Figure 1
UMAP on 7 MOFA factors
## UMAP
plot.umap.data <- plot_dimred(mofa, method="UMAP", color_by = "condition",stroke = 0.001, dot_size =1, alpha = 0.2, return_data = T)
plot.umap.all <- ggplot(plot.umap.data, aes(x=x, y = y, fill = color_by))+
geom_point(size = 0.8, alpha = 0.6, shape = 21, stroke = 0) +
theme_half_open() +
scale_fill_manual(values = colorgradient6_manual, labels = c(labels), name = "Time aIg",)+
theme(legend.position="none")+
scale_x_reverse()+
scale_y_reverse()+
add.textsize +
labs(title = "UMAP on MOFA+ factors shows minute and \nhour timescale signal transductions", x = "UMAP 1", y = "UMAP 2")
## UMAP legend
legend.umap <- get_legend( ggplot(plot.umap.data, aes(x=x, y = y, fill = color_by))+
geom_point(size = 2, alpha = 1, shape = 21, stroke = 0) +
theme_half_open() +
scale_fill_manual(values = colorgradient6_manual, labels = c(labels), name = "Time aIg \n(minutes)",)+
add.textsize +
labs(title = "UMAP on MOFA+ factors shows minute and \nhour timescale signal transductions", x = "UMAP 1", "y = UMAP 2"))
legend.umap <- as_ggplot(legend.umap)plot_fig1_umap <- plot_grid(plot.umap.all,legend.umap, labels = c(""), label_size = 10, ncol = 2, rel_widths = c(1, 0.2))
ggsave(plot_fig1_umap, filename = "output/paper_figures/Fig1_UMAP.pdf", width = 68, height = 62, units = "mm", dpi = 300, useDingbats = FALSE)
ggsave(plot_fig1_umap, filename = "output/paper_figures/Fig1_UMAP.png", width = 68, height = 62, units = "mm", dpi = 300)
plot_fig1_umapFigure 1. UMAP of 7 MOFA factors, integrating phospho-protein and RNA measurements
Suppl PCA
seu_combined_selectsamples <- RunPCA(seu_combined_selectsamples,assay = "SCT.RNA", features = genes.variable, verbose = FALSE, ndims.print = 0, reduction.name = "pca.RNA")
seu_combined_selectsamples <- RunPCA(seu_combined_selectsamples, assay = "PROT", features = proteins.all, verbose = FALSE, ndims.print = 0,reduction.name = "pca.PROT")## PCA analysis
plot.PCA.RNA <- DimPlot(seu_combined_selectsamples, reduction = "pca.RNA", group.by = "condition", pt.size =0.2) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg",)+
labs(title = "RNA PCA separates cells \nat hour timescale", x= "RNA PC 1", y = " RNA PC 2") +
add.textsize +
theme(legend.position = "none")
plot.PCA.PROT <- DimPlot(seu_combined_selectsamples, reduction = "pca.PROT", group.by = "condition", pt.size = 0.2) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg",)+
labs(title = "(Phospho)protein PCA separates cells\nat minutes timescale", x= "Protein PC 1", y = "Protein PC 2") +
add.textsize +
# scale_x_reverse()+
theme(legend.position = "none")
legend <- get_legend(DimPlot(seu_combined_selectsamples, reduction = "pca.RNA", group.by = "condition", pt.size =0.1) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg",)+
add.textsize)
legend <- as_ggplot(legend)
PCA.PROTPC1.data <- data.frame(rank = 1:80,
protein = names(sort(seu_combined_selectsamples@reductions$pca.PROT@feature.loadings[,1])),
weight.PC1 = sort(seu_combined_selectsamples@reductions$pca.PROT@feature.loadings[,1]),
highlights = c(names(sort(seu_combined_selectsamples@reductions$pca.PROT@feature.loadings[,1]))[1:4],rep("",76))
)
PCA.PROTPC1.data <- PCA.PROTPC1.data %>%
mutate(highlights = as.character(highlights)) %>%
mutate(highlights = case_when(as.character(highlights) == "p-Syk" ~ "p-SYK",
as.character(highlights) == "p-Src" ~ "p-SRC",
TRUE ~ highlights))
plot.PCA.PROTweightsPC1 <- ggplot(PCA.PROTPC1.data, aes(x=weight.PC1, y = rank, label = highlights)) +
geom_point(size=0.1) +
labs(title = "Top 4 protein loadings \n", x= "Weight Protein PC1") +
geom_point(color = ifelse(PCA.PROTPC1.data$highlights == "", "grey50", "red")) +
geom_text_repel(size = 2, segment.size = 0.25)+
theme_half_open()+
add.textsize +
theme(axis.title.y=element_blank(),
axis.text.y=element_blank(),
axis.ticks.y=element_blank()
) +
scale_x_reverse()+
scale_y_reverse()
PCA.RNAPC1.data <- data.frame(rank = 1:length(genes.variable),
