1 Introduction

This tutorial demonstrates how to visualize and interpret the results from a GSFA run.

The results are a bit different from what was reported in the biorxiv version of GSFA manuscript due to slight changes in random sampling and calibration of negative control effects.

We have described how to run GSFA on LUHMES CROP-seq data here.

To recapitulate, the processed dataset consists of 8708 neural progenitor cells that belong to one of the 15 perturbation conditions (CRISPR knock-down of 14 neurodevelopmental genes, and negative control). Top 6000 genes ranked by deviance statistics were kept. And GSFA was performed on the data with 20 factors specified.

1.1 Load necessary packages and data

library(data.table)
library(Matrix)
library(tidyverse)
library(ggplot2)
theme_set(theme_bw() + theme(plot.title = element_text(size = 14, hjust = 0.5),
                             axis.title = element_text(size = 14),
                             axis.text = element_text(size = 12),
                             legend.title = element_text(size = 13),
                             legend.text = element_text(size = 12),
                             panel.grid.minor = element_blank())
)
library(gridExtra)
library(ComplexHeatmap)
library(kableExtra)
library(WebGestaltR)

source("/project2/xinhe/yifan/Factor_analysis/analysis_website_for_Kevin/scripts/plotting_functions.R")
wkdir <- "/project2/xinhe/yifan/Factor_analysis/LUHMES/"
result_folder <- "gsfa_output_detect_01/use_negctrl/"

The first thing we need is the output of GSFA fit_gsfa_multivar() run. The lighter version containing just the posterior mean estimates and LFSR of perturbation-gene effects is enough.

fit <- readRDS(paste0(wkdir, result_folder,
                      "All.gibbs_obj_k20.svd_negctrl.seed_14314.light.rds"))
gibbs_PM <- fit$posterior_means
lfsr_mat <- fit$lfsr[, -ncol(fit$lfsr)]
total_effect <- fit$total_effect[, -ncol(fit$total_effect)]
KO_names <- colnames(lfsr_mat)

We also need the cell by perturbation matrix which was used as input \(G\) for GSFA.

metadata <- readRDS(paste0(wkdir, "processed_data/merged_metadata.rds"))
G_mat <- metadata[, 4:18]

Finally, we load the mapping from gene name to ENSEMBL ID for all 6k genes used in GSFA, as well as selected neuronal marker genes. This is specific to this study and analysis.

feature.names <- data.frame(fread(paste0(wkdir, "GSE142078_raw/GSM4219576_Run2_genes.tsv.gz"),
                                  header = FALSE), stringsAsFactors = FALSE)
genes_df <- feature.names[match(rownames(lfsr_mat), feature.names$V1), ]
names(genes_df) <- c("ID", "Name")
interest_df <- readRDS(paste0(wkdir, "processed_data/selected_neuronal_markers.rds"))

2 Factor ~ Perturbation Association

2.1 Perturbation effects on factors

First of all, we look at the estimated effects of gene perturbations on factors inferred by GSFA.

We found that targeting of 7 genes, ADNP, ARID1B, ASH1L, CHD2, DYRK1A, PTEN, and SETD5, has significant effects (PIP > 0.95) on at least 1 of the 20 inferred factors.

All targets and factors (Figure S6A):

dotplot_beta_PIP(t(gibbs_PM$Gamma_pm), t(gibbs_PM$beta_pm),
                 marker_names = KO_names,
                 reorder_markers = c(KO_names[KO_names!="Nontargeting"], "Nontargeting"),
                 inverse_factors = F) +
  coord_flip()

## Similar visualization using GSFA built-in functions:
GSFA::dotplot_beta_PIP(fit,
                       target_names = KO_names,
                       reorder_targets = c(KO_names[KO_names!="Nontargeting"], "Nontargeting"))

Here is a closer look at the estimated effects of selected perturbations on selected factors (Figure 5A):

targets <- c("ADNP", "ARID1B", "ASH1L", "CHD2", "DYRK1A", "PTEN", "SETD5")
complexplot_perturbation_factor(gibbs_PM$Gamma_pm[-nrow(gibbs_PM$Gamma_pm), ],
                                gibbs_PM$beta_pm[-nrow(gibbs_PM$beta_pm), ],
                                marker_names = KO_names,
                                reorder_markers = targets,
                                reorder_factors = c(6, 9, 15))

