---

title: "COVID19: DGE visualisation"

output: html_notebook

---

This is to visualise the output from DE testing run on each T cell subset, either comparing across linear and quadratic trends.

```{r, warning=FALSE, message=FALSE}

library(ggplot2)

library(ggthemes)

library(ggsci)

library(scales)

library(enrichR)

library(ggrepel)

library(cowplot)

library(RColorBrewer)

```

# DGE testing across COVID19 severity - linear trends

Testing for a linear trend across disease severity, from healthy through to critical.

```{r}

dge.files <- list.files("~/Dropbox/COVID19/Data/Updated/DEResults.dir/", pattern="\\.L\\.csv")

linear.dge.list <- list()

for(x in seq_along(dge.files)){

    x.csv <- read.csv(paste0("~/Dropbox/COVID19/Data/Updated/DEResults.dir/", dge.files[x]),

                      header=TRUE, stringsAsFactors=FALSE)

    x.annot <- gsub(dge.files[x], pattern="DE_(\\S*)_Severity\\.L\\.csv", replacement="\\1")

    x.csv$Sub.Annotation <- x.annot

    linear.dge.list[[dge.files[x]]] <- x.csv

}

linear.dge.df <- do.call(rbind.data.frame, linear.dge.list)

linear.dge.df$Diff <- sign(linear.dge.df$logFC)

linear.dge.df$Diff[linear.dge.df$FDR >= 0.01] <- 0

table(linear.dge.df$Diff, linear.dge.df$Sub.Annotation)

```

There are variable numbers of gene DE across categories, but generally quite a small number. 

```{r}

# collect all de genes for plotting

de.genes.all <- unique(linear.dge.df$X[linear.dge.df$FDR < 0.01])

write.table(de.genes.all,

            file="~/Dropbox/COVID19/Data/Updated/DEResults.dir/ALL_linear_DEgenes.tsv",

            quote=FALSE, row.names=FALSE, sep="\t")

```

## CD4.CM

```{r}

enrich.dbs <- c("Transcription_Factor_PPIs", "MSigDB_Computational", "MSigDB_Hallmark_2020", 

                "KEGG_2019_Human", "GO_Biological_Process_2018")

```

```{r, fig.height=3.95, fig.width=4.95}

cd4.cm.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.CM"), ]$X

cd4.cm.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.CM"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.CM"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd4.cm.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD4.CM-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd4.cm.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.CM") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd4.cm.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.CM") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd4.msigdb.up <- cd4.cm.enriched.up[["MSigDB_Hallmark_2020"]]

cd4.msigdb.up$Dir <- "Up"

cd4.msigdb.down <- cd4.cm.enriched.down[["MSigDB_Hallmark_2020"]]

cd4.msigdb.down$Dir <- "Down"

cd4.msigdb <- do.call(rbind.data.frame, list(cd4.msigdb.up, cd4.msigdb.down))

cd4.msigdb$logP <- -log10(cd4.msigdb$P.value)

cd4.msigdb$logP[cd4.msigdb$Dir %in% c("Down")] <- -1 * cd4.msigdb$logP[cd4.msigdb$Dir %in% c("Down")]

ggplot(cd4.msigdb[cd4.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD4.CM-linear_MSigDB_enriched.pdf",

           height=4.15, width=4.15, useDingbats=FALSE) +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd4.tfppi.up <- cd4.cm.enriched.up[["Transcription_Factor_PPIs"]]

cd4.tfppi.up$Dir <- "Up"

cd4.tfppi.down <- cd4.cm.enriched.down[["Transcription_Factor_PPIs"]]

cd4.tfppi.down$Dir <- "Down"

cd4.tfppi <- do.call(rbind.data.frame, list(cd4.tfppi.up, cd4.tfppi.down))

cd4.tfppi$logP <- -log10(cd4.tfppi$P.value)

cd4.tfppi$logP[cd4.tfppi$Dir %in% c("Down")] <- -1 * cd4.tfppi$logP[cd4.tfppi$Dir %in% c("Down")]

ggplot(cd4.tfppi[cd4.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD4.Naive