RNA = names(sort(seu_combined_selectsamples@reductions$pca.RNA@feature.loadings[,1])),
weight.PC1 = sort(seu_combined_selectsamples@reductions$pca.RNA@feature.loadings[,1]),
highlights = c(rep("",(length(genes.variable)-4)),names(sort(seu_combined_selectsamples@reductions$pca.RNA@feature.loadings[,1]))[(length(genes.variable)-3):length(genes.variable)])
)
## Loadings RNA
plot.PCA.RNAweightsPC1 <- ggplot(PCA.RNAPC1.data, aes(x=weight.PC1, y = rank, label = highlights)) +
geom_point(size=0.1) +
labs(title = "Top 4 RNA loadings \n", x= "Weight RNA PC1") +
geom_point(color = ifelse(PCA.RNAPC1.data$highlights == "", "grey50", "red")) +
geom_text_repel(size = 2, segment.size = 0.25)+
theme_half_open()+
add.textsize +
theme(axis.title.y=element_blank(),
axis.text.y=element_blank(),
axis.ticks.y=element_blank()
)
## ridgeplots
PC1.data <- data.frame(sample = rownames(seu_combined_selectsamples@reductions$pca.PROT@cell.embeddings),
PC1_PROT = seu_combined_selectsamples@reductions$pca.PROT@cell.embeddings[,1],
PC1_RNA = seu_combined_selectsamples@reductions$pca.RNA@cell.embeddings[,1]) %>%
left_join(meta.allcells)
plot_ridge_PC1Prot <- ggplot(PC1.data, aes(x = PC1_PROT, y = condition, fill = condition)) +
scale_fill_manual(values = colorgradient6_manual, labels = c(labels), name = "Time aIg \n(minutes)")+
geom_density_ridges2() +
scale_y_discrete(labels = labels, name = "Time aIg (minutes)")+
scale_x_continuous(name = "PC1 Proteins") +
theme_half_open() +
add.textsize+
theme(legend.position = "none")
plot_ridge_PC1RNA <- ggplot(PC1.data, aes(x = PC1_RNA, y = condition, fill = condition)) +
scale_fill_manual(values = colorgradient6_manual, labels = c(labels), name = "Time aIg \n(minutes)")+
geom_density_ridges2() +
scale_y_discrete(labels = labels, name = "Time aIg (minutes)")+
scale_x_continuous(name = "PC1 RNA") +
theme_half_open() +
add.textsize+
theme(legend.position = "none")Fig1.pca <- plot_grid(plot.PCA.PROT,plot.PCA.RNA,legend, plot_ridge_PC1Prot, plot_ridge_PC1RNA,NULL, plot.PCA.PROTweightsPC1, plot.PCA.RNAweightsPC1, labels = c(panellabels[1:2], "",panellabels[3:4],"",panellabels[5:7]), label_size = 10, ncol = 3, rel_widths = c(0.9,0.9,0.2,0.9,0.9), rel_heights = c(1,0.8,1.3))
ggsave(filename = "output/paper_figures/Suppl_PCA_aIg.pdf", width = 143, height = 170, units = "mm", dpi = 300, useDingbats = FALSE)
ggsave(filename = "output/paper_figures/Suppl_PCA_aIg.png", width = 143, height = 170, units = "mm", dpi = 300)
Fig1.pcaSupplementary Figure. PCA analysis on RNA and Protein datasets separately.
Suppl MOFA model properties
## Variance per factor
plot.variance.perfactor.all <- plot_variance_explained(mofa, x="factor", y="view") +
add.textsize +
labs(title = "Variance explained by each factor per modality")
## variance total
plot.variance.total <- plot_variance_explained(mofa, x="view", y="factor", plot_total = T)
plot.variance.total <- plot.variance.total[[2]] +
add.textsize +
labs(title = "Total \nvariance") +
geom_text(aes(label=round(R2,1)), vjust=1.6, color="white", size=2.5)
## Significance correlation covariates
plot.heatmap.pval.covariates <- as.ggplot(correlate_factors_with_covariates(mofa,
covariates = c("time"),
factors = 1:mofa@dimensions$K,
fontsize = 7,
cluster_row = F,
cluster_col = F
))+
add.textsize +
theme(axis.text.y=element_blank(),
axis.text.x=element_blank(),
plot.title = element_text(size=7, face = "plain"),
)## Factor values over time
plot.violin.factorall <- plot_factor(mofa,
factor = c(2,4:mofa@dimensions$K),
color_by = "condition",
dot_size = 0.2, # change dot size
dodge = T, # dodge points with different colors
legend = F, # remove legend
add_violin = T, # add violin plots,
violin_alpha = 0.9 # transparency of violin plots
) +
add.textsize +
scale_color_manual(values=c(colorgradient6_manual2, labels = c(labels), name = "Time aIg")) +
scale_fill_manual(values=c(colorgradient6_manual2, labels = c(labels), name = "Time aIg"))+
labs(title = "Factor values per time-point of additional factors not correlating with time." ) +
