Differential Gene Expression (DGE) Analysis
Last updated: 2024-03-13
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Knit directory: mecfs-dge-analysis/
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DGE Analysis Setup
Ensure you have all necessary libraries installed and load the helper code.
At a later date, renv will be integrated to ensure
reproducibility of this analysis.
Use the below code to install these packages:
# Install packages from CRAN
install.packages(c("tidyverse", "RColorBrewer", "pheatmap", "gprofiler2", "plotly", "ggupset"))
# Install packages from Bioconductor
install.packages("BiocManager")
BiocManager::install(c("DESeq2", "genefilter", "limma", "biomaRt", "mygene"))library(tidyverse) # Available via CRAN
library(DESeq2) # Available via Bioconductor
library(RColorBrewer) # Available via CRAN
library(pheatmap) # Available via CRAN
library(genefilter) # Available via Bioconductor
library(limma) # Available via Bioconductor
library(gprofiler2) # Available via CRAN
library(biomaRt) # Available via Bioconductor
library(plotly) # Available via CRAN
library(ggpubr)
library(rmarkdown)
library(ggupset)
library(clusterProfiler)
library(DOSE)
library(org.Hs.eg.db) # Available via Bioconductor
library(UpSetR)
library(ggrepel)Data Import
We will be importing counts data from the star-salmon pipeline and our metadata for the project which is hosted on Box. This also ensures data is properly ordered by sample id.
counts <- read_tsv("data/star-salmon/salmon.merged.gene_counts_length_scaled.tsv")
# Import variants of interest
genes_of_interest <- read_csv("data/Patient_Genes_2024_03_05.csv")
genes_of_interest <- unique(genes_of_interest$Genes)
genes_of_interest <- genes_of_interest[!is.na(genes_of_interest)]
# Use first column (gene_id) for row names
counts <- data.frame(counts, row.names = 1)
counts$Ensembl_ID <- row.names(counts)
drop <- c("Ensembl_ID", "gene_name")
gene_info <- counts[, drop]
counts <- counts[, !(names(counts) %in% drop)] # remove both columns
# Import metadata
sample_metadata <- read_csv("data/MECFS_RNAseq_metadata_2024_03_05.csv")
row.names(sample_metadata) <- sample_metadata$ID
# Assuming counts is your counts dataframe and sample_metadata is your metadata dataframe
# Call the function with the appropriate column names
counts <- rename_counts_columns(counts, sample_metadata, "ID", "RNA_Samples_id")
# Check that data is ordered properly
sample_metadata <- check_order(sample_metadata = sample_metadata, counts = counts)
genes_biomart <- retrieve_gene_info(values = gene_info$Ensembl_ID, filters = "ensembl_gene_id_version")DESeq2 Analysis
sample_metadata$Family <- factor(sample_metadata$Family)
sample_metadata$Affected <- factor(sample_metadata$Affected)
sample_metadata$Batch <- factor(sample_metadata$Batch)
sample_metadata$Sex <- factor(sample_metadata$Sex)
sample_metadata$Ancestry <- factor(sample_metadata$Ancestry)
sample_metadata$SubCategory <- factor(sample_metadata$SubCategory)
sample_metadata$CombinedCategory <- factor(sample_metadata$CombinedCategory)
# Account for Family later but batch is accounted for
# Accounting for another factor seems to be an issue.
dds <- DESeqDataSetFromMatrix(
countData = round(counts), colData = sample_metadata,
design = ~ Batch + Affected
)
# Pre-filtering: Keep only rows that have at least 10 reads total
keep <- rowSums(counts(dds)) >= 10
dds <- dds[keep, ]
# Remove male samples
remove_multiple_fam <- c("8", "12", "19", "21", "23", "27")
# Create a logical vector to index the columns you want to keep
multiple_fam_members <- !(colnames(dds) %in% remove_multiple_fam)
dds <- dds[, multiple_fam_members]
# Run DESeq function
dds <- DESeq(dds)
# Normalize gene counts for differences in seq. depth/global differences
counts_norm <- counts(dds, normalized = TRUE)Data transformation and visualization
Perform count data transformation by variance stabilizing transformation (vst) on normalized counts.