## Similar visualization using GSFA built-in functions:
GSFA::dotplot_beta_PIP(fit,
                       target_names = KO_names,
                       reorder_targets = targets, reorder_factors = c(6, 9, 15))

2.2 Factor-perturbation association p values

We can also assess the correlations between each pair of perturbation and inferred factor.
The distribution of correlation p values show significant signals.

gibbs_res_tb <- make_gibbs_res_tb(gibbs_PM, G_mat, compute_pve = F)
heatmap_matrix <- gibbs_res_tb %>% select(starts_with("pval"))
rownames(heatmap_matrix) <- 1:nrow(heatmap_matrix)
colnames(heatmap_matrix) <- colnames(G_mat)

summ_pvalues(unlist(heatmap_matrix),
             title_text = "GSFA\n(15 Targets x 20 Factors)")

3 Factor Interpretation

3.1 Correlation within factors

Since the GSFA model does not enforce orthogonality among factors, we first inspect the pairwise correlation within them to see if there is any redundancy. As we can see below, the inferred factors are mostly independent of each other.

plot_pairwise.corr_heatmap(input_mat_1 = gibbs_PM$Z_pm,
                           corr_type = "pearson",
                           name_1 = "Pairwise correlation within factors (Z)",
                           label_size = 10)

plot_pairwise.corr_heatmap(input_mat_1 = (gibbs_PM$F_pm > 0.95) * 1,
                           corr_type = "jaccard",
                           name_1 = "Pairwise correlation within \nbinarized gene loadings (F_pm > 0.95)",
                           label_size = 10)

3.2 Gene loading in factors

To understand these latent factors, we inspect the loadings (weights) of several marker genes for neuron maturation and differentiation in them.

protein_name gene_name type gene_ID
TP53 TP53 Cell proliferation ENSG00000141510
CDK4 CDK4 Cell proliferation ENSG00000135446
Nestin NES Neural progenitor cell ENSG00000132688
STMN2 STMN2 Mature neuron ENSG00000104435
MAP2 MAP2 Mature neuron ENSG00000078018
DPYSL3 DPYSL3 Mature neuron ENSG00000113657
MAP1B MAP1B Mature neuron ENSG00000131711
CRABP2 CRABP2 Mature neuron ENSG00000143320
NEFL NEFL Mature neuron ENSG00000277586
ZEB2 ZEB2 Mature neuron ENSG00000169554
ITM2C ITM2C Negative regulation of neuron projection ENSG00000135916
CNTN2 CNTN2 Negative regulation of neuron projection ENSG00000184144
DRAXIN DRAXIN Negative regulation of neuron projection ENSG00000162490
HDAC2 HDAC2 Negative regulation of neuron projection ENSG00000196591

We visualize both the gene PIPs (dot size) and gene weights (dot color) in all factors (Figure S6B):

plt <- complexplot_gene_factor(genes_df, interest_df, gibbs_PM$F_pm, gibbs_PM$W_pm)

plt

A closer look at some factors that are associated with perturbations (Figure 5C):

complexplot_gene_factor(genes_df, interest_df, gibbs_PM$F_pm, gibbs_PM$W_pm,
                        reorder_factors = c(6, 9, 15))

3.3 GO enrichment analysis in factors

To further characterize these latent factors, we perform GO (gene ontology) enrichment analysis of genes loaded on the factors using WebGestalt.

Foreground genes: genes w/ non-zero loadings in each factor (gene PIP > 0.95);
Background genes: all 6000 genes used in GSFA;
Statistical test: hypergeometric test (over-representation test);
Gene sets: GO Slim “Biological Process” (non-redundant).

## The "WebGestaltR" tool needs Internet connection.
enrich_db <- "geneontology_Biological_Process_noRedundant"
PIP_mat <- gibbs_PM$F_pm
enrich_res_by_factor <- list()
for (i in 1:ncol(PIP_mat)){
  enrich_res_by_factor[[i]] <- 
    WebGestaltR::WebGestaltR(enrichMethod = "ORA",
                             organism = "hsapiens",
                             enrichDatabase = enrich_db,
                             interestGene = genes_df[PIP_mat[, i] > 0.95, ]$ID,
                             interestGeneType = "ensembl_gene_id",
                             referenceGene = genes_df$ID,
                             referenceGeneType = "ensembl_gene_id",
                             isOutput = F)
}