theme(axis.text.x=element_blank())
## Loadings factors stable protein
plot.rank.PROT.2.4to7 <- plot_weights(mofa,
view = "PROT",
factors = c(1:mofa@dimensions$K),
nfeatures = 3,
text_size = 1.5
) +
add.textsize +
labs(title = "Top 3 Protein loadings per factor") +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank()
)
## Loadings factors stable protein
plot.rank.RNA.2.4to7 <- plot_weights(mofa,
view = "RNA",
factors = c(1:mofa@dimensions$K),
nfeatures = 3,
text_size = 1.5
) +
add.textsize +
labs(title = "Top 3 RNA loadings per factor") +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank()
)
## correlation time and factors
plot.correlation.covariates <- correlate_factors_with_covariates(mofa,
covariates = c("time"),
factors = mofa@dimensions$K:1,
plot = "r"
)plot.correlation.covariates <- ggcorrplot(plot.correlation.covariates, tl.col = "black", method = "square", lab = TRUE, ggtheme = theme_void, colors = c("#11304C", "white", "#11304C"), lab_size = 2.5) +
add.textsize +
labs(title = "Correlation of \nfactors with \ntime of treatment", y = "") +
scale_y_discrete(labels = "") +
coord_flip() +
theme(axis.text.x=element_text(angle =0,hjust = 0.5),
axis.text.y=element_text(size = 5),
legend.position="none",
plot.title = element_text(hjust = 0.5))Coordinate system already present. Adding new coordinate system, which will replace the existing one.Fig.1.suppl.mofa.row1 <- plot_grid(plot.variance.perfactor.all, plot.variance.total,NULL, plot.heatmap.pval.covariates, labels = c(panellabels[1:3]), label_size = 10, ncol = 4, rel_widths = c(1.35, 0.3,0.25,0.38))
Fig.1.suppl.mofa.row2 <- plot_grid(plot.violin.factorall,legend.umap, labels = panellabels[4], label_size = 10, ncol = 2, rel_widths = c(1,0.1))
Suppl_mofa <- plot_grid(Fig.1.suppl.mofa.row1, Fig.1.suppl.mofa.row2, plot.rank.PROT.2.4to7, plot.rank.RNA.2.4to7, labels = c("","", panellabels[5:6]),label_size = 10, ncol = 1, rel_heights = c(1.45,1,1.1,1.1))
ggsave(Suppl_mofa, filename = "output/paper_figures/Fig2.Suppl_MOFAaIg.pdf", width = 183, height = 220, units = "mm", dpi = 300, useDingbats = FALSE)
ggsave(Suppl_mofa, filename = "output/paper_figures/Fig2.Suppl_MOFAaIg.png", width = 183, height = 220, units = "mm", dpi = 300)
Suppl_mofaSupplementary Figure. MOFA model additional information
Figure 2
Prepare main panels
meta.allcells <- seu_combined_selectsamples@meta.data %>%
mutate(sample = rownames(seu_combined_selectsamples@meta.data))
proteindata <- as.data.frame(t(seu_combined_selectsamples@assays$PROT@scale.data)) %>%
mutate(sample = rownames(t(seu_combined_selectsamples@assays$PROT@scale.data))) %>%
left_join(meta.allcells)
MOFAfactors<- as.data.frame(mofa@expectations$Z) %>%
mutate(sample = rownames(as.data.frame(mofa@expectations$Z)[,1:mofa@dimensions$K]))
MOFAfactors <- left_join(as.data.frame(MOFAfactors), meta.allcells)
weights.prot <- get_weights(mofa, views = "PROT",as.data.frame = TRUE)
topnegprots.factor1 <- weights.prot %>%
filter(factor == "Factor1" & value <= 0) %>%
arrange(value)
topposprots.factor3 <- weights.prot %>%
filter(factor == "Factor3" & value >= 0) %>%
arrange(-value)
weights.RNA <- get_weights(mofa, views = "RNA",as.data.frame = TRUE)## violin plots phospho-proteins highlighted in main
f.violin.fact <- function(data , protein){
ggplot(subset(data) , aes(x = as.factor(condition), y =get(noquote(protein)))) +
annotate("rect",
xmin = 5 - 0.45,
xmax = 6 + 0.6,
ymin = -4.5, ymax = 4.5, fill = "lightblue",
alpha = .4
)+
geom_hline(yintercept=0, linetype='dotted', col = 'black')+
geom_violin(alpha = 0.9,aes(fill = condition))+
geom_jitter(width = 0.05,size = 0.1, fill = "black", shape = 21)+
stat_summary(fun=median, geom="point", shape=95, size=2, inherit.aes = T, position = position_dodge(width = 0.9), color = "red")+
theme_few()+
ylab(paste0(protein)) +
scale_x_discrete(labels = labels, expand = c(0.1,0.1), name = "Time aIg (minutes)") +
scale_y_continuous(expand = c(0,0), name = strsplit(protein, split = "\\.")[[1]][2]) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",)+