vsd <- vst(dds, blind = FALSE)Batch correction with limma
counts_vst <- assay(vsd)
write.csv(counts_vst, file = "output/counts_vst_subsetted..csv")
mm <- model.matrix(~ Batch + Affected, colData(vsd))
counts_vst_limma <- limma::removeBatchEffect(counts_vst, batch = vsd$Batch, design = mm)Coefficients not estimable: batch1 batch2 write.csv(counts_vst_limma, file = "output/counts_vst_limma_subsetted.csv")
vsd_limma <- vsd
assay(vsd_limma) <- counts_vst_limmaSample distances heatmap
sample_dists_all <- dist(t(assay(vsd_limma)))
sample_dist_matrix_all <- as.matrix(sample_dists_all)
rownames(sample_dist_matrix_all) <- paste(vsd_limma$Batch, vsd_limma$Affected, sep = " | ")
colnames(sample_dist_matrix_all) <- paste(vsd_limma$ID, vsd_limma$Affected, sep = " | ")
colors <- colorRampPalette(rev(brewer.pal(9, "Blues")))(255)
pheatmap(sample_dist_matrix_all, clustering_distance_rows = sample_dists_all, clustering_distance_cols = sample_dists_all, col = colors)
Principal Components Analysis
Our below PCA shows that there does not seem to be a batch-related effect occurring after using limma. However, we can see that our 3 male samples are grouping. Given this knowledge, removing them from downstream analyses is the best option.
pca_data_all <- plotPCA(vsd_limma, intgroup = c("Batch", "Affected", "Sex"), returnData = TRUE)
percent_var_all <- round(100 * attr(pca_data_all, "percentVar"))
ggplot(pca_data_all, aes(PC1, PC2)) +
geom_point(aes(colour = Affected, fill = Sex, shape = Batch), size = 4) +
scale_shape_manual(values = c(21, 22, 23)) +
scale_fill_manual(values = c("white", "gray")) +
scale_color_manual(values = c("blue", "red")) +
geom_text(aes(label = name),
data = subset(pca_data_all, PC2 < -18 | PC1 < -30),
vjust = -1, hjust = 0.5, size = 2.5
) +
xlab(paste0("PC1: ", percent_var_all[1], "% variance")) +
ylab(paste0("PC2: ", percent_var_all[2], "% variance")) +
coord_fixed()
pca_data_all <- plotPCA(vsd_limma, intgroup = c("Affected", "Sex", "CombinedCategory"), returnData = TRUE)
percent_var_all <- round(100 * attr(pca_data_all, "percentVar"))
ggplot(pca_data_all, aes(PC1, PC2)) +
geom_point(aes(colour = CombinedCategory, fill = Sex, shape = Affected), size = 4) +
scale_shape_manual(values = c(21, 22, 23)) +
scale_fill_manual(values = c("white", "gray")) +
geom_text(aes(label = name), vjust = -1, hjust = 0.65, size = 5) +
# geom_text_repel(aes(label = name), size = 5, vjust = -1, hjust = -0.65) +
xlab(paste0("PC1: ", percent_var_all[1], "% variance")) +
ylab(paste0("PC2: ", percent_var_all[2], "% variance")) +
coord_fixed()
pca_data <- plotPCA(vsd_limma, intgroup = c("Sex", "Affected"), returnData = TRUE)
percent_var <- round(100 * attr(pca_data, "percentVar"))
ggplot(pca_data, aes(PC1, PC2)) +
geom_point(aes(shape = Sex, colour = Affected), size = 5) +
scale_color_manual(
values = c("blue", "red"),
name = "Affected Status",
labels = c("Unaffected", "Affected")
) +
scale_shape_manual(
name = "Sex",
labels = c("Male", "Female"),
values = c(16, 17)
) + # Example shapes, change as needed
geom_text_repel(aes(label = name), size = 5, vjust = -2, hjust = -0.65) +
xlab(paste0("PC1: ", percent_var[1], "% variance")) +
ylab(paste0("PC2: ", percent_var[2], "% variance")) +
coord_fixed()
pca_data_all <- plotPCA(vsd_limma, intgroup = c("CombinedCategory"), returnData = TRUE)