Several GO “biological process” terms related to neuronal development are enriched in factors 4, 9, and 16 (Figure 5D):

factor_indx <- 6
terms_of_interest <- c("regulation of ion transmembrane transport",
                       "regulation of trans-synaptic signaling",
                       "axon development",
                       "regulation of neuron projection development")
barplot_top_enrich_terms(enrich_res_by_factor[[factor_indx]],
                         terms_of_interest = terms_of_interest,
                         str_wrap_length = 35, pval_max = 8, FC_max = 6) +
  labs(title = paste0("Factor ", factor_indx),
       x = "Fold of enrichment")

factor_indx <- 9
terms_of_interest <- c("actin filament organization",
                       "cell fate commitment",
                       "axon development",
                       "regulation of cell morphogenesis")
barplot_top_enrich_terms(enrich_res_by_factor[[factor_indx]],
                         terms_of_interest = terms_of_interest,
                         str_wrap_length = 35, pval_max = 8, FC_max = 6) +
  labs(title = paste0("Factor ", factor_indx),
       x = "Fold of enrichment")

factor_indx <- 15
terms_of_interest <- c("developmental growth involved in morphogenesis",
                       "axon development")
barplot_top_enrich_terms(enrich_res_by_factor[[factor_indx]],
                         terms_of_interest = terms_of_interest, 
                         str_wrap_length = 35, pval_max = 8, FC_max = 6) +
  labs(title = paste0("Factor ", factor_indx),
       x = "Fold of enrichment")

4 DEG Interpretation

In GSFA, differential expression analysis can be performed based on the LFSR method. Here we evaluate the specific downstream genes affected by the perturbations detected by GSFA.

We also performed several other differential expression methods for comparison, including scMAGeCK-LR, MAST, and DESeq.

4.1 Number of DEGs detected by different methods

fdr_cutoff <- 0.05
lfsr_cutoff <- 0.05
Number of DEGs detected by GSFA under each perturbation:
KO ADNP ARID1B ASH1L CHD2 CHD8
Num_genes 795 310 322 756 0
KO CTNND2 DYRK1A HDAC5 MECP2 MYT1L
Num_genes 0 23 0 0 0
KO Nontargeting POGZ PTEN RELN SETD5
Num_genes 0 0 895 0 466
guides <- KO_names[KO_names!="Nontargeting"]
deseq_list <- list()
for (m in guides){
  fname <- paste0(wkdir, "processed_data/DESeq2/dev_top6k_negctrl/gRNA_", 
                  m, ".dev_res_top6k.vs_negctrl.rds")
  res <- readRDS(fname)
  res <- as.data.frame(res@listData, row.names = res@rownames)
  res$geneID <- rownames(res)
  res <- res %>% dplyr::rename(FDR = padj, PValue = pvalue)
  deseq_list[[m]] <- res
}
deseq_signif_counts <- sapply(deseq_list, function(x){filter(x, FDR < fdr_cutoff) %>% nrow()})
mast_list <- list()
for (m in guides){
  fname <- paste0(wkdir, "processed_data/MAST/dev_top6k_negctrl/gRNA_", 
                  m, ".dev_res_top6k.vs_negctrl.rds")
  tmp_df <- readRDS(fname)
  tmp_df$geneID <- rownames(tmp_df)
  tmp_df <- tmp_df %>% dplyr::rename(FDR = fdr, PValue = pval)
  mast_list[[m]] <- tmp_df
}
mast_signif_counts <- sapply(mast_list, function(x){filter(x, FDR < fdr_cutoff) %>% nrow()})
scmageck_res <- readRDS(paste0(wkdir, "scmageck/scmageck_lr.LUHMES.dev_res_top_6k.rds"))
colnames(scmageck_res$fdr)[colnames(scmageck_res$fdr) == "NegCtrl"] <- "Nontargeting"
scmageck_signif_counts <- colSums(scmageck_res$fdr[, KO_names] < fdr_cutoff)
scmageck_signif_counts <- scmageck_signif_counts[names(scmageck_signif_counts) != "Nontargeting"]
sceptre_res <- readRDS("/project2/xinhe/kevinluo/GSFA/sceptre_analysis/LUHMES_data_updated/sceptre_output/sceptre.result.rds")
sceptre_count_df <- data.frame(matrix(nrow = length(guides), ncol = 2))
colnames(sceptre_count_df) <- c("target", "num_DEG")
for (i in 1:length(guides)){
  sceptre_count_df$target[i] <- guides[i]
  tmp_pval <- sceptre_res %>% filter(gRNA_id == guides[i]) %>% pull(p_value)
  tmp_fdr <- p.adjust(tmp_pval, method = "fdr")
  sceptre_count_df$num_DEG[i] <- sum(tmp_fdr < 0.05)
}
dge_comparison_df <- data.frame(Perturbation = guides,
                                GSFA = lfsr_signif_num[guides],
                                scMAGeCK = scmageck_signif_counts,
                                DESeq2 = deseq_signif_counts,
                                MAST = mast_signif_counts,
                                SCEPTRE = sceptre_count_df$num_DEG)
# dge_comparison_df$Perturbation[dge_comparison_df$Perturbation == "Nontargeting"] <- "NegCtrl"