scale_fill_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",) +
add.textsize +
theme(#axis.title.x=element_blank(),
#axis.text.x=element_blank(),
#axis.ticks.x=element_blank(),
legend.position="none")
}
factors_toplot <- c(colnames(MOFAfactors)[c(1,3)])
for(i in factors_toplot) {
assign(paste0("plot.factor.", i), f.violin.fact(data = MOFAfactors,protein = i))
}
legend.violinfactor <- as_ggplot( get_legend( ggplot(subset(MOFAfactors) , aes(x = as.factor(condition), y =get(noquote(factors_toplot[1])))) +
annotate("rect",
xmin = 5 - 0.45,
xmax = 6 + 0.6,
ymin = -4.5, ymax = 4.5, fill = "lightblue",
alpha = .4
)+
geom_hline(yintercept=0, linetype='dotted', col = 'black')+
geom_violin(alpha = 0.9,aes(fill = condition))+
geom_jitter(width = 0.05,size = 0.1, aes(col = condition), fill = "black", shape = 21)+
stat_summary(fun=median, geom="point", shape=23, size=2, inherit.aes = T, position = position_dodge(width = 0.9), color = "black")+
theme_few()+
ylab(paste0(factors_toplot[1])) +
scale_x_discrete(labels = labels, expand = c(0.1,0.1), name = "Time aIg (minutes)") +
scale_y_continuous(expand = c(0,0), name = strsplit(factors_toplot[1], split = "\\.")[[1]][2]) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg \n(minutes)",)+
scale_fill_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg \n(minutes)",) +
add.textsize ) )
plot.violin.factor1 <- plot.factor.group1.Factor1 +
scale_y_reverse(expand = c(0,0), name = "Factor 1 value")+
annotate(geom="text", x=1.5, y=-4.3, label="minutes", size = 2,
color="grey3") +
annotate(geom="text", x=5.5, y=-4.3, label="hours", size = 2,
color="grey3") +
labs(title = "Factor 1 captures \nminute times-scale signal transduction")
plot.violin.factor3 <- plot.factor.group1.Factor3 +
annotate(geom="text", x=5.5, y=4.3, label="hours", size = 2,
color="grey3")+
annotate(geom="text", x=1.5, y=4.3, label="minutes", size = 2,
color="grey3") +
scale_y_continuous(expand = c(0,0), name = "Factor 3 value")+
labs(title = "Factor 3 captures \nhour timescale signal transduction")
#### protein Loadings factor 1
list.bcellpathway.protein <- c("p-CD79a", "p-SYK", "p-SRC", "p-ERK1/2", "p-PLC-y2", "p-BLNK","p-PLC-y2Y759","p-PKC-b1", "p-p38", "p-AKT", "p-S6", "p-TOR", "CD79a") # , "p-PLC-y2", "p-PKC-b1", "p-IKKa/b","p-JNK", "p-p38", "p-p65","p-Akt", "p-S6", "p-TOR"
list.top20 <- topnegprots.factor1$feature[1:20]
## protein factor 1
plotdata.rank.PROT.1 <-plot_weights(mofa,
view = "PROT",
factors = c(1),
nfeatures = 15,
text_size = 1,
manual = list(list.top20, list.bcellpathway.protein, "p-JAK1" ),
color_manual = list("grey","blue4","red"),
return_data = TRUE
)
plotdata.rank.PROT.1 <- plotdata.rank.PROT.1 %>%
mutate(Rank = 1:80,
Weight = value,
colorvalue = ifelse(labelling_group == 2 & value <= -0.2,"grey", ifelse(labelling_group == 3 & value <= -0.2, "blue4", ifelse(labelling_group == 4 & value <= -0.2, "red", "grey"))),
highlights = ifelse(labelling_group >= 1 & value <= -0.25, as.character(feature), "")
)
plot.rank.PROT.1 <- ggplot(plotdata.rank.PROT.1, aes(x=Weight, y = Rank, label = highlights)) +
labs(title = "(Phospho)proteins: <p><span style='color:blue4'>BCR </span>&<span style='color:red'> JAK1</span> signaling", #<span style='color:blue4'>BCR signaling</span> and <span style='color:red'>p-JAK1</span>
x= "Factor 1 loading value",
y= "Factor 1 loading rank") +
geom_point(size=0.1, color =plotdata.rank.PROT.1$colorvalue, size =1) +
geom_text_repel(size = 2,
segment.size = 0.2,
color =plotdata.rank.PROT.1$colorvalue,
nudge_x = 1 - plotdata.rank.PROT.1$Weight,
direction = "y",
hjust = 1,
segment.color = "grey50")+
theme_few()+
add.textsize +
scale_y_continuous(trans = "reverse") +
add.textsize +
theme(
plot.title = element_markdown()
) +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank()
)+
xlim(c(-1,1))
## violin plots phospho-proteins highlighted in main
f.violin.prot <- function(data , protein){