```{r, fig.height=3.95, fig.width=4.95}

cd4.naive.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Naive"), ]$X

cd4.naive.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Naive"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Naive"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd4.naive.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD4.Naive-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd4.naive.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Naive") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd4.naive.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Naive") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd4.naive.msigdb.up <- cd4.naive.enriched.up[["MSigDB_Hallmark_2020"]]

cd4.naive.msigdb.up$Dir <- "Up"

cd4.naive.msigdb.down <- cd4.naive.enriched.down[["MSigDB_Hallmark_2020"]]

cd4.naive.msigdb.down$Dir <- "Down"

cd4.naive.msigdb <- do.call(rbind.data.frame, list(cd4.naive.msigdb.up, cd4.naive.msigdb.down))

cd4.naive.msigdb$logP <- -log10(cd4.naive.msigdb$P.value)

cd4.naive.msigdb$logP[cd4.naive.msigdb$Dir %in% c("Down")] <- -1 * cd4.naive.msigdb$logP[cd4.naive.msigdb$Dir %in% c("Down")]

ggplot(cd4.naive.msigdb[cd4.naive.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

        ggsave("~/Dropbox/COVID19/Updated_plots/CD4.Naive-linear_MSigDB_enriched.pdf",

           height=4.15, width=4.15, useDingbats=FALSE) +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd4.naive.tfppi.up <- cd4.naive.enriched.up[["Transcription_Factor_PPIs"]]

cd4.naive.tfppi.up$Dir <- "Up"

cd4.naive.tfppi.down <- cd4.naive.enriched.down[["Transcription_Factor_PPIs"]]

cd4.naive.tfppi.down$Dir <- "Down"

cd4.naive.tfppi <- do.call(rbind.data.frame, list(cd4.naive.tfppi.up, cd4.naive.tfppi.down))

cd4.naive.tfppi$logP <- -log10(cd4.naive.tfppi$P.value)

cd4.naive.tfppi$logP[cd4.naive.tfppi$Dir %in% c("Down")] <- -1 * cd4.naive.tfppi$logP[cd4.naive.tfppi$Dir %in% c("Down")]

ggplot(cd4.naive.tfppi[cd4.naive.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD8.Naive

```{r, fig.height=3.95, fig.width=4.95}

cd8.naive.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.Naive"), ]$X

cd8.naive.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.Naive"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.Naive"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd8.naive.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD8.Naive-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd8.naive.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.Naive") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd8.naive.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.Naive") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd8.naive.msigdb.up <- cd8.naive.enriched.up[["MSigDB_Hallmark_2020"]]

cd8.naive.msigdb.up$Dir <- "Up"

cd8.naive.msigdb.down <- cd8.naive.enriched.down[["MSigDB_Hallmark_2020"]]

cd8.naive.msigdb.down$Dir <- "Down"

cd8.naive.msigdb <- do.call(rbind.data.frame, list(cd8.naive.msigdb.up, cd8.naive.msigdb.down))

cd8.naive.msigdb$logP <- -log10(cd8.naive.msigdb$P.value)

cd8.naive.msigdb$logP[cd8.naive.msigdb$Dir %in% c("Down")] <- -1 * cd8.naive.msigdb$logP[cd8.naive.msigdb$Dir %in% c("Down")]

ggplot(cd8.naive.msigdb[cd8.naive.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd8.naive.tfppi.up <- cd8.naive.enriched.up[["Transcription_Factor_PPIs"]]

cd8.naive.tfppi.up$Dir <- "Up"

cd8.naive.tfppi.down <- cd8.naive.enriched.down[["Transcription_Factor_PPIs"]]

cd8.naive.tfppi.down$Dir <- "Down"

cd8.naive.tfppi <- do.call(rbind.data.frame, list(cd8.naive.tfppi.up, cd8.naive.tfppi.down))

cd8.naive.tfppi$logP <- -log10(cd8.naive.tfppi$P.value)

cd8.naive.tfppi$logP[cd8.naive.tfppi$Dir %in% c("Down")] <- -1 * cd8.naive.tfppi$logP[cd8.naive.tfppi$Dir %in% c("Down")]

ggplot(cd8.naive.tfppi[cd8.naive.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD8.EM

```{r, fig.height=3.95, fig.width=4.95}

cd8.em.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.EM"), ]$X

cd8.em.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.EM"), ]$FDR > 1e-2] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.EM"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd8.em.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD8.EM-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd8.em.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.EM") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd8.em.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.EM") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd8.em.msigdb.up <- cd8.em.enriched.up[["MSigDB_Hallmark_2020"]]

cd8.em.msigdb.up$Dir <- "Up"

cd8.em.msigdb.down <- cd8.em.enriched.down[["MSigDB_Hallmark_2020"]]

if(nrow(cd8.em.msigdb.down) > 0){

    cd8.em.msigdb.down$Dir <- "Down"

    cd8.em.msigdb <- do.call(rbind.data.frame, list(cd8.em.msigdb.up, cd8.em.msigdb.down))