percent_var_all <- round(100 * attr(pca_data_all, "percentVar"))
ggplot(pca_data_all, aes(PC1, PC2)) +
geom_point(aes(colour = CombinedCategory), size = 5) +
scale_colour_manual(
values = c(
"#B3DE69", "#FFFFB3", "#BEBADA", "#FB8072", "#80B1D3",
"#FCCDE5", "#8DD3C7", "#000000"
),
name = "Category"
) +
# geom_text(aes(label = name), vjust = -.75, hjust = -.5, size = 3) +
# geom_text_repel(aes(label = name), size = 5, vjust = -2, hjust = -0.65) +
geom_text_repel(aes(label = name), size = 3) +
xlab(paste0("PC1: ", percent_var_all[1], "% variance")) +
ylab(paste0("PC2: ", percent_var_all[2], "% variance")) +
ggthemes::theme_clean()
Heatmap of all genes, top 50 & top 100 genes
# Specify annotation colors by columns
# Use RColorBrewer::brewer.pal(n=10, name="Set1")
disease_colors <- c(
"Cryohydrocytosis" = "#8DD3C7",
"Gitelman Syndrome" = "#FFFFB3",
"Gitelman Syndrome & Thyrotoxic Periodic Paralysis" = "#BEBADA",
"Glycogen Storage Disease" = "#FB8072",
"Immunodeficiency/Autoinflammatory Diseases" = "#80B1D3",
"Immunodeficiency/Autoinflammatory Diseases & Mitochondrial Disorder" = "#FDB462",
"Mitochondrial Disorder" = "#B3DE69",
"N/A" = "#FCCDE5",
"Neurological/Neuromuscular Disorders" = "#D9D9D9",
"Stromme syndrome" = "#BC80BD",
"Thyrotoxic Periodic Paralysis" = "#CCEBC5",
"Unknown" = "#FFED6F"
)
category_colors <- c(
"Autoimmune disorder" = "#B3DE69",
"Immunodeficiency" = "#FFFFB3",
"Channelopathy" = "#BEBADA",
"Inflammatory disorder" = "#FB8072",
"Metabolic disorder" = "#80B1D3",
"Mitochondrial disorder" = "#FCCDE5",
"Neurologic disorder" = "#D9D9D9",
"Myasthenic disorder" = "#FDB462",
"Neuropathy" = "#8DD3C7",
"None" = "#001111"
)
combined_category_colors <- c(
"Transporter-Metabolism" = "#E41A1C", # Dark green
"Metabolism" = "#377EB8", # Dark orange
"Metabolism-Immune" = "#4DAF4A", # Light purple
"Immune-Transporter" = "#984EA3", # Magenta
"Transporter" = "#FF7F00", # Light green
"Cytoskeletal" = "#FFFF33", # Mustard yellow
"Transporter-Immune-Metabolism" = "#F781BF", # Brown
"NA" = "#000000" # Dark grey
)
# Specify colors
ann_colors <- list(
Batch = c(B1 = "purple", B2 = "firebrick", B3 = "yellow"),
Affected = c(Yes = "green", No = "navy"),
Disease = disease_colors,
SubCategory = category_colors,
CombinedCategory = combined_category_colors
)
ann_colors2 <- list(
Batch = c(B1 = "purple", B2 = "firebrick", B3 = "yellow"),
Affected = c(Yes = "green", No = "navy"),
SubCategory = category_colors,
Disease = disease_colors,
CombinedCategory = combined_category_colors
)This is a heatmap of the top 50 genes with the highest variance across samples
top_var_genes <- head(order(-rowVars(assay(vsd_limma))), 50)
mat <- assay(vsd_limma)[top_var_genes, ]
mat <- mat - rowMeans(mat)
df <- as.data.frame(colData(vsd_limma)[, c("Batch", "Affected", "CombinedCategory")])
ensembl_to_gene <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids <- rownames(mat)
# Find the corresponding gene names for the Ensembl IDs
new_row_names <- ensembl_to_gene[current_ensembl_ids]
# Set the new row names for the matrix 'mat'
rownames(mat) <- new_row_names
pheatmap(mat, annotation_col = df, annotation_colors = ann_colors, fontsize = 5)
This is a heatmap of the top 50 genes with the highest variance across all samples.
