forked from MelinaKlostermann/Molitor-et-al-2022
-
Notifications
You must be signed in to change notification settings - Fork 0
/
6_Compare_RNAseq_CLIP.Rmd
220 lines (165 loc) · 8.59 KB
/
6_Compare_RNAseq_CLIP.Rmd
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
---
title: "6 Comparison of PURA RNA binding to expression changes in PURA knockdown "
author: "Melina"
date: "`r format(Sys.time(), '%d %B, %Y')`"
output:
BiocStyle::html_document:
toc_float: TRUE
code_folding: hide
toc: TRUE
---
```{r setup, include=FALSE}
require("knitr")
knitr::opts_chunk$set(warning=FALSE, message=FALSE, cache=TRUE) #, fig.pos = "!H", out.extra = ""
```
```{r libraries, include=FALSE}
library(GenomicRanges)
library(knitr)
library(dplyr)
library(ggpubr)
library(DESeq2)
library(purrr)
report_color <- (pals::ocean.solar(15))
source("/Users/melinaklostermann/Documents/projects/PURA/02_R_new_pip/XX-helpful-chunks/theme_paper.R")
outpath <- "/Users/melinaklostermann/Documents/projects/PURA/Molitor-et-al-2022/"
```
# What was done?
- Boxplot comparisons of RNA Fold changes between bound and unbound regions
- coloring of vulcano by region of binding site
# Input
```{r}
# binding sites
BS <- readRDS("/Users/melinaklostermann/Documents/projects/PURA/Molitor-et-al-2022/binding_sites_characterized.rds")
# deseq analysis from PURA knockdown
res_RNAseq <- readRDS("/Users/melinaklostermann/Documents/projects/PURA/Molitor-et-al-2022/deseq.rds")
```
# Combine RNAseq and CLIP
```{r}
############################
# combine RNAseq and both CLIPs
###########################
# strongest BS per gene
strongest_Bs_bound_gene <- BS %>% as.data.frame(.) %>%
group_by(gene_id) %>%
arrange(desc(score), .by_group = T)%>%
dplyr::slice(1) %>%
mutate(gene_id = substring(gene_id,1,15))
# mark bound and unbound
res_RNAseq_CLIP <- mutate(res_RNAseq, gene_id = substr(gene_id, 1, 15),
CLIP_bound=case_when(gene_id %in% strongest_Bs_bound_gene$gene_id ~ "bound",
T ~ "unbound"),
changing = case_when(log2FoldChange==0 ~ F,
is.na(log2FoldChange) ~ F,
T~T))
# add matching scores
res_RNAseq_CLIP <- merge(res_RNAseq_CLIP, strongest_Bs_bound_gene, by = "gene_id", all.x=T)
# remove unexpressed genes
unexpressed_genes <- res_RNAseq_CLIP[res_RNAseq_CLIP$baseMean == 0,]
# unexpressed genes that where bound?
unexpressed_genes_unbound <- unexpressed_genes[unexpressed_genes$CLIP_bound=="unbound",]
unexpressed_genes_bound <- unexpressed_genes[unexpressed_genes$CLIP_bound=="bound",]
# n number of bound "unexpressed " genes ( -> polyA selection)
unexpressed_genes_bound %>% summarise(n_distinct(gene_id))
# keep expressed as results
res_RNAseq_CLIP <- res_RNAseq_CLIP[!(res_RNAseq_CLIP$gene_id %in% unexpressed_genes$gene_id),]
# remove empty rows
res_RNAseq_CLIP <- res_RNAseq_CLIP[!is.na(res_RNAseq_CLIP$baseMean),]
# sign change
res_RNAseq_CLIP <- res_RNAseq_CLIP %>%
mutate(sig.change = case_when(padj<=0.01 ~T,
T~F))
table(res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change==T,]$region)
```
# Boxplot comparison
```{r}
plot_box_all <- ggplot(res_RNAseq_CLIP, aes(x=CLIP_bound, y=log2FoldChange, fill = CLIP_bound))+
geom_boxplot()+
coord_cartesian(ylim = c(-3.5,3.5))+
ggpubr::stat_compare_means(method = "t.test", vjust = 4)+
stat_summary(fun = "mean", geom="point",colour="black", size=3) +
stat_summary(fun= "mean", geom="text", show.legend = FALSE,
vjust=-1.3, hjust=-0.3, aes(label=round(..y.., digits=3)))+
theme_paper()+
scale_fill_manual(values= c("darkred", "grey"), name = "iClip Hit")+
geom_hline(yintercept = 0)+
theme(legend.position = "None")
plot_box_all
ggsave(plot_box_all, file=paste0(outpath,"rnaseq_clip_box_all.pdf"), width = 4, height = 4, units = "cm")
# boxplot regions
res_RNAseq_CLIP$region <- factor(res_RNAseq_CLIP$region, levels = c("three_prime_UTR", "five_prime_UTR", "CDS", "non_coding"))
plot_box_region <- ggplot(res_RNAseq_CLIP, aes(x=region, y=log2FoldChange, fill = region, levels))+
geom_boxplot()+
theme_paper()+
scale_fill_manual(values= c( "darkred","orange", "#2AB7CA","purple", "darkgreen"), name = "iClip Hit")+
geom_hline(yintercept = 0)+
theme(legend.position = "None")
plot_box_region
ggsave(plot_box_region, file=paste0(outpath,"rnaseq_clip_box_region.pdf"), width = 4, height = 4, units = "cm")
table(res_RNAseq_CLIP$region)
```
# Color vulcano by bound region
```{r}
####################
# vulcanos with one region of BS colored each
###################
# 3"UTR
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj), ), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$region =="three_prime_UTR",], aes(x=log2FoldChange, y= -log10(padj)), color = "darkred", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_3UTR.pdf"), width = 4, height = 4, units = "cm")
# 5'UTR
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj)), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$region =="five_prime_UTR",], aes(x=log2FoldChange, y= -log10(padj)), color = "orange", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_5UTR.pdf"), width = 4, height = 4, units = "cm")
# CDS
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj)), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$region =="CDS",], aes(x=log2FoldChange, y= -log10(padj)), color = "#2AB7CA", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_CDS.pdf"), width = 4, height = 4, units = "cm")
# non coding
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj)), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$region =="non_coding",], aes(x=log2FoldChange, y= -log10(padj)), color = "purple", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_non_cod.pdf"), width = 4, height = 4, units = "cm")
# intron
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj)), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$region =="intron",], aes(x=log2FoldChange, y= -log10(padj)), color = "blue", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_intron.pdf"), width = 4, height = 4, units = "cm")
# unbound
ggplot()+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==F,], aes(x=log2FoldChange, y= -log10(padj)), color = "grey", shape = 1, size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T,], aes(x=log2FoldChange, y= -log10(padj)), size = 0.5), dpi = 300)+
ggrastr::rasterise(geom_point(data = res_RNAseq_CLIP[res_RNAseq_CLIP$sig.change ==T & res_RNAseq_CLIP$CLIP_bound == "unbound",], aes(x=log2FoldChange, y= -log10(padj)), color = "darkgreen", size = 0.5), dpi = 300)+
theme_bw()+
xlim(c(-2,2))+
ylim(c(0,40))+
theme_paper()
ggsave(paste0(outpath, "rnaseq_vulcano_unbound.pdf"), width = 4, height = 4, units = "cm")
```