} else{

    cd8.em.msigdb <- do.call(rbind.data.frame, list(cd8.em.msigdb.up))

}

cd8.em.msigdb$logP <- -log10(cd8.em.msigdb$P.value)

cd8.em.msigdb$logP[cd8.em.msigdb$Dir %in% c("Down")] <- -1 * cd8.em.msigdb$logP[cd8.em.msigdb$Dir %in% c("Down")]

ggplot(cd8.em.msigdb[cd8.em.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd8.em.tfppi.up <- cd8.em.enriched.up[["Transcription_Factor_PPIs"]]

cd8.em.tfppi.up$Dir <- "Up"

cd8.em.tfppi.down <- cd8.em.enriched.down[["Transcription_Factor_PPIs"]]

if(nrow(cd8.em.tfppi.down) > 0){

    cd8.em.tfppi.down$Dir <- "Down"

    cd8.em.tfppi <- do.call(rbind.data.frame, list(cd8.em.tfppi.up, cd8.em.tfppi.down))

} else{

    cd8.em.tfppi <- do.call(rbind.data.frame, list(cd8.em.tfppi.up))

}

cd8.em.tfppi$logP <- -log10(cd8.em.tfppi$P.value)

cd8.em.tfppi$logP[cd8.em.tfppi$Dir %in% c("Down")] <- -1 * cd8.em.tfppi$logP[cd8.em.tfppi$Dir %in% c("Down")]

ggplot(cd8.em.tfppi[cd8.em.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD8.TE

```{r, fig.height=3.95, fig.width=4.95}

cd8.te.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.TE"), ]$X

cd8.te.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.TE"), ]$FDR > 1e-2] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.TE"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd8.te.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD8.TE-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd8.te.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.TE") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd8.te.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD8.TE") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd8.te.msigdb.up <- cd8.te.enriched.up[["MSigDB_Hallmark_2020"]]

cd8.te.msigdb.up$Dir <- "Up"

cd8.te.msigdb.down <- cd8.te.enriched.down[["MSigDB_Hallmark_2020"]]

if(nrow(cd8.te.msigdb.down) > 0){

    cd8.te.msigdb.down$Dir <- "Down"

}

cd8.te.msigdb <- do.call(rbind.data.frame, list(cd8.te.msigdb.up, cd8.te.msigdb.down))

cd8.te.msigdb$logP <- -log10(cd8.te.msigdb$P.value)

cd8.te.msigdb$logP[cd8.te.msigdb$Dir %in% c("Down")] <- -1 * cd8.te.msigdb$logP[cd8.te.msigdb$Dir %in% c("Down")]

ggplot(cd8.te.msigdb[cd8.te.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd8.te.tfppi.up <- cd8.te.enriched.up[["Transcription_Factor_PPIs"]]

if(nrow(cd8.te.tfppi.up) > 0){

    cd8.te.tfppi.up$Dir <- "Up"

}

cd8.te.tfppi.down <- cd8.te.enriched.down[["Transcription_Factor_PPIs"]]

if(nrow(cd8.te.tfppi.down) > 0){

    cd8.te.tfppi.down$Dir <- "Down"

}

cd8.te.tfppi <- do.call(rbind.data.frame, list(cd8.te.tfppi.up, cd8.te.tfppi.down))

cd8.te.tfppi$logP <- -log10(cd8.te.tfppi$P.value)

cd8.te.tfppi$logP[cd8.te.tfppi$Dir %in% c("Down")] <- -1 * cd8.te.tfppi$logP[cd8.te.tfppi$Dir %in% c("Down")]

cd8.te.tfppi$Sub

ggplot(cd8.te.tfppi[cd8.te.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD4.Tfh

```{r, fig.height=3.95, fig.width=4.95}

tfh.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Tfh"), ]$X

tfh.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Tfh"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Tfh"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=tfh.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD4.Tfh-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

tfh.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Tfh") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

tfh.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("CD4.Tfh") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

tfh.msigdb.up <- tfh.enriched.up[["MSigDB_Hallmark_2020"]]

tfh.msigdb.up$Dir <- "Up"

tfh.msigdb.down <- tfh.enriched.down[["MSigDB_Hallmark_2020"]]

if(!is.null(tfh.msigdb.down)){

tfh.msigdb.down$Dir <- "Down"

tfh.msigdb <- do.call(rbind.data.frame, list(tfh.msigdb.up, tfh.msigdb.down))