top_var_genes_all <- head(order(-rowVars(assay(vsd_limma))), 50)
mat_all <- assay(vsd_limma)[top_var_genes_all, ]
mat_all <- mat_all - rowMeans(mat_all)
df_all <- as.data.frame(colData(vsd_limma)[, c("Batch", "Affected", "SubCategory")])
df_affcat <- as.data.frame(colData(vsd_limma)[, c("Affected", "CombinedCategory")])
ensembl_to_gene <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_all <- rownames(mat_all)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_all <- ensembl_to_gene[current_ensembl_ids_all]
# Set the new row names for the matrix 'mat'
rownames(mat_all) <- new_row_names_all
pheatmap(mat_all, annotation_col = df_all, annotation_colors = ann_colors, fontsize = 5)
pheatmap(mat_all, annotation_col = df_affcat, annotation_colors = ann_colors2, fontsize = 5)
This is a heatmap of the top 100 genes with the highest variance across
top_var_genes_100_all <- head(order(-rowVars(assay(vsd_limma))), 100)
mat_100_all <- assay(vsd_limma)[top_var_genes_100_all, ]
mat_100_all <- mat_100_all - rowMeans(mat_100_all)
df_100_all <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory")])
ensembl_to_gene <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_100_all <- rownames(mat_100_all)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_100_all <- ensembl_to_gene[current_ensembl_ids_100_all]
# Set the new row names for the matrix 'mat'
rownames(mat_100_all) <- new_row_names_100_all
pheatmap(mat_100_all, annotation_col = df_100_all, annotation_colors = ann_colors2, fontsize = 6)
Comparison/Contrast of Affected_Yes_vs_No
res_aff_vs_unaff_sub <- results(dds, contrast = c("Affected", "Yes", "No"))
res_aff_vs_unaff_df_sub <- process_and_save_results(
res_aff_vs_unaff_sub,
"output/res_aff_vs_unaff_sub.csv"
)
res_aff_vs_unaff_df_sub <- arrange(res_aff_vs_unaff_df_sub, padj)
res_aff_vs_unaff_df_05_sub <- subset(res_aff_vs_unaff_df_sub, padj < 0.05)
res_aff_vs_unaff_df_1_sub <- subset(res_aff_vs_unaff_df_sub, padj < 0.05)
summary(res_aff_vs_unaff_sub)
out of 29623 with nonzero total read count
adjusted p-value < 0.1
LFC > 0 (up) : 22, 0.074%
LFC < 0 (down) : 35, 0.12%
outliers [1] : 219, 0.74%
low counts [2] : 0, 0%
(mean count < 0)
[1] see 'cooksCutoff' argument of ?results
[2] see 'independentFiltering' argument of ?resultstopgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 100)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 3.5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 100)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 3.5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 50)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- df <- colData(vsd_limma) %>%
as.data.frame() %>%
dplyr::select(CombinedCategory)
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 50)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- df <- colData(vsd_limma) %>%
as.data.frame() %>%
dplyr::select(Affected, SubCategory, CombinedCategory)
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 57)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- df <- colData(vsd_limma) %>%
as.data.frame() %>%
dplyr::select(CombinedCategory)
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 57)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- df <- colData(vsd_limma) %>%
as.data.frame() %>%
dplyr::select(Affected, SubCategory, CombinedCategory)
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 75)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 75)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 200)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 1.5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 200)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 1.5)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 150)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
# topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 3.75)
topgenes_byensemblid_sub <- head(rownames(res_aff_vs_unaff_df_sub), 150)
topgenes_aff_vs_unaff_05_sub <- assay(vsd_limma)[topgenes_byensemblid_sub, ]
topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub - rowMeans(topgenes_aff_vs_unaff_05_sub)