Number of DEGs detected under each perturbation using 4 different methods (Figure 5E):

Compared with other differential expression analysis methods, GSFA detected the most DEGs for all 7 gene targets that have significant effects.

dge_plot_df <- reshape2::melt(dge_comparison_df, id.var = "Perturbation",
                              variable.name = "Method", value.name = "Num_DEGs")
dge_plot_df$Perturbation <- factor(dge_plot_df$Perturbation,
                                   levels = guides)
                                   # levels = c("NegCtrl", KO_names[KO_names!="Nontargeting"]))
dge_plt <- ggplot(dge_plot_df,
                  aes(x = Perturbation, y = Num_DEGs+1, fill = Method)) +
  geom_bar(position = "dodge", stat = "identity") +
  geom_text(aes(label = Num_DEGs), position=position_dodge(width=0.9), vjust=-0.25) +
  scale_y_log10() +
  scale_fill_brewer(palette = "Set2") +
  labs(x = "Target gene",
       y = "Number of DEGs",
       title = "Number of DEGs detected by different methods") +
  theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
        legend.position = "bottom",
        legend.text = element_text(size = 13))
dge_plt

4.2 Perturbation effects on marker genes

To better understand the functions of these 7 target genes, we examined their effects on marker genes for neuron maturation and differentiation.

4.2.1 GSFA

Here are the summarized effects of perturbations on marker genes estimated by GSFA (Figure 5G).

As we can see, knockdown of ADNP, ASH1L, CHD2, and DYRK1A has mostly negative effects on mature neuronal markers, and positive effects on negative regulators of neuron projection, indicating delayed neuron maturation.

Knockdown of PTEN and SETD5 has the opposite pattern, which indicates accelerated neuron maturation.

targets <- c("ADNP", "ARID1B", "ASH1L", "CHD2", "DYRK1A", "PTEN", "SETD5")
complexplot_gene_perturbation(genes_df, interest_df,
                              targets = targets,
                              lfsr_mat = lfsr_mat,
                              effect_mat = total_effect)

## Similar visualization using GSFA built-in functions:
GSFA::dotplot_total_effect(fit,
                           gene_indices = match(interest_df$gene_ID, rownames(lfsr_mat)),
                           gene_names = interest_df$gene_name,
                           reorder_targets = targets, 
                           plot_max_score = 0.2)

4.2.2 scMAGeCK

Here are scMAGeCK estimated effects of perturbations on marker genes (Figure 5H):

score_mat <- scmageck_res$score
fdr_mat <- scmageck_res$fdr
complexplot_gene_perturbation(genes_df, interest_df,
                              targets = targets,
                              lfsr_mat = fdr_mat, lfsr_name = "FDR",
                              effect_mat = score_mat, effect_name = "scMAGeCK\nselection score", 
                              score_break = c(-0.2, 0, 0.2),
                              color_break = c("blue3", "grey90", "red3"))

4.2.3 DESeq2

FC_mat <- matrix(nrow = nrow(interest_df), ncol = length(targets))
rownames(FC_mat) <- interest_df$gene_name
colnames(FC_mat) <- targets
fdr_mat <- FC_mat
for (m in targets){
  FC_mat[, m] <- deseq_list[[m]]$log2FoldChange[match(interest_df$gene_ID,
                                                      deseq_list[[m]]$geneID)]
  fdr_mat[, m] <- deseq_list[[m]]$FDR[match(interest_df$gene_ID, deseq_list[[m]]$geneID)]
}