} else{

    tfh.msigdb <- do.call(rbind.data.frame, list(tfh.msigdb.up))

}

tfh.msigdb$logP <- -log10(tfh.msigdb$P.value)

tfh.msigdb$logP[tfh.msigdb$Dir %in% c("Down")] <- -1 * tfh.msigdb$logP[tfh.msigdb$Dir %in% c("Down")]

ggplot(tfh.msigdb[tfh.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

tfh.tfppi.up <- tfh.enriched.up[["Transcription_Factor_PPIs"]]

tfh.tfppi.up$Dir <- "Up"

tfh.tfppi.down <- tfh.enriched.down[["Transcription_Factor_PPIs"]]

if(!is.null(tfh.tfppi.down)){

    tfh.tfppi.down$Dir <- "Down"

    tfh.tfppi <- do.call(rbind.data.frame, list(tfh.tfppi.up, tfh.tfppi.down))

} else{

    tfh.tfppi <- do.call(rbind.data.frame, list(tfh.tfppi.up))

}

tfh.tfppi <- do.call(rbind.data.frame, list(tfh.tfppi.up, tfh.tfppi.down))

tfh.tfppi$logP <- -log10(tfh.tfppi$P.value)

tfh.tfppi$logP[tfh.tfppi$Dir %in% c("Down")] <- -1 * tfh.tfppi$logP[tfh.tfppi$Dir %in% c("Down")]

ggplot(tfh.tfppi[tfh.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## gdT

```{r, fig.height=3.95, fig.width=4.95}

gdt.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("gdT"), ]$X

gdt.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("gdT"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("gdT"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=gdt.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/gdT-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

gdt.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("gdT") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

gdt.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("gdT") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

gdt.msigdb.up <- gdt.enriched.up[["MSigDB_Hallmark_2020"]]

if(!is.null(gdt.msigdb.up)){

    gdt.msigdb.up$Dir <- "Up"    

}

gdt.msigdb.down <- gdt.enriched.down[["MSigDB_Hallmark_2020"]]

if(!is.null(gdt.msigdb.down)){

    gdt.msigdb.down$Dir <- "Down"

}

if(!is.null(gdt.msigdb.up) & !is.null(gdt.msigdb.down)){

    gdt.msigdb <- do.call(rbind.data.frame, list(gdt.msigdb.up, gdt.msigdb.down))

    gdt.msigdb$logP <- -log10(gdt.msigdb$P.value)

    gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")] <- -1 * gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")]

} else if(!is.null(gdt.msigdb.up) & is.null(gdt.msigdb.down)){

    gdt.msigdb <- do.call(rbind.data.frame, list(gdt.msigdb.up))

    gdt.msigdb$logP <- -log10(gdt.msigdb$P.value)

    gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")] <- -1 * gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")]

} else if(is.null(gdt.msigdb.up) & !is.null(gdt.msigdb.down)){

    gdt.msigdb <- do.call(rbind.data.frame, list(gdt.msigdb.down))

    gdt.msigdb$logP <- -log10(gdt.msigdb$P.value)

    gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")] <- -1 * gdt.msigdb$logP[gdt.msigdb$Dir %in% c("Down")]

} else{

    gdt.msigdb <- NULL

}

if(!is.null(gdt.msigdb)){

    ggplot(gdt.msigdb[gdt.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

}

```

```{r, fig.height=4.15, fig.width=4.15}

gdt.tfppi.up <- gdt.enriched.up[["Transcription_Factor_PPIs"]]

gdt.tfppi.up$Dir <- "Up"

gdt.tfppi.down <- gdt.enriched.down[["Transcription_Factor_PPIs"]]

if(!is.null(gdt.tfppi.down)){

    gdt.tfppi.down$Dir <- "Down"

    gdt.tfppi <- do.call(rbind.data.frame, list(gdt.tfppi.up, gdt.tfppi.down))

    gdt.tfppi$logP <- -log10(gdt.tfppi$P.value)

    gdt.tfppi$logP[gdt.tfppi$Dir %in% c("Down")] <- -1 * gdt.tfppi$logP[gdt.tfppi$Dir %in% c("Down")]

} else{

    gdt.tfppi <- do.call(rbind.data.frame, list(gdt.tfppi.up))

    gdt.tfppi$logP <- -log10(gdt.tfppi$P.value)

    gdt.tfppi$logP[gdt.tfppi$Dir %in% c("Down")] <- -1 * gdt.tfppi$logP[gdt.tfppi$Dir %in% c("Down")]

}

ggplot(gdt.tfppi[gdt.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## MAIT