ensembl_to_gene_sub <- setNames(gene_info$gene_name, gene_info$Ensembl_ID)
# Get the current row names of the matrix 'mat'
current_ensembl_ids_sub <- rownames(topgenes_aff_vs_unaff_05_sub)
# Find the corresponding gene names for the Ensembl IDs
new_row_names_sub <- ensembl_to_gene_sub[current_ensembl_ids_sub]
# Set the new row names for the matrix 'mat'
rownames(topgenes_aff_vs_unaff_05_sub) <- new_row_names_sub
# topgenes_aff_vs_unaff_05_sub <- topgenes_aff_vs_unaff_05_sub[order(row.names(topgenes_aff_vs_unaff_05_sub)), ]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory", "CombinedCategory")])
pheatmap(topgenes_aff_vs_unaff_05_sub, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 3.75)
gb_df <- genes_biomart[, c(1, ncol(genes_biomart))]
res_aff_vs_unaff_df_sub_genename <- res_aff_vs_unaff_df_sub
res_aff_vs_unaff_df_sub_genename$Ensembl_ID <- row.names(res_aff_vs_unaff_df_sub)
res_aff_vs_unaff_df_sub_genename <- merge(x = res_aff_vs_unaff_df_sub_genename, y = gene_info, by.x = "Ensembl_ID", by.y = "Ensembl_ID", all.x = TRUE)
res_aff_vs_unaff_df_sub_genename <- res_aff_vs_unaff_df_sub_genename[, c(dim(res_aff_vs_unaff_df_sub_genename)[2], 1:dim(res_aff_vs_unaff_df_sub_genename)[2] - 1)]
res_aff_vs_unaff_df_sub_genename <- res_aff_vs_unaff_df_sub_genename[order(res_aff_vs_unaff_df_sub_genename[, "padj"]), ]
write.csv(res_aff_vs_unaff_df_sub_genename, file = "output/res_aff_vs_unaff_df_sub_genename.csv")res_aff_vs_unaff_df_genename_05 <- subset(res_aff_vs_unaff_df_sub_genename, padj < 0.05)
res_aff_vs_unaff_df_genename_05 <- res_aff_vs_unaff_df_genename_05[order(res_aff_vs_unaff_df_genename_05$padj), ]
write.csv(res_aff_vs_unaff_df_genename_05, file = "output/res_aff_vs_unaff_df_sub_genename_05.csv")
res_aff_vs_unaff_df_genename_1 <- subset(res_aff_vs_unaff_df_sub_genename, padj < 0.1)
res_aff_vs_unaff_df_genename_1 <- res_aff_vs_unaff_df_genename_1[order(res_aff_vs_unaff_df_genename_1$padj), ]
write.csv(res_aff_vs_unaff_df_genename_1, file = "output/res_aff_vs_unaff_df_sub_genename_padj1.csv")Below is a table of information about the top genes.
library(mygene)
genes <- res_aff_vs_unaff_df_genename_05$gene_name
my_gene_data <- queryMany(genes, scopes = "symbol", fields = c("symbol", "name", "summary", species = "human"))Finished
Pass returnall=TRUE to return lists of duplicate or missing query terms.my_gene_data_unique <- as.data.frame(my_gene_data) %>% dplyr::distinct(query, .keep_all = TRUE)
# paged_table(my_gene_data, options = list(rows.print = 15))filtered_gene_names <- res_aff_vs_unaff_df_sub_genename$gene_name[!grepl("^ENS", res_aff_vs_unaff_df_sub_genename$gene_name)]
# Select specific genes to show
# set top = 0, then specify genes using label.select argument
maplot <- ggmaplot(res_aff_vs_unaff_df_sub,
main = "Affected vs Unaffected MA Plot",
fdr = .1, fc = 1, size = 0.4, # Same used for others.
genenames = as.vector(res_aff_vs_unaff_df_sub_genename$gene_name),
ggtheme = ggplot2::theme_minimal(),
legend = "top", top = 30,
label.select = c("KCNQ5"),
font.label = c("bold", 6), label.rectangle = TRUE,
font.legend = "bold", font.main = "bold"
)
maplot
significant_data <- maplot$data %>%
filter(grepl("Up|Down", sig)) %>%
mutate(direction = ifelse(grepl("Up", sig), "Up", "Down")) %>%
dplyr::select(-sig) # This removes the 'sig' column
# Combine DataFrames based on matching 'query' in my_gene_data_unique to 'gene' in significant_data
combined_data <- inner_join(my_gene_data_unique, significant_data, by = c("query" = "name"))
combined_data <- combined_data %>%
dplyr::select(-notfound, -X_id, -X_score) %>%
rename(gene = query)
paged_table(as.data.frame(significant_data), options = list(rows.print = 30))# Save significant genes
write.csv(significant_data, file = "output/res_aff_vs_unaff_significant_subsetted_samples.csv", row.names = FALSE)
# Save significant genes
write.csv(combined_data, file = "output/res_aff_vs_unaff_significant_subsetted_mygene.csv", row.names = FALSE)Expression of Candidate Genes
Below is a table of expression of the genes identified during our WGS analysis.