ggplot(subset(data) , aes(x = as.factor(condition), y =get(noquote(protein)))) +
annotate("rect",
xmin = 5 - 0.45,
xmax = 6 + 0.6,
ymin = -5.5, ymax = 5.5, fill = "lightblue",
alpha = .4
)+
geom_violin(alpha = 0.9,aes(fill = condition))+
geom_jitter(width = 0.05,size = 0.1, color = "black")+
stat_summary(fun=median, geom="point", shape=95, size=2, inherit.aes = T, position = position_dodge(width = 0.9), color = "red")+
theme_few()+
ylab(paste0(protein)) +
scale_x_discrete(labels = labels, expand = c(0.1,0.1), name = "Time aIg (minutes)") +
scale_y_continuous(expand = c(0,0), name = protein) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",)+
scale_fill_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",) +
add.textsize +
theme(#axis.title.x=element_blank(),
#axis.text.x=element_blank(),
#axis.ticks.x=element_blank(),
legend.position="none")
}
proteins_toplot <- c("p-CD79a","p-Syk","p-JAK1", "IgM")
for(i in proteins_toplot) {
assign(paste0("plot.violin.", i), f.violin.prot(data = proteindata ,protein = i))
}
## Fig 2 bottom row panels
`plot.violin.p-CD79a` <- `plot.violin.p-CD79a` +
labs(title = "BCR signaling pathway and \nJAK1 activation")+
add.textsize+
theme(axis.title.x=element_blank(),
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),)
`plot.violin.p-Syk` <- `plot.violin.p-Syk` +
add.textsize+
theme(axis.title.x=element_blank(),
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),) +
scale_y_continuous(name = "p-SYK")
`plot.violin.p-JAK1` <-`plot.violin.p-JAK1` +
add.textsize +
labs(y = "<span style='color:red'>p-JAK1</span>") +
theme(axis.title.y = element_markdown()) Enrichment analysis of Factor 3 positive loadings
topposrna.factor3 <- weights.RNA %>%
filter(factor == "Factor3" & value >= 0) %>%
arrange(-value)
rownames(topposrna.factor3) <- topposrna.factor3$feature
### Convert Gene-names to gene IDs (using 'org.Hs.eg.db' library)
topposrna.factor3 <- topposrna.factor3 %>%
mutate(ENTREZID = mapIds(org.Hs.eg.db, as.character(topposrna.factor3$feature), 'ENTREZID', 'SYMBOL'))
listgenes.factor3 <- topposrna.factor3$value
names(listgenes.factor3) <- topposrna.factor3$ENTREZID
go.pb.fct3.pos <- enrichGO(gene = topposrna.factor3$feature,
OrgDb = org.Hs.eg.db,
keyType = 'SYMBOL',
ont = "BP",
pAdjustMethod = "BH")
go.pb.fct3.pos <- simplify(go.pb.fct3.pos, cutoff=0.6, by="p.adjust", select_fun=min)
go.pb.fct3.pos.dplyr <- mutate(go.pb.fct3.pos, richFactor = Count / as.numeric(sub("/\\d+", "", BgRatio)))
## plot main panel
plot.enriched.go.bp.factor3 <- ggplot(go.pb.fct3.pos.dplyr, showCategory = 10,
aes(-log10(p.adjust), fct_reorder(Description, -log10(p.adjust)))) + geom_segment(aes(xend=0, yend = Description)) +
geom_point(aes(size = Count)) +
scale_color_viridis_c(guide=guide_colorbar(reverse=TRUE)) +
scale_size_continuous(range=c(0.5, 3)) +
theme_minimal() +
add.textsize +
xlab("-log adj pval") +
ylab(NULL) +
ggtitle("Enriched Biological Processes") +
theme(legend.position="none")
legend.plot.enriched.go.bp.factor3 <- as.ggplot(get_legend(ggplot(go.pb.fct3.pos.dplyr, showCategory = 10,
aes(-log10(p.adjust), fct_reorder(Description, -log10(p.adjust)))) + geom_segment(aes(xend=0, yend = Description)) +
geom_point(aes(size = Count)) +
scale_color_viridis_c(guide=guide_colorbar(reverse=TRUE)) +
scale_size_continuous(range=c(0.5, 3)) +
theme_minimal() +
add.textsize +
xlab("-log adj pval") +
ylab(NULL) +
ggtitle("Enriched Biological Processes") +
theme(legend.position="right")))
## Plot supplementary
plot.enriched.go.bp.factor3_50 <- ggplot(go.pb.fct3.pos.dplyr, showCategory = 50,
aes(-log10(p.adjust), fct_reorder(Description, -log10(p.adjust)))) + geom_segment(aes(xend=0, yend = Description)) +
geom_point(aes(size = Count)) +
scale_color_viridis_c(guide=guide_colorbar(reverse=TRUE)) +
scale_size_continuous(range=c(0.5, 3)) +
theme_minimal() +
add.textsize +
xlab("-log adj pval") +
ylab(NULL) +
ggtitle("Enriched Biological Processes") message(c("Significance protein folding GO term: \n",go.pb.fct3.pos.dplyr@result[which(go.pb.fct3.pos.dplyr@result$Description == "protein folding"),"p.adjust"]))Significance protein folding GO term:
0.0457794707358827##For RNA loadings panels
topgeneset.fct3<- unlist(str_split(go.pb.fct3.pos.dplyr@result[1:10,"geneID"], pattern = "/"))
topgeneset.fct3 = bitr(topgeneset.fct3, fromType="SYMBOL", toType="ENTREZID", OrgDb="org.Hs.eg.db")
## RNA factor 1 loadings
plotdata.rank.RNA.3 <-plot_weights(mofa,
view = "RNA",
factors = c(3),
nfeatures = 5,
text_size = 1,
manual = list(topposrna.factor3$feature[c(1:5)] ,topgeneset.fct3$SYMBOL),
color_manual = list("grey","blue4"),
return_data = TRUE
)
plotdata.rank.RNA.3 <- plotdata.rank.RNA.3 %>%
mutate(Rank = 1:nrow(plotdata.rank.RNA.3),
Weight = value,
colorvalue = ifelse(labelling_group == 3 &value >= 0.25,"blue4", ifelse(labelling_group == 2&value >= 0.25, "grey2", "grey")),
highlights = ifelse(labelling_group >= 1&value >= 0.25, as.character(feature), "")
)
plot.rank.RNA.3 <- ggplot(plotdata.rank.RNA.3, aes(x=Weight, y = Rank, label = highlights)) +
labs(title = "Contributing genes <p><span style='color:blue4'><span style='color:blue4'>GO-term vesicle related </span> ", #<span style='color:blue4'>BCR signaling regulation (find GO term+list todo)</span>
x= "Factor 3 loading value",
y= "Factor 3 loading rank") +
geom_point(size=0.1, color =plotdata.rank.RNA.3$colorvalue) +
geom_text_repel(size = 2,
segment.size = 0.2,
color =plotdata.rank.RNA.3$colorvalue,
nudge_x = -1 - plotdata.rank.RNA.3$Weight,
direction = "y",
hjust = 0,
segment.color = "grey50")+
theme_few()+
add.textsize +
scale_y_continuous() +
add.textsize +
theme(
plot.title = element_markdown()
) +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank(),
axis.title.y = element_blank())+
xlim(c(-1,1))
#### Protein loadings factor 3
list.highlight.prot.fct3 <- c("p-p38", "p-AKT", "p-S6", "p-TOR", "XBP1_PROT", "p-STAT5")
list.top20 <- topnegprots.factor1$feature[1:20]
plotdata.rank.PROT.3 <-plot_weights(mofa,
view = "PROT",
factors = c(3),
nfeatures = 30,
text_size = 1,
manual = list(topposprots.factor3$feature[c(1:8)] ,list.highlight.prot.fct3),
color_manual = list("grey","blue4"),
return_data = TRUE
)
plotdata.rank.PROT.3 <- plotdata.rank.PROT.3 %>%
mutate(Rank = 1:80,
Weight = value,
colorvalue = ifelse(labelling_group == 3 &value >= 0.25,"blue4", ifelse(labelling_group == 2&value >= 0.4, "grey", "grey")),
highlights = ifelse(labelling_group >= 1&value >= 0.25, as.character(feature), "")
) %>%
mutate(highlights = case_when(as.character(highlights) == "XBP1_PROT" ~ "XBP1",
TRUE ~ highlights))
plot.rank.PROT.3 <- ggplot(plotdata.rank.PROT.3, aes(x=Weight, y = Rank, label = highlights)) +
labs(title = "Signaling implicated in <p><span style='color:blue4'>Unfolded protein response</span>",
x= "Factor 3 loading value",
y= "Factor 3 loading rank") +
geom_point(size=0.1, color =plotdata.rank.PROT.3$colorvalue, size =1) +
geom_text_repel(size = 2,
segment.size = 0.2,
color =plotdata.rank.PROT.3$colorvalue,
nudge_x = -1 - plotdata.rank.PROT.3$Weight,
direction = "y",
hjust = 0,
segment.color = "grey50")+
theme_few()+
add.textsize +
scale_y_continuous() +
add.textsize +
theme(
plot.title = element_markdown()
) +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank()
)+
xlim(c(-1,1))
#### RNA loadings factor 1
plotdata.rank.RNA.1 <-plot_weights(mofa,
view = "RNA",
factors = c(1),
nfeatures = 2,
text_size = 1,
return_data = TRUE
)
plotdata.rank.RNA.1 <- plotdata.rank.RNA.1 %>%
mutate(Rank = 1:nrow(plotdata.rank.RNA.1),
Weight = value,
colorvalue = ifelse(labelling_group == 1,"blue4", ifelse(labelling_group == 1, "blue4", "grey")),
highlights = ifelse(labelling_group >= 1, as.character(feature), "")
)
plot.rank.RNA.1 <- ggplot(plotdata.rank.RNA.1, aes(x=Weight, y = Rank, label = highlights)) +
labs(title = " Contributing genes<p><span style='color:blue4'>BCR activation</span>",
x= "Factor 1 loading value",
y= "Factor 1 loading rank") +
geom_point(size=0.1, color =plotdata.rank.RNA.1$colorvalue) +