Here are DESeq2 estimated effects of perturbations on marker genes (Figure S6D):

plt <- complexplot_gene_perturbation(genes_df, interest_df,
                              targets = targets,
                              lfsr_mat = fdr_mat, lfsr_name = "FDR",
                              effect_mat = FC_mat, effect_name = "DESeq2 log2FC",
                              score_break = c(-0.4, 0, 0.4),
                              color_break = c("blue3", "grey90", "red3"))

4.2.4 MAST

FC_mat <- matrix(nrow = nrow(interest_df), ncol = length(targets))
rownames(FC_mat) <- interest_df$gene_name
colnames(FC_mat) <- targets
fdr_mat <- FC_mat
for (m in targets){
  FC_mat[, m] <- mast_list[[m]]$logFC[match(interest_df$gene_ID, mast_list[[m]]$geneID)]
  fdr_mat[, m] <- mast_list[[m]]$FDR[match(interest_df$gene_ID, mast_list[[m]]$geneID)]
}

MAST estimated effects of perturbations on marker genes (Figure S6E):

plt <- complexplot_gene_perturbation(genes_df, interest_df,
                              targets = targets,
                              lfsr_mat = fdr_mat, lfsr_name = "FDR",
                              effect_mat = FC_mat, effect_name = "MAST logFC",
                              score_break = c(-0.4, 0, 0.4),
                              color_break = c("blue3", "grey90", "red3"))

4.2.5 SCEPTRE

FC_mat <- matrix(nrow = nrow(interest_df), ncol = length(targets))
rownames(FC_mat) <- interest_df$gene_name
colnames(FC_mat) <- targets
fdr_mat <- FC_mat
for (m in targets){
  sceptre_tmp_res <- sceptre_res %>% filter(gRNA_id == m)
  tmp_pval <- sceptre_tmp_res %>% pull(p_value)
  sceptre_tmp_res$FDR <- p.adjust(tmp_pval, method = "fdr")
  FC_mat[, m] <- sceptre_tmp_res$log_fold_change[match(interest_df$gene_ID, 
                                                       sceptre_tmp_res$gene_id)]
  fdr_mat[, m] <- sceptre_tmp_res$FDR[match(interest_df$gene_ID, 
                                            sceptre_tmp_res$gene_id)]
}

SCEPTRE estimated effects of perturbations on marker genes (Figure S6F):

plt <- complexplot_gene_perturbation(genes_df, interest_df,
                              targets = targets,
                              lfsr_mat = fdr_mat, lfsr_name = "FDR",
                              effect_mat = FC_mat, effect_name = "SCEPTRE logFC",
                              score_break = c(-0.4, 0, 0.4),
                              color_break = c("blue3", "grey90", "red3"))

4.3 GO enrichment in DEGs

We further examine these DEGs for enrichment of relevant biological processes through GO enrichment analysis.

Foreground genes: Genes w/ GSFA LFSR < 0.05 under each perturbation;
Background genes: all 6000 genes used in GSFA;
Statistical test: hypergeometric test (over-representation test);
Gene sets: Gene ontology “Biological Process” (non-redundant).

## The "WebGestaltR" tool needs Internet connection.
targets <- names(lfsr_signif_num)[lfsr_signif_num > 0]
enrich_db <- "geneontology_Biological_Process_noRedundant"
enrich_res <- list()
for (i in targets){
  print(i)
  interest_genes <- genes_df %>% mutate(lfsr = lfsr_mat[, i]) %>%
    filter(lfsr < lfsr_cutoff) %>% pull(ID)
  enrich_res[[i]] <- 
    WebGestaltR::WebGestaltR(enrichMethod = "ORA",
                             organism = "hsapiens",
                             enrichDatabase = enrich_db,
                             interestGene = interest_genes,
                             interestGeneType = "ensembl_gene_id",
                             referenceGene = genes_df$ID,
                             referenceGeneType = "ensembl_gene_id",
                             isOutput = F)
}
signif_GO_list <- list()
for (i in names(enrich_res)) {
  signif_GO_list[[i]] <- enrich_res[[i]] %>%
    dplyr::filter(FDR < 0.05) %>%
    dplyr::select(geneSet, description, size, enrichmentRatio, pValue) %>%
    mutate(target = i)
}
signif_term_df <- do.call(rbind, signif_GO_list) %>%
  group_by(geneSet, description, size) %>%
  summarise(pValue = min(pValue)) %>%
  ungroup()