8 of the genes were not included using DESeq2’s count filtering method (these genes had no counts):
“DPEP1”, “RERGL”, “TDO2”, “CCDC178”, “ADRA1D”, “AVPR1B”, “LRCOL1”, “KCNJ18”
# Subset gene_info using genes of interest
subset_gene_info <- gene_info[gene_info$gene_name %in% genes_of_interest, ]
filtered_by_interest <- filter(res_aff_vs_unaff_df_sub_genename, Ensembl_ID %in% subset_gene_info$Ensembl_ID)
# Filtering the dataframe by row names
filtered_counts <- counts[row.names(counts) %in% subset_gene_info$Ensembl_ID, ]
# Match and update row names
matching_indices <- match(rownames(filtered_counts), subset_gene_info$Ensembl_ID)
# Update row names based on matching_column values from metadata
rownames(filtered_counts) <- subset_gene_info$gene_name[matching_indices]
missing_genes <- setdiff(subset_gene_info$Ensembl_ID, filtered_by_interest$Ensembl_ID)
corresponding_gene_names <- subset_gene_info$gene_name[subset_gene_info$Ensembl_ID %in% missing_genes]
missing_gene_counts <- counts[row.names(counts) %in% missing_genes, ]
paged_table(filtered_by_interest, options = list(rows.print = 15))paged_table(filtered_counts, options = list(rows.print = 15))Below is a heatmap highlighting the expression of our genes of interest across batch, disease group, and affected status.
mat3 <- assay(vsd_limma)[filtered_by_interest$Ensembl_ID, ]
rownames(mat3) <- gene_info$gene_name[match(filtered_by_interest$Ensembl_ID, gene_info$Ensembl_ID)]
df_sub <- as.data.frame(colData(vsd_limma)[, c("Affected", "SubCategory")])
pheatmap(mat3, annotation_col = df_sub, annotation_colors = ann_colors2, fontsize = 4)
Enrichment analysis
res <- gost(query = significant_data$name, organism = "hsapiens", significant = FALSE) # should significant be true or false?
gostplot(res, capped = FALSE, interactive = FALSE)
# Your existing code for obtaining geneList with Ensembl IDs
rownames(res_aff_vs_unaff_df_sub) <- gsub("\\..*", "", rownames(res_aff_vs_unaff_df_sub))
geneList <- res_aff_vs_unaff_df_sub$log2FoldChange
names(geneList) <- rownames(res_aff_vs_unaff_df_sub)
# Initialize BioMart
ensembl <- useMart("ensembl", dataset = "hsapiens_gene_ensembl")
# Convert all Ensembl IDs in geneList to Entrez IDs
attributes <- c("ensembl_gene_id", "entrezgene_id")
filters <- "ensembl_gene_id"
results <- getBM(attributes = attributes, filters = filters, values = names(geneList), mart = ensembl)
# Remove rows with NA or empty Entrez IDs
results <- results[!is.na(results$entrezgene_id) & results$entrezgene_id != "", ]
# Create a new geneList with Entrez IDs
geneList_entrez <- geneList[results$ensembl_gene_id]
names(geneList_entrez) <- results$entrezgene_id
# Filter genes with abs(log2FoldChange) > 2
gene_entrez <- names(geneList_entrez)[abs(geneList_entrez) > 2]
# Run enrichGO with Entrez IDs
ego_cc <- enrichGO(
gene = gene_entrez,
