leading edge genes found for category: ", category_name))
    return(NULL)
  }
  
  category_genes <- unique(category_data$gene)
  
  # Get expression values from vst_matrix for these genes
  expression_df <- vst_matrix[category_genes, ] %>%
    as.data.frame() %>%
    rownames_to_column("Gene") %>%
    pivot_longer(-Gene, names_to = "Sample", values_to = "Expression") %>%
    left_join(meta, by = c("Sample" = "label")) %>%
    # Add pathway annotation
    left_join(
      category_data %>% dplyr::select(gene, pathway) %>% distinct(),
      by = c("Gene" = "gene"),
      relationship = "many-to-many"
    )
  
  # Compute mean expression by condition
  expression_summary <- expression_df %>%
    group_by(Gene, pathway, condition, Bac.Vir_label, Bac.Vir_label_symbols, species, virus) %>%
    summarise(MeanExpression = mean(Expression), .groups = "drop")
  
  # Boxplot  
  boxplot <- ggplot(expression_df, aes(x = Bac.Vir_label_symbols, y = Expression, color = Bac.Vir_label)) +
    geom_boxplot(outliers = FALSE) +
    geom_jitter(size = 0.8, shape = 16, width = 0.2, alpha = 0.3) +
    scale_color_manual(values = Bac.Vir_colors) +
    labs(
      title = paste0(category_name, " (", length(category_genes), " genes)"),
      x = "Condition",
      y = "VST Expression"
    ) +
    theme_bw() +
    theme(axis.text = element_text(color = "black"),
          axis.text.x = element_markdown(size = 10),
          legend.text = element_markdown(size = 10),
          panel.grid.major = element_blank(),
          panel.grid.minor = element_blank())
  
  # Show mean expression per pathway (averaging across all genes in pathway) and sd
  pathway_summary <- expression_summary %>%
    group_by(pathway, condition, Bac.Vir_label_symbols, species, virus) %>%
    summarise(PathwayMeanExpression = mean(MeanExpression), 
              PathwaySDExpression = sd(MeanExpression),
              .groups = "drop") %>%
    mutate(pathway = factor(pathway, levels = pathway_groups$pathway[pathway_groups$category == category_name]))
  
  # Plot of pathway means
  plot <-  ggplot(pathway_summary, aes(x = Bac.Vir_label_symbols, y = PathwayMeanExpression, 
                                       color = pathway, group = pathway)) +
    geom_line(size = 0.5, linetype = "dotted") +
    geom_point(size = 2) +
    scale_color_manual(values = pathway_colors) +
    labs(
      title = paste0(category_name),
      x = "",
      y = "Mean VST Expression",
      color = "Gene Set"
    ) +
    theme_bw() +
    theme(axis.text = element_text(color = "black"),
          axis.text.x = element_markdown(size = 10),
          panel.grid.major = element_blank(),
          panel.grid.minor = element_blank())
  
  return(list(boxplot = boxplot, plot = plot))
}

# Pathway Overlap Analysis
le_genes_VirusEffect_diff_vsNB  <- extract_le_genes_from_gsea(multi_gsea_VirusEffect_diff_vsNB, msigdbr_human, collection = "H")
le_genes_overlap_VirusEffect_diff_vsNB <- le_genes_VirusEffect_diff_vsNB$overlap_table

kable(le_genes_VirusEffect_diff_vsNB$le_summary)

pathway	Genes_In_Pathway	LE_Genes	Grouped_LE_Genes	Genes_Removed	Pct_Retained	Pct_LE_of_Total
TNFA_SIGNALING_VIA_NFKB	200	155	154	1	99.4	77.0
OXIDATIVE_PHOSPHORYLATION	201	131	129	2	98.5	64.2
PROTEIN_SECRETION	97	71	64	7	90.1	66.0
E2F_TARGETS	200	150	126	24	84.0	63.0
INTERFERON_GAMMA_RESPONSE	200	170	142	28	83.5	71.0
APICAL_JUNCTION	201	134	103	31	76.9	51.2
CHOLESTEROL_HOMEOSTASIS	74	50	38	12	76.0	51.4
FATTY_ACID_METABOLISM	158	105	74	31	70.5	46.8
MYC_TARGETS_V2	58	46	32	14	69.6	55.2
KRAS_SIGNALING_UP	201	113	77	36	68.1	38.3
ANDROGEN_RESPONSE	101	59	39	20	66.1	38.6
UNFOLDED_PROTEIN_RESPONSE	113	76	49	27	64.5	43.4
G2M_CHECKPOINT	200	139	88	51	63.3	44.0
ESTROGEN_RESPONSE_EARLY	200	115	72	43	62.6	36.0
MITOTIC_SPINDLE	199	119	72	47	60.5	36.2
IL2_STAT5_SIGNALING	200	119	70	49	58.8	35.0
COMPLEMENT	200	116	68	48	58.6	34.0
UV_RESPONSE_DN	144	98	57	41	58.2	39.6
EPITHELIAL_MESENCHYMAL_TRANSITION	200	116	67	49	57.8	33.5
TGF_BETA_SIGNALING	54	45	26	19	57.8	48.1
MYC_TARGETS_V1	200	134	76	58	56.7	38.0
ALLOGRAFT_REJECTION	200	114	64	50	56.1	32.0
MTORC1_SIGNALING	200	135	75	60	55.6	37.5
P53_PATHWAY	200	119	64	55	53.8	32.0
PI3K_AKT_MTOR_SIGNALING	105	74	38	36	51.4	36.2
INFLAMMATORY_RESPONSE	201	124	63	61	50.8	31.3
MYOGENESIS	200	104	52	52	50.0	26.0
IL6_JAK_STAT3_SIGNALING	91	64	31	33	48.4	34.1
UV_RESPONSE_UP	158	78	37	41	47.4	23.4
HYPOXIA	200	112	52	60	46.4	26.0
APOPTOSIS	161	91	40	51	44.0	24.8
COAGULATION	138	57	19	38	33.3	13.8
GLYCOLYSIS	200	110	36	74	32.7	18.0
ANGIOGENESIS	36	21	6	15	28.6	16.7
ESTROGEN_RESPONSE_LATE	200	98	27	71	27.6	13.5
INTERFERON_ALPHA_RESPONSE	97	95	25	70	26.3	25.8