```{r, fig.height=3.95, fig.width=4.95}

mait.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("MAIT"), ]$X

mait.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("MAIT"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("MAIT"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=mait.labels)) +

        ggsave("~/Dropbox/COVID19/Updated_plots/MAIT-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

mait.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("MAIT") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

mait.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("MAIT") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

mait.msigdb.up <- mait.enriched.up[["MSigDB_Hallmark_2020"]]

if(!is.null(mait.msigdb.up)){

    mait.msigdb.up$Dir <- "Up"

}

mait.msigdb.down <- mait.enriched.down[["MSigDB_Hallmark_2020"]]

if(!is.null(mait.msigdb.down)){

    mait.msigdb.down$Dir <- "Down"

}

if(!is.null(mait.msigdb.down) & !is.null(mait.msigdb.up)){

    mait.msigdb <- do.call(rbind.data.frame, list(mait.msigdb.up, mait.msigdb.down))

} else if(is.null(mait.msigdb.down) & !is.null(mait.msigdb.up)){

    mait.msigdb <- do.call(rbind.data.frame, list(mait.msigdb.up))

} else if(!is.null(mait.msigdb.down) & is.null(mait.msigdb.up)){

    mait.msigdb <- do.call(rbind.data.frame, list(mait.msigdb.down))

}

mait.msigdb$logP <- -log10(mait.msigdb$P.value)

mait.msigdb$logP[mait.msigdb$Dir %in% c("Down")] <- -1 * mait.msigdb$logP[mait.msigdb$Dir %in% c("Down")]

ggplot(mait.msigdb[mait.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

mait.tfppi.up <- mait.enriched.up[["Transcription_Factor_PPIs"]]

if(!is.null(mait.tfppi.up)){

    mait.tfppi.up$Dir <- "Up"

}

mait.tfppi.down <- mait.enriched.down[["Transcription_Factor_PPIs"]]

if(!is.null(mait.tfppi.down)){

    mait.tfppi.down$Dir <- "Down"

}

if(!is.null(mait.tfppi.up) & !is.null(mait.tfppi.down)){

    mait.tfppi <- do.call(rbind.data.frame, list(mait.tfppi.up, mait.tfppi.down))

} else if(!is.null(mait.tfppi.up) & is.null(mait.tfppi.down)){

    mait.tfppi <- do.call(rbind.data.frame, list(mait.tfppi.up))

} else if(is.null(mait.tfppi.up) & !is.null(mait.tfppi.down)){

    mait.tfppi <- do.call(rbind.data.frame, list(mait.tfppi.down))

}

mait.tfppi$logP <- -log10(mait.tfppi$P.value)

mait.tfppi$logP[mait.tfppi$Dir %in% c("Down")] <- -1 * mait.tfppi$logP[mait.tfppi$Dir %in% c("Down")]

ggplot(mait.tfppi[mait.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## Treg

```{r, fig.height=3.95, fig.width=4.95}

treg.labels <- linear.dge.df[linear.dge.df$Sub.Annotation %in% c("Treg"), ]$X

treg.labels[linear.dge.df[linear.dge.df$Sub.Annotation %in% c("Treg"), ]$FDR > 1e-3] <- ""

ggplot(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("Treg"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=treg.labels)) +

        ggsave("~/Dropbox/COVID19/Updated_plots/Treg-linear_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

treg.enriched.up <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("Treg") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

treg.enriched.down <- enrichr(linear.dge.df[linear.dge.df$Sub.Annotation %in% c("Treg") &

                                                linear.dge.df$FDR < 0.01 &

                                                linear.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

treg.msigdb.up <- treg.enriched.up[["MSigDB_Hallmark_2020"]]

treg.msigdb.up$Dir <- "Up"

treg.msigdb.down <- treg.enriched.down[["MSigDB_Hallmark_2020"]]

if(!is.null(treg.msigdb.down)){

    treg.msigdb.down$Dir <- "Down"

}

treg.msigdb <- do.call(rbind.data.frame, list(treg.msigdb.up, treg.msigdb.down))

treg.msigdb$logP <- -log10(treg.msigdb$P.value)

treg.msigdb$logP[treg.msigdb$Dir %in% c("Down")] <- -1 * treg.msigdb$logP[treg.msigdb$Dir %in% c("Down")]

ggplot(treg.msigdb[treg.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

treg.tfppi.up <- treg.enriched.up[["Transcription_Factor_PPIs"]]

treg.tfppi.up$Dir <- "Up"

treg.tfppi.down <- treg.enriched.down[["Transcription_Factor_PPIs"]]

if(!is.null(treg.tfppi.down)){

    treg.tfppi.down$Dir <- "Down"

}

treg.tfppi <- do.call(rbind.data.frame, list(treg.tfppi.up, treg.tfppi.down))

treg.tfppi$logP <- -log10(treg.tfppi$P.value)

treg.tfppi$logP[treg.tfppi$Dir %in% c("Down")] <- -1 * treg.tfppi$logP[treg.tfppi$Dir %in% c("Down")]

ggplot(treg.tfppi[treg.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

Aggregate all results from the linear trend analysis.