universe = names(geneList_entrez),
OrgDb = org.Hs.eg.db,
ont = "CC",
pAdjustMethod = "BH",
pvalueCutoff = 0.01,
qvalueCutoff = 0.05,
readable = TRUE
)
# Plot results
barplot(ego_cc, showCategory = 10) + ggthemes::theme_clean()
ego_bp <- enrichGO(
gene = gene_entrez,
universe = names(geneList_entrez),
OrgDb = org.Hs.eg.db,
ont = "BP",
pAdjustMethod = "BH",
pvalueCutoff = 0.01,
qvalueCutoff = 0.05,
readable = TRUE
)
# Plot results
barplot(ego_bp, showCategory = 10) + ggthemes::theme_clean()
ego_mf <- enrichGO(
gene = gene_entrez,
universe = names(geneList_entrez),
OrgDb = org.Hs.eg.db,
ont = "MF",
pAdjustMethod = "BH",
pvalueCutoff = 0.01,
qvalueCutoff = 0.05,
readable = TRUE
)
# Plot results
barplot(ego_mf, showCategory = 10) + ggthemes::theme_clean()
top_genes_df <- res_aff_vs_unaff_df_sub[order(-abs(res_aff_vs_unaff_df_sub$log2FoldChange)), ]
top_20_genes <- rownames(top_genes_df)[1:20]
# Enriched via DisGeNet
#edo <- enrichDGN(gene_entrez)
#barplot(edo, showCategory = 20)# upsetplot(ego_mff)# For loop for plot counts
for (ensembl_id in filtered_by_interest$Ensembl_ID) {
d <- plotCounts(dds, gene = ensembl_id, intgroup = "Affected", returnData = TRUE)
gene_name <- filtered_by_interest$gene_name[filtered_by_interest$Ensembl_ID == ensembl_id]
ggplot_box <- ggboxplot(d, x = "Affected", y = "count", add = "jitter",
color = "Affected", palette = c("navy", "red"),
title = gene_name) +
geom_text_repel(aes(label = rownames(d))) +
scale_color_manual(name = "Affected Status",
labels = c("Unaffected", "Affected"),
values = c("navy", "red"))
print(ggplot_box) # Ensure each plot is printed during the loop
ggsave(
filename = paste0(gene_name, "-subsetted-plot-counts.png"),
path = "output/batch-correction-limma/plot-counts",
plot = ggplot_box, dpi = 450
)
}
Save data
save.image(file = "output/subsetted-condition-analysis.RData")
sessionInfo()R version 4.3.2 (2023-10-31)
Platform: x86_64-apple-darwin20 (64-bit)
Running under: macOS Sonoma 14.3.1
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.3-x86_64/Resources/lib/libRlapack.dylib; LAPACK version 3.11.0
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: America/Chicago
tzcode source: internal
attached base packages:
[1] stats4 stats graphics grDevices utils datasets methods
[8] base
other attached packages:
[1] mygene_1.36.0 GenomicFeatures_1.52.2
[3] ggrepel_0.9.4 UpSetR_1.4.0
[5] org.Hs.eg.db_3.17.0 AnnotationDbi_1.62.2
[7] DOSE_3.26.2 clusterProfiler_4.8.3
[9] ggupset_0.3.0.9002 rmarkdown_2.25
[11] ggpubr_0.6.0 plotly_4.10.3
[13] biomaRt_2.56.1 gprofiler2_0.2.2
[15] limma_3.56.2 genefilter_1.82.1
[17] pheatmap_1.0.12 RColorBrewer_1.1-3
[19] DESeq2_1.40.2 SummarizedExperiment_1.30.2
[21] Biobase_2.60.0 MatrixGenerics_1.12.3
[23] matrixStats_1.2.0 GenomicRanges_1.52.1
[25] GenomeInfoDb_1.36.4 IRanges_2.34.1