abs_FC_colormap <- circlize::colorRamp2(breaks = c(0, 3, 6),
                                        colors = c("grey95", "#77d183", "#255566"))
targets <- names(enrich_res)
enrich_table <- data.frame(matrix(nrow = nrow(signif_term_df),
                                  ncol = length(targets)),
                           row.names = signif_term_df$geneSet)
colnames(enrich_table) <- targets
for (i in 1:ncol(enrich_table)){
  m <- colnames(enrich_table)[i]
  enrich_df <- enrich_res[[m]] %>% dplyr::filter(FDR < 0.05)
  enrich_table[enrich_df$geneSet, i] <- enrich_df$enrichmentRatio
}
rownames(enrich_table) <- signif_term_df$description

Here are selected GO “biological process” terms and their folds of enrichment in DEGs detected by GSFA (Figure S6F):
(In the code below, we omitted the content in terms_of_interest_df as one can subset the enrich_table with any terms of their choice.)

interest_enrich_table <- enrich_table[terms_of_interest_df$description, ]
interest_enrich_table[is.na(interest_enrich_table)] <- 0
# Convert only the first letter of each GO term to upper case.
rownames(interest_enrich_table) <-
  str_replace(rownames(interest_enrich_table),  "^\\w{1}", toupper)

map <- Heatmap(abs(interest_enrich_table),
               name = "Fold of enrichment",
               col = abs_FC_colormap,
               na_col = "grey90",
               row_title = NULL, column_title = NULL,
               cluster_rows = F, cluster_columns = F,
               show_row_dend = F, show_column_dend = F,
               show_heatmap_legend = T,
               row_names_gp = gpar(fontsize = 10.5),
               column_names_rot = 45,
               column_names_side = "top",
               width = unit(6, "cm"))
draw(map, heatmap_legend_side = "bottom")

Similar GO enrichment analyses were performed on DEGs detected by alternative methods (FDR < 0.05).
Below summarizes the number of significant GO terms in DEGs by each method:

enrich_thres <- 0.05
enrich_dir <- paste0(wkdir, result_folder, "WebGestalt_ORA/")
enrich_res <- readRDS(paste0(enrich_dir,
                             "GO_enrich_in_GSFA_DEGs.rds"))
gsfa_term_count <-
  sapply(enrich_res, function(x){ x %>% dplyr::filter(FDR < enrich_thres) %>% nrow()})
# Skip low count target genes to simplify plot.
gsfa_term_count <- gsfa_term_count[gsfa_term_count > 10]

term_count_df <- data.frame(Perturbation = names(gsfa_term_count),
                            GSFA = as.numeric(gsfa_term_count))
for (method in c("scmageck", "DESeq2", "MAST", "sceptre")) {
  enrich_res <- readRDS(paste0(enrich_dir,
                               method, "_DEGs_fdr_0.05.go_bp_thres_1.rds"))
  term_count <-
    sapply(enrich_res, function(x){ x %>% dplyr::filter(FDR < enrich_thres) %>% nrow()})
  term_count_df[[method]] <- term_count[term_count_df$Perturbation]
}
names(term_count_df)[c(3, 6)] <- c("scMAGeCK", "SCEPTRE")
term_plot_df <- term_count_df %>%
  tidyr::pivot_longer(cols = !Perturbation,
                      names_to = "Method", values_to = "Num_GO_terms") %>%
  dplyr::mutate(Method = factor(Method, levels = names(term_count_df)[-1]))
term_plot_df$Num_GO_terms <-
  tidyr::replace_na(term_plot_df$Num_GO_terms, 0)

num_terms_plt <-
  ggplot(term_plot_df, aes(x = Perturbation, y = Num_GO_terms, fill = Method)) +
  geom_bar(position = "dodge", stat = "identity") +
  geom_text(aes(label = Num_GO_terms),
            position = position_dodge(width = 0.9), vjust = -0.25) +
  scale_fill_brewer(palette = "Set2") +
  labs(x = "Target gene",
       y = "Number of enriched GO terms",
       title = "Number of GO terms enriched in DEGs detected by different methods") +
  theme(axis.text.x = element_text(angle = 45, hjust = 1, size = 12),
        legend.text = element_text(size = 13))
num_terms_plt