```{r}

cd4.msigdb$Sub.Annotation <- "CD4.CM"

cd4.naive.msigdb$Sub.Annotation <- "CD4.Naive"

cd8.naive.msigdb$Sub.Annotation <- "CD8.Naive"

cd8.em.msigdb$Sub.Annotation <- "CD8.EM"

cd8.te.msigdb$Sub.Annotation <- "CD8.TE"

tfh.msigdb$Sub.Annotation <- "CD4.Tfh"

# treg.msigdb$Sub.Annotation <- "Treg"

mait.msigdb$Sub.Annotation <- "MAIT"

# gdt.msigdb$Sub.Annotation <- "gdT"

msigdb.linear.enrichments <- list(cd4.msigdb, cd4.naive.msigdb, cd8.naive.msigdb, cd8.em.msigdb, cd8.te.msigdb,

                                  tfh.msigdb, mait.msigdb)

msigdb.linear.enrich.df <- do.call(rbind.data.frame,

                                   msigdb.linear.enrichments)

```

```{r}

cell.order <- c("CD4.Naive", "CD4.CM", "CD4.EM", "CD4.IL22", "CD4.Prolif", "CD4.Th1", "CD4.Th2", "CD4.Th17", "CD4.Tfh",

                "Treg", "CD8.Naive", "CD8.Activated", "CD8.Prolif", "CD8.CM", "CD8.TE", "CD8.EM", "gdT", "MAIT", "NKT")

all.qual.cols <- brewer.pal.info[brewer.pal.info$category %in% c("qual") & brewer.pal.info$colorblind, ]

col_vector = unlist(mapply(brewer.pal, all.qual.cols$maxcolors, rownames(all.qual.cols)))

# cell.cols <- col_vector[c(1:7, 9:19)]

cell.cols <- col_vector[c(1:17, 19, 20)]

names(cell.cols) <- cell.order

```

```{r, fig.height=5.95, fig.width=6.95}

ggplot(msigdb.linear.enrich.df[msigdb.linear.enrich.df$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP, colour=Sub.Annotation)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point(size=3.5, shape=18) +

    scale_colour_manual(values=cell.cols) +

    #facet_wrap(~Term, scales="free_y", ncol=1) +

    labs(x="Pathway", y="Odds Rato") +

    guides(colour=guide_legend(title="Cell Type", override.aes=list(size=5, shape=18))) +

    theme_cowplot() +

    coord_flip() +

    theme(panel.spacing=unit(0, "lines"),

          strip.text=element_blank(),

          strip.background=element_blank()) +

    ggsave("~/Dropbox/COVID19/Updated_plots/Tcell-linear_MSigDB_enriched.pdf",

           height=5.95, width=6.95, useDingbats=FALSE) +

    NULL

```

And for the enriched TFs. Aggregate all results from the linear trend analysis.

```{r}

cd4.tfppi$Sub.Annotation <- "CD4.CM"

cd4.naive.tfppi$Sub.Annotation <- "CD4.Naive"

cd8.naive.tfppi$Sub.Annotation <- "CD8.Naive"

cd8.em.tfppi$Sub.Annotation <- "CD8.EM"

cd8.te.tfppi$Sub.Annotation <- "CD8.TE"

tfh.tfppi$Sub.Annotation <- "CD4.Tfh"

# treg.tfppi$Sub.Annotation <- "Treg"

mait.tfppi$Sub.Annotation <- "MAIT"

# gdt.tfppi$Sub.Annotation <- "gdT"

tfppi.linear.enrichments <- list(cd4.tfppi, cd4.naive.tfppi, cd8.naive.tfppi, cd8.em.tfppi, cd8.te.tfppi,