[27] S4Vectors_0.38.2 BiocGenerics_0.46.0
[29] lubridate_1.9.3 forcats_1.0.0
[31] stringr_1.5.1 dplyr_1.1.4
[33] purrr_1.0.2 readr_2.1.4
[35] tidyr_1.3.0 tibble_3.2.1
[37] ggplot2_3.4.4 tidyverse_2.0.0
[39] workflowr_1.7.1
loaded via a namespace (and not attached):
[1] fs_1.6.3 bitops_1.0-7 enrichplot_1.20.3
[4] HDO.db_0.99.1 httr_1.4.7 tools_4.3.2
[7] backports_1.4.1 utf8_1.2.4 R6_2.5.1
[10] lazyeval_0.2.2 withr_2.5.2 prettyunits_1.2.0
[13] gridExtra_2.3 textshaping_0.3.7 cli_3.6.2
[16] scatterpie_0.2.1 labeling_0.4.3 sass_0.4.8
[19] systemfonts_1.0.5 Rsamtools_2.16.0 yulab.utils_0.1.1
[22] gson_0.1.0 foreign_0.8-86 rstudioapi_0.15.0
[25] RSQLite_2.3.4 generics_0.1.3 gridGraphics_0.5-1
[28] BiocIO_1.10.0 vroom_1.6.5 car_3.1-2
[31] GO.db_3.17.0 Matrix_1.6-4 fansi_1.0.6
[34] abind_1.4-5 lifecycle_1.0.4 whisker_0.4.1
[37] yaml_2.3.8 carData_3.0-5 qvalue_2.32.0
[40] BiocFileCache_2.11.1 grid_4.3.2 blob_1.2.4
[43] promises_1.2.1 crayon_1.5.2 lattice_0.22-5
[46] cowplot_1.1.2 annotate_1.78.0 KEGGREST_1.40.1
[49] pillar_1.9.0 knitr_1.45 fgsea_1.26.0
[52] rjson_0.2.21 codetools_0.2-19 fastmatch_1.1-4
[55] glue_1.6.2 getPass_0.2-4 downloader_0.4
[58] ggfun_0.1.3 data.table_1.14.10 vctrs_0.6.5
[61] png_0.1-8 treeio_1.24.3 gtable_0.3.4
[64] gsubfn_0.7 cachem_1.0.8 xfun_0.41
[67] S4Arrays_1.0.6 tidygraph_1.3.0 survival_3.5-7
[70] nlme_3.1-164 ggtree_3.8.2 bit64_4.0.5
[73] progress_1.2.3 filelock_1.0.3 rprojroot_2.0.4
[76] bslib_0.6.1 rpart_4.1.23 colorspace_2.1-0
[79] DBI_1.1.3 Hmisc_5.1-1 nnet_7.3-19
[82] tidyselect_1.2.0 processx_3.8.3 chron_2.3-61
[85] bit_4.0.5 compiler_4.3.2 curl_5.2.0
[88] git2r_0.33.0 htmlTable_2.4.2 xml2_1.3.6
[91] DelayedArray_0.26.7 shadowtext_0.1.2 rtracklayer_1.60.1
[94] checkmate_2.3.1 scales_1.3.0 callr_3.7.3
[97] rappdirs_0.3.3 digest_0.6.33 XVector_0.40.0
[100] htmltools_0.5.7 pkgconfig_2.0.3 base64enc_0.1-3
[103] highr_0.10 dbplyr_2.4.0 fastmap_1.1.1
[106] rlang_1.1.2 htmlwidgets_1.6.4 ggthemes_5.0.0
[109] farver_2.1.1 jquerylib_0.1.4 jsonlite_1.8.8
[112] BiocParallel_1.34.2 GOSemSim_2.26.1 RCurl_1.98-1.13
[115] magrittr_2.0.3 Formula_1.2-5 GenomeInfoDbData_1.2.10
[118] ggplotify_0.1.2 patchwork_1.1.3 munsell_0.5.0
[121] Rcpp_1.0.11 proto_1.0.0 ape_5.7-1
[124] viridis_0.6.4 sqldf_0.4-11 stringi_1.8.3
[127] ggraph_2.1.0 zlibbioc_1.46.0 MASS_7.3-60
[130] plyr_1.8.9 parallel_4.3.2 Biostrings_2.68.1
[133] graphlayouts_1.0.2 splines_4.3.2 hms_1.1.3
[136] locfit_1.5-9.8 ps_1.7.5 igraph_1.6.0
[139] ggsignif_0.6.4 reshape2_1.4.4 XML_3.99-0.16
[142] evaluate_0.23 tzdb_0.4.0 tweenr_2.0.2
[145] httpuv_1.6.13 polyclip_1.10-6 ggforce_0.4.1
[148] broom_1.0.5 xtable_1.8-4 restfulr_0.0.15
[151] tidytree_0.4.6 rstatix_0.7.2 later_1.3.2
[154] ragg_1.2.7 viridisLite_0.4.2 aplot_0.2.2
[157] memoise_2.0.1 GenomicAlignments_1.36.0 cluster_2.1.6
[160] timechange_0.2.0