geom_text_repel(size = 2,
segment.size = 0.2,
color =plotdata.rank.RNA.1$colorvalue,
nudge_x = 1 - plotdata.rank.RNA.1$Weight,
direction = "y",
hjust = 1,
segment.color = "grey50") +
theme_few()+
add.textsize +
scale_y_continuous(trans = "reverse") +
add.textsize +
theme(
plot.title = element_markdown()
) +
theme(axis.text.y=element_blank(),
axis.ticks.y=element_blank(),
axis.title.y=element_blank()
)+
xlim(c(-1,1))
#### Violins genes
f.violin.rna <- function(data , protein){
ggplot(subset(data) , aes(x = as.factor(condition), y =get(noquote(protein)))) +
annotate("rect",
xmin = 5 - 0.45,
xmax = 6 + 0.6,
ymin = -3, ymax = 15, fill = "lightblue",
alpha = .4
)+
geom_violin(alpha = 0.9,aes(fill = condition))+
geom_jitter(width = 0.05,size = 0.1, color = "black")+
stat_summary(fun=median, geom="point", shape=95, size=2, inherit.aes = T, position = position_dodge(width = 0.9), color = "red")+
theme_few()+
ylab(paste0(protein)) +
scale_x_discrete(labels = labels, expand = c(0.1,0.1), name = "Time aIg (minutes)") +
scale_y_continuous(expand = c(0,0), name = protein) +
scale_color_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",)+
scale_fill_manual(values = colorgradient6_manual2, labels = c(labels), name = "Time aIg ",) +
add.textsize +
theme(#axis.title.x=element_blank(),
#axis.text.x=element_blank(),
#axis.ticks.x=element_blank(),
legend.position="none")
}
rnadata <- as.data.frame(t(seu_combined_selectsamples@assays$SCT.RNA@scale.data)) %>%
mutate(sample = rownames(t(seu_combined_selectsamples@assays$SCT.RNA@scale.data))) %>%
left_join(meta.allcells)
enriched.geneset.posregBcell.fact1 <- c("NPM1", "NEAT1", "BTF3", "IGHM", "IGKC")
##Violin genes
for(i in enriched.geneset.posregBcell.fact1) {
assign(paste0("plot.violin.rna.", i), f.violin.rna(data = rnadata ,protein = i))
}
plot.violin.rna.NEAT1 <- plot.violin.rna.NEAT1 +
labs(title = "Expression of upregulated genes\n")+
add.textsize+
theme(axis.title.x=element_blank(),
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),)
plot.violin.rna.NPM1 <- plot.violin.rna.NPM1 +
add.textsize+
theme(axis.title.x=element_blank(),
axis.text.x=element_blank(),
axis.ticks.x=element_blank(),)
Main figure 2
Fig2.row1.violinsprot <- plot_grid(`plot.violin.p-CD79a`, `plot.violin.p-Syk`, `plot.violin.p-JAK1`,labels = c(panellabels[4]), label_size = 10, ncol = 1, rel_heights = c(1.2,0.95,1.2))
Fig2.row1 <- plot_grid(plot.violin.factor1,plot.rank.PROT.1, NULL, Fig2.row1.violinsprot, labels = c(panellabels[1:3], ""), label_size = 10, ncol =4, rel_widths = c(0.8,0.55,0.5,0.8))
Fig2.row2.violinsrna <- plot_grid(plot.violin.rna.NEAT1,plot.violin.rna.NPM1, plot.violin.rna.BTF3,labels = "", label_size = 10, ncol = 1, rel_heights = c(1.2,0.95,1.2))
Fig2.row2 <- plot_grid(plot.violin.factor3,plot.rank.PROT.3,plot.rank.RNA.3,Fig2.row2.violinsrna, labels = c(panellabels[5:6], "", panellabels[8]), label_size = 10, ncol =4, rel_widths = c(0.8,0.55,0.5,0.8))
Fig2.row3 <- plot_grid(plot.enriched.go.bp.factor3,legend.plot.enriched.go.bp.factor3,NULL, labels = panellabels[7], label_size = 10, ncol =3, rel_widths = c(1.8,0.2,0.6))
plot_fig2 <- plot_grid(Fig2.row1, Fig2.row2,Fig2.row3, labels = c("", "", ""), label_size = 10, ncol = 1, rel_heights = c(1,1,0.55))
ggsave(plot_fig2, filename = "output/paper_figures/Fig2.pdf", width = 183, height = 183, units = "mm", dpi = 300, useDingbats = FALSE)
ggsave(plot_fig2, filename = "output/paper_figures/Fig2.png", width = 183, height = 183, units = "mm", dpi = 300)
plot_fig2Figure 2. Factor 1 and 3 exploration.
Suppl Enriched Fact 3
ggsave(plot.enriched.go.bp.factor3_50, filename = "output/paper_figures/Suppl_Enrichm_Fct3.pdf", width = 183, height = 183, units = "mm", dpi = 300, useDingbats = FALSE)
ggsave(plot.enriched.go.bp.factor3_50, filename = "output/paper_figures/Suppl_Enrichm_Fct3.png", width = 183, height = 183, units = "mm", dpi = 300)
plot.enriched.go.bp.factor3_50Supplementary Figure. Top 50 enriched gene-sets in postive loadings factor 3.