                                  tfh.tfppi,  mait.tfppi)

tfppi.linear.enrich.df <- do.call(rbind.data.frame,

                                   tfppi.linear.enrichments)

```

```{r, fig.height=8.95, fig.width=6.95}

ggplot(tfppi.linear.enrich.df[tfppi.linear.enrich.df$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP, colour=Sub.Annotation)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point(size=3.5, shape=18) +

    scale_colour_manual(values=cell.cols) +

    #facet_wrap(~Term, scales="free_y", ncol=1) +

    labs(x="Pathway", y="Odds Rato") +

    guides(colour=guide_legend(title="Cell Type", override.aes=list(size=5, shape=18))) +

    theme_cowplot() +

    coord_flip() +

    theme(panel.spacing=unit(0, "lines"),

          strip.text=element_blank(),

          strip.background=element_blank()) +

ggsave("~/Dropbox/COVID19/Updated_plots/Tcell-linear_TFPPI_enriched.pdf",

           height=5.95, width=6.95, useDingbats=FALSE) +

    NULL

```

# DGE testing across COVID19 severity - quadratic trends

As the model above, but testing for a linear trend across disease severity, from healthy through to critical.

```{r}

dge.files <- list.files("~/Dropbox/COVID19/Data/Updated/DEResults.dir/", pattern="\\.Q\\.csv")

quadratic.dge.list <- list()

for(x in seq_along(dge.files)){

    x.csv <- read.csv(paste0("~/Dropbox/COVID19/Data/Updated/DEResults.dir/", dge.files[x]),

                      header=TRUE, stringsAsFactors=FALSE)

    x.annot <- gsub(dge.files[x], pattern="DE_(\\S*)_Severity\\.Q\\.csv", replacement="\\1")

    x.csv$Sub.Annotation <- x.annot

    quadratic.dge.list[[dge.files[x]]] <- x.csv

}

quadratic.dge.df <- do.call(rbind.data.frame, quadratic.dge.list)

quadratic.dge.df$Diff <- sign(quadratic.dge.df$logFC)

quadratic.dge.df$Diff[quadratic.dge.df$FDR >= 0.01] <- 0

table(quadratic.dge.df$Diff, quadratic.dge.df$Sub.Annotation)

```

There are variable numbers of gene DE across categories, but generally quite a small number. Somehow this doesn't feel right...

## CD4.CM

```{r}

enrich.dbs <- c("Transcription_Factor_PPIs", "MSigDB_Computational", "MSigDB_Hallmark_2020", 

                "UK_Biobank_GWAS_v1", "KEGG_2019_Human", "GO_Biological_Process_2018")

```

```{r, fig.height=3.95, fig.width=4.95}

cd4.cm.labels <- quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.CM"), ]$X

cd4.cm.labels[quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.CM"), ]$FDR > 1e-3] <- ""

ggplot(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.CM"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd4.cm.labels)) +

        ggsave("~/Dropbox/COVID19/Updated_plots/CD4.CM-quadratic_volcano.pdf",

           height=3.95, width=4.95, useDingbats=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd4.cm.enriched.up <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.CM") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd4.cm.enriched.down <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.CM") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd4.msigdb.up <- cd4.cm.enriched.up[["MSigDB_Hallmark_2020"]]

cd4.msigdb.up$Dir <- "Up"

cd4.msigdb.down <- cd4.cm.enriched.down[["MSigDB_Hallmark_2020"]]

cd4.msigdb.down$Dir <- "Down"

cd4.msigdb <- do.call(rbind.data.frame, list(cd4.msigdb.up, cd4.msigdb.down))

cd4.msigdb$logP <- -log10(cd4.msigdb$P.value)

cd4.msigdb$logP[cd4.msigdb$Dir %in% c("Down")] <- -1 * cd4.msigdb$logP[cd4.msigdb$Dir %in% c("Down")]

ggplot(cd4.msigdb[cd4.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd4.tfppi.up <- cd4.cm.enriched.up[["Transcription_Factor_PPIs"]]

cd4.tfppi.up$Dir <- "Up"

cd4.tfppi.down <- cd4.cm.enriched.down[["Transcription_Factor_PPIs"]]

cd4.tfppi.down$Dir <- "Down"

cd4.tfppi <- do.call(rbind.data.frame, list(cd4.tfppi.up, cd4.tfppi.down))

cd4.tfppi$logP <- -log10(cd4.tfppi$P.value)

cd4.tfppi$logP[cd4.tfppi$Dir %in% c("Down")] <- -1 * cd4.tfppi$logP[cd4.tfppi$Dir %in% c("Down")]