write_csv(le_genes_overlap_VirusEffect_diff_vsNB,  file.path(outputs_folder, "Pathway_overlap_GSEA_VirusEffectDiff.csv"))
saveRDS(le_genes_VirusEffect_diff_vsNB, file.path(outputs_folder, "GSEA_VirusEffectDiff_LE.rds"))

The overlap table quantifies gene sharing between pathways at two levels:

• Basal overlap: All genes annotated to each pathway
• Leading-edge overlap: Only the core genes driving enrichment

This helps identify functionally related pathway clusters and potential redundancy.

Individual Pathway Expression Heatmaps

For each significant pathway, we generate Z-score normalized heatmaps showing the expression of all leading-edge genes across conditions. Genes are hierarchically clustered to reveal co-expression patterns.

We also generate category_plots showing average expression patterns of leading-edge genes grouped by pathway categories defined earlier. Each category plot includes a boxplot of individual gene expressions alongside the mean pathway expression trends.

# Loop through all categories using map to run plot_expression_by_category()
category_plots <- map(
  unique(pathway_groups_HALLMARK$category),
  ~ {
    plot_obj <- plot_expression_by_category(
      pathway_groups = pathway_groups_HALLMARK,
      category_name = .x,
      leading_edges = le_genes_VirusEffect_diff_vsNB$grouped_leading_edge,
      vst_matrix = vst_matrix,
      meta = meta
    )
    
    # Combine boxplot and main plot side by side
    combined_plot <- ggarrange(
      plot_obj$boxplot,
      plot_obj$plot,
      ncol = 2,
      widths = c(1, 1)
    )
    
    # Add title using annotate_figure
    annotate_figure(combined_plot, top = text_grob(.x, face = "bold", size = 18))

  }
)

# Create a combined multi-page PDF with all plots
CairoPDF(file = file.path(outputs_folder, paste0("GSEA_VirusEffectDiff_GenSets_LE.pdf")),
         width = 15, height = 6)
walk(category_plots, ~ print(.x))
dev.off()

quartz_off_screen 
                2 

Custom Pathway Selection: Key Biological Processes

For focused visualization, we can select specific pathways of interest and plot their expression patterns together. This example highlights pathways central to immune response, cell cycle, and stress response.

Note: This visualization uses all_leading_edge, meaning genes that are leading-edge members of multiple selected pathways will be included for each pathway they contribute to, providing a complete view of pathway membership.

# We select specific gen sets to plot together with genes that are LE in more than one pathway plotted duplicated as needed.
pathway_groups_selected <- bind_rows(
  tibble(
    pathway = c("INFLAMMATORY_RESPONSE", "INTERFERON_ALPHA_RESPONSE", 
                "INTERFERON_GAMMA_RESPONSE", "TNFA_SIGNALING_VIA_NFKB",
                "E2F_TARGETS", "TGF_BETA_SIGNALING"),
    category = "SelectedFig",
    color = "grey"  
  )
)
colors_groups_selected <- c("#FA8072", "#FF0000", "#D40118", "#65000b", "#32CD32", "#006400")


# Loop through all categories using map to run plot_expression_by_category()
selected_gene_sets <- map(
  unique(pathway_groups_selected$category),
  ~ {
    plot_obj <- plot_expression_by_category(
      pathway_groups = pathway_groups_selected,
      pathway_colors = colors_groups_selected,
      category_name = .x,
      leading_edges = le_genes_VirusEffect_diff_vsNB$all_leading_edge,
      vst_matrix = vst_matrix,
      meta = meta
    )
    
    # Save combined plot as PDF
    ggsave(
      filename = paste0(outputs_folder, "/GSEA_VirusEffectDiff_LE_", .x, ".png"),
      plot = plot_obj$plot,
      width = 8, height = 4, dpi = 300
    )
    