sessionInfo()R version 3.6.3 (2020-02-29)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 17763)
Matrix products: default
locale:
[1] LC_COLLATE=English_Netherlands.1252 LC_CTYPE=English_Netherlands.1252
[3] LC_MONETARY=English_Netherlands.1252 LC_NUMERIC=C
[5] LC_TIME=English_Netherlands.1252
attached base packages:
[1] parallel stats4 grid stats graphics grDevices utils
[8] datasets methods base
other attached packages:
[1] png_0.1-7 forcats_0.5.0
[3] clusterProfiler_3.14.3 clusterProfiler.dplyr_0.0.2
[5] enrichplot_1.6.1 org.Hs.eg.db_3.10.0
[7] AnnotationDbi_1.48.0 IRanges_2.20.2
[9] S4Vectors_0.24.4 Biobase_2.46.0
[11] BiocGenerics_0.32.0 ggridges_0.5.2
[13] cowplot_1.1.0 ggtext_0.1.0
[15] ggplotify_0.0.5 ggcorrplot_0.1.3
[17] ggrepel_0.8.2 ggpubr_0.4.0
[19] scico_1.2.0 MOFA2_1.1
[21] extrafont_0.17 patchwork_1.0.1
[23] RColorBrewer_1.1-2 viridis_0.5.1
[25] viridisLite_0.3.0 ggsci_2.9
[27] sctransform_0.3.1 ggthemes_4.2.0
[29] matrixStats_0.57.0 kableExtra_1.2.1
[31] gridExtra_2.3 Seurat_3.2.2
[33] ggplot2_3.3.2 scales_1.1.1
[35] tidyr_1.1.2 dplyr_1.0.2
[37] stringr_1.4.0 workflowr_1.6.1
loaded via a namespace (and not attached):
[1] reticulate_1.16 tidyselect_1.1.0 RSQLite_2.2.1
[4] htmlwidgets_1.5.2 BiocParallel_1.20.1 Rtsne_0.15
[7] munsell_0.5.0 codetools_0.2-16 ica_1.0-2
[10] future_1.19.1 miniUI_0.1.1.1 withr_2.3.0
[13] GOSemSim_2.12.1 colorspace_1.4-1 knitr_1.30
[16] rstudioapi_0.11 ROCR_1.0-11 ggsignif_0.6.0
[19] tensor_1.5 DOSE_3.12.0 Rttf2pt1_1.3.8
[22] listenv_0.8.0 labeling_0.4.2 git2r_0.27.1
[25] urltools_1.7.3 mnormt_2.0.2 polyclip_1.10-0
[28] farver_2.0.3 bit64_4.0.5 pheatmap_1.0.12
[31] rhdf5_2.30.1 rprojroot_1.3-2 vctrs_0.3.4
[34] generics_0.0.2 xfun_0.18 markdown_1.1
[37] R6_2.4.1 graphlayouts_0.7.0 rsvd_1.0.3
[40] fgsea_1.12.0 spatstat.utils_1.17-0 gridGraphics_0.5-0
[43] DelayedArray_0.12.3 promises_1.1.0 ggraph_2.0.3
[46] gtable_0.3.0 globals_0.13.1 goftest_1.2-2
[49] tidygraph_1.2.0 rlang_0.4.8 splines_3.6.3
[52] rstatix_0.6.0 extrafontdb_1.0 lazyeval_0.2.2
[55] europepmc_0.4 broom_0.7.1 BiocManager_1.30.10
[58] yaml_2.2.1 reshape2_1.4.4 abind_1.4-5
[61] backports_1.1.10 httpuv_1.5.2 qvalue_2.18.0
[64] gridtext_0.1.1 tools_3.6.3 psych_2.0.9
[67] ellipsis_0.3.1 Rcpp_1.0.4.6 plyr_1.8.6
[70] progress_1.2.2 purrr_0.3.4 prettyunits_1.1.1
[73] rpart_4.1-15 deldir_0.1-29 pbapply_1.4-3
[76] zoo_1.8-8 haven_2.3.1 cluster_2.1.0
[79] fs_1.4.1 magrittr_1.5 data.table_1.13.0
[82] DO.db_2.9 openxlsx_4.2.2 triebeard_0.3.0
[85] lmtest_0.9-38 RANN_2.6.1 tmvnsim_1.0-2
[88] whisker_0.4 fitdistrplus_1.1-1 hms_0.5.3
[91] mime_0.9 evaluate_0.14 xtable_1.8-4
[94] rio_0.5.16 readxl_1.3.1 compiler_3.6.3
[97] tibble_3.0.4 KernSmooth_2.23-16 crayon_1.3.4
[100] htmltools_0.5.0 mgcv_1.8-31 later_1.0.0
[103] DBI_1.1.0 tweenr_1.0.1 corrplot_0.84
[106] MASS_7.3-53 rappdirs_0.3.1 Matrix_1.2-18
[109] car_3.0-10 igraph_1.2.6 pkgconfig_2.0.3
[112] rvcheck_0.1.8 foreign_0.8-75 plotly_4.9.2.1
[115] xml2_1.3.2 webshot_0.5.2 rvest_0.3.6
[118] digest_0.6.26 RcppAnnoy_0.0.16 spatstat.data_1.4-3
[121] fastmatch_1.1-0 rmarkdown_2.4 cellranger_1.1.0
[124] leiden_0.3.3 uwot_0.1.8 curl_4.3
[127] shiny_1.5.0 lifecycle_0.2.0 nlme_3.1-144
[130] jsonlite_1.7.1 Rhdf5lib_1.8.0 carData_3.0-4
[133] pillar_1.4.6 lattice_0.20-38 GO.db_3.10.0
[136] fastmap_1.0.1 httr_1.4.2 survival_3.1-8
[139] glue_1.4.2 zip_2.1.1 spatstat_1.64-1
[142] bit_4.0.4 ggforce_0.3.2 stringi_1.4.6
[145] HDF5Array_1.14.4 blob_1.2.1 memoise_1.1.0
[148] irlba_2.3.3 future.apply_1.6.0
Figure 1. UMAP of 7 MOFA factors, integrating phospho-protein and RNA measurements
Supplementary Figure. PCA analysis on RNA and Protein datasets separately.
Supplementary Figure. MOFA model additional information
Figure 2. Factor 1 and 3 exploration.
Supplementary Figure. Top 50 enriched gene-sets in postive loadings factor 3.