5 Session Information

sessionInfo()
R version 4.2.0 (2022-04-22)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: CentOS Linux 7 (Core)

Matrix products: default
BLAS/LAPACK: /software/openblas-0.3.13-el7-x86_64/lib/libopenblas_haswellp-r0.3.13.so

locale:
 [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C         LC_TIME=C           
 [4] LC_COLLATE=C         LC_MONETARY=C        LC_MESSAGES=C       
 [7] LC_PAPER=C           LC_NAME=C            LC_ADDRESS=C        
[10] LC_TELEPHONE=C       LC_MEASUREMENT=C     LC_IDENTIFICATION=C 

attached base packages:
[1] grid      stats     graphics  grDevices utils     datasets  methods  
[8] base     

other attached packages:
 [1] lattice_0.20-45       WebGestaltR_0.4.4     kableExtra_1.3.4     
 [4] ComplexHeatmap_2.12.1 gridExtra_2.3         forcats_0.5.1        
 [7] stringr_1.4.0         dplyr_1.0.9           purrr_0.3.4          
[10] readr_2.1.2           tidyr_1.2.0           tibble_3.1.7         
[13] ggplot2_3.3.6         tidyverse_1.3.2       Matrix_1.4-1         
[16] data.table_1.14.2    

loaded via a namespace (and not attached):
 [1] matrixStats_0.62.0  fs_1.5.2            lubridate_1.8.0    
 [4] webshot_0.5.3       doParallel_1.0.17   RColorBrewer_1.1-3 
 [7] httr_1.4.3          doRNG_1.8.2         tools_4.2.0        
[10] backports_1.4.1     bslib_0.3.1         utf8_1.2.2         
[13] R6_2.5.1            DBI_1.1.2           BiocGenerics_0.42.0
[16] colorspace_2.0-3    GetoptLong_1.0.5    withr_2.5.0        
[19] tidyselect_1.1.2    compiler_4.2.0      cli_3.3.0          
[22] rvest_1.0.2         xml2_1.3.3          labeling_0.4.2     
[25] sass_0.4.1          scales_1.2.0        apcluster_1.4.10   
[28] systemfonts_1.0.4   digest_0.6.29       R.utils_2.11.0     
[31] svglite_2.1.0       rmarkdown_2.14      pkgconfig_2.0.3    
[34] htmltools_0.5.2     highr_0.9           dbplyr_2.1.1       
[37] fastmap_1.1.0       rlang_1.0.5         GlobalOptions_0.1.2
[40] readxl_1.4.0        rstudioapi_0.13     farver_2.1.0       
[43] shape_1.4.6         jquerylib_0.1.4     generics_0.1.2     
[46] jsonlite_1.8.0      R.oo_1.24.0         googlesheets4_1.0.0
[49] magrittr_2.0.3      Rcpp_1.0.8.3        munsell_0.5.0      
[52] S4Vectors_0.34.0    fansi_1.0.3         R.methodsS3_1.8.1  
[55] lifecycle_1.0.1     whisker_0.4         stringi_1.7.6      
[58] yaml_2.3.5          plyr_1.8.7          parallel_4.2.0     
[61] crayon_1.5.1        haven_2.5.0         pander_0.6.5       
[64] circlize_0.4.15     hms_1.1.1           knitr_1.39         
[67] pillar_1.7.0        igraph_1.3.1        rjson_0.2.21       
[70] rngtools_1.5.2      reshape2_1.4.4      codetools_0.2-18   
[73] stats4_4.2.0        reprex_2.0.1        glue_1.6.2         
[76] evaluate_0.15       modelr_0.1.8        png_0.1-7          
[79] vctrs_0.4.1         tzdb_0.3.0          foreach_1.5.2      
[82] cellranger_1.1.0    gtable_0.3.0        clue_0.3-61        
[85] assertthat_0.2.1    xfun_0.30           broom_0.8.0        
[88] viridisLite_0.4.0   googledrive_2.0.0   gargle_1.2.0       
[91] iterators_1.0.14    IRanges_2.30.0      cluster_2.1.3      
[94] ellipsis_0.3.2