   # Save plot object as RDS
    saveRDS(
      plot_obj$plot,
      file = paste0(outputs_folder, "/GSEA_VirusEffectDiff_LE_", .x, ".rds")
    )
    
    plot_obj$plot  # Return combined plot for reference
  }
)

selected_gene_sets[[1]] 

# For those same gene sets we generate individual Z-score heatmaps and save them as one object
selected_gene_sets <- map(
  unique(pathway_groups_selected$pathway),
  ~ {
    plot_obj <- plot_expression_by_pathway(
      pathway_name = .x,
      leading_edges = le_genes_VirusEffect_diff_vsNB$all_leading_edge,
      vst_matrix = vst_matrix,
      meta = meta
    )
    return(list(plot = plot_obj$heatmap_Z,
                n_rows = n_distinct(plot_obj$heatmap_data$Gene)
    ))
  }
)

# Name the list elements by pathway
names(selected_gene_sets) <- pathway_groups_selected$pathway

# Save the entire list as well for easy access
saveRDS(selected_gene_sets, file = file.path(outputs_folder, "GSEA_VirusEffectDiff_ZHeatmaps_SelectedFig.rds"))

# For each pathway, calculate mean expression of leading-edge genes and perform mixed-effects modeling
analyze_pathway_mean_expression <- function(pathway_name, leading_edges, 
                                           vst_matrix, meta) {
  
  # Get all genes in this pathway's leading edge
  pathway_genes <- leading_edges %>%
    filter(pathway == pathway_name) %>%
    pull(gene)                      
  
  # Get expression values and calculate mean per sample
  expression_df <- vst_matrix[pathway_genes, ] %>%
    as.data.frame() %>%
    rownames_to_column("Gene") %>%
    pivot_longer(-Gene, names_to = "Sample", values_to = "Expression") %>%
    left_join(meta, by = c("Sample" = "label")) %>%
    group_by(Sample, condition, Bac.Vir_label, Bac.Vir_label_symbols, 
             species, virus, line, date) %>%
    summarise(MeanExpression = mean(Expression, na.rm = TRUE), 
              .groups = "drop") %>%
    mutate(pathway = pathway_name)
  
  # Fit mixed-effects model
  model <- lmer(MeanExpression ~ condition + (1|line) + (1|line:date), 
                data = expression_df)
  
  # Check for singularity
  singular <- isSingular(model)
  if (singular) {
    model <- lmer(MeanExpression ~ condition + (1|date), 
                  data = expression_df)
  }
  
  # Get ANOVA results
  anova_result <- anova(model)
  anova_pval <- anova_result$`Pr(>F)`[1]
  
  # Extract random effects
  random_effects_df <- as.data.frame(VarCorr(model)) %>%
    mutate(proportion = round(100 * (vcov / sum(vcov)), 2),
           singular = singular,
           pathway = pathway_name)
  
  # Extract fixed effects
  fixed_effects_df <- as.data.frame(summary(model)$coefficients) %>%
    rownames_to_column(var = "term") %>%
    mutate(pathway = pathway_name)
  
  # Get emmeans contrasts vs NB.NV with Holm adjustment
  emmeans_model <- emmeans(model, ~ condition)
  contrasts <- pairs(emmeans_model, adjust = "none")
  
  contrast_df <- as.data.frame(summary(contrasts)) %>%
    separate(contrast, into = c("condition1", "condition2"), 
             sep = " - ", remove = FALSE) %>%
    mutate(pathway = pathway_name,
           anova_pval = anova_pval)
  
  return(list(
    expression_data = expression_df,
    anova = anova_result,
    random_effects = random_effects_df,
    fixed_effects = fixed_effects_df,
    contrasts = contrast_df
  ))
}

# Analyze all pathways
pathway_stats_list <- map(
  pathway_groups_selected$pathway,
  ~ analyze_pathway_mean_expression(
    pathway_name = .x,
    leading_edges = le_genes_VirusEffect_diff_vsNB$grouped_leading_edge,
    vst_matrix = vst_matrix,
    meta = meta
  )
)
names(pathway_stats_list) <- pathway_groups_selected$pathway

# Combine all contrasts 
selected_gene_sets_contrasts <- bind_rows(
  map(pathway_stats_list, "contrasts"),
  .id = "pathway"
) %>%
  mutate(across(where(is.character), as.factor))

# Adjust across all contrasts 
selected_gene_sets_contrasts <- selected_gene_sets_contrasts %>%
  mutate(adj.p.FDR = p.adjust(p.value, method = "fdr")) %>%
  mutate(sign.FDR = ifelse(adj.p.FDR < 0.05, T, F)) %>%
  mutate(sign = case_when(
    adj.p.FDR < 0.05 ~ "*",
    TRUE ~ ""  
  )) 

selected_gene_sets_contrasts$adj.p.FDR = format(selected_gene_sets_contrasts$adj.p.FDR, digits = 2, scientific = TRUE)

write_csv(selected_gene_sets_contrasts, file.path(outputs_folder, "GSEA_VirusEffectDiff_LE_Selected_statistics.csv"))
saveRDS(selected_gene_sets_contrasts, file.path(outputs_folder, "GSEA_VirusEffectDiff_LE_Selected_statistics.rds"))