ggplot(cd4.tfppi[cd4.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD4.Naive

```{r, fig.height=3.95, fig.width=4.95}

cd4.naive.labels <- quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.Naive"), ]$X

cd4.naive.labels[quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.Naive"), ]$FDR > 1e-3] <- ""

ggplot(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.Naive"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd4.naive.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD4.Naive-quadratic_volcano.pdf",

           height=3.95, width=4.95, useDingbat=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd4.naive.enriched.up <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.Naive") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd4.naive.enriched.down <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD4.Naive") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd4.naive.msigdb.up <- cd4.naive.enriched.up[["MSigDB_Hallmark_2020"]]

cd4.naive.msigdb.up$Dir <- "Up"

cd4.naive.msigdb.down <- cd4.naive.enriched.down[["MSigDB_Hallmark_2020"]]

cd4.naive.msigdb.down$Dir <- "Down"

cd4.naive.msigdb <- do.call(rbind.data.frame, list(cd4.naive.msigdb.up, cd4.naive.msigdb.down))

cd4.naive.msigdb$logP <- -log10(cd4.naive.msigdb$P.value)

cd4.naive.msigdb$logP[cd4.naive.msigdb$Dir %in% c("Down")] <- -1 * cd4.naive.msigdb$logP[cd4.naive.msigdb$Dir %in% c("Down")]

ggplot(cd4.naive.msigdb[cd4.naive.msigdb$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

```{r, fig.height=4.15, fig.width=4.15}

cd4.naive.tfppi.up <- cd4.naive.enriched.up[["Transcription_Factor_PPIs"]]

cd4.naive.tfppi.up$Dir <- "Up"

cd4.naive.tfppi.down <- cd4.naive.enriched.down[["Transcription_Factor_PPIs"]]

cd4.naive.tfppi.down$Dir <- "Down"

cd4.naive.tfppi <- do.call(rbind.data.frame, list(cd4.naive.tfppi.up, cd4.naive.tfppi.down))

cd4.naive.tfppi$logP <- -log10(cd4.naive.tfppi$P.value)

cd4.naive.tfppi$logP[cd4.naive.tfppi$Dir %in% c("Down")] <- -1 * cd4.naive.tfppi$logP[cd4.naive.tfppi$Dir %in% c("Down")]

ggplot(cd4.naive.tfppi[cd4.naive.tfppi$P.value < 0.01, ],

       aes(x=reorder(Term, logP), y=logP)) +

    geom_hline(yintercept=0, lty=2, colour='grey80') +

    geom_point() +

    coord_flip() +

    labs(x="Pathway", y="Odds Rato") +

    theme_cowplot()

```

## CD8.Naive

```{r, fig.height=3.95, fig.width=4.95}

cd8.naive.labels <- quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD8.Naive"), ]$X

cd8.naive.labels[quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD8.Naive"), ]$FDR > 1e-3] <- ""

ggplot(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD8.Naive"), ],

       aes(x=logFC, y=-log10(FDR))) +

    geom_point() +

    theme_cowplot() +

    geom_text_repel(aes(label=cd8.naive.labels)) +

    ggsave("~/Dropbox/COVID19/Updated_plots/CD8.Naive-quadratic_volcano.pdf",

           height=3.95, width=4.95, useDingbat=FALSE) +

    NULL

```

What are the enriched pathways amongst these genes?

```{r}

cd8.naive.enriched.up <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD8.Naive") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC > 0, ]$X,

                              enrich.dbs)

cd8.naive.enriched.down <- enrichr(quadratic.dge.df[quadratic.dge.df$Sub.Annotation %in% c("CD8.Naive") &

                                                quadratic.dge.df$FDR < 0.01 &

                                                quadratic.dge.df$logFC < 0, ]$X,

                              enrich.dbs)

```

## MSigDB 2020 enrichments

```{r, fig.height=4.15, fig.width=4.15}

cd8.naive.msigdb.up <- cd8.naive.enriched.up[["MSigDB_Hallmark_2020"]]

cd8.naive.msigdb.up$Dir <- "Up"

cd8.naive.msigdb.down <- cd8.naive.enriched.down[["MSigDB_Hallmark_2020"]]

cd8.naive.msigdb.down$Dir <- "Down"

cd8.naive.msigdb <- do.call(rbind.data.frame, list(cd8.naive.msigdb.up, cd8.naive.msigdb.down))

cd8.naive.msigdb$logP <- -log10(cd8.naive.msigdb$P.value)