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3_DApeaksInNonTBC.Rmd
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3_DApeaksInNonTBC.Rmd
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---
title: "scATAC-seq Rat Metrial Glands"
author: "Ha T. H. Vu"
output: html_document
---
```{r setup, include=FALSE}
options(max.print = "75")
knitr::opts_chunk$set(
echo = TRUE,
collapse = TRUE,
comment = "#>",
fig.path = "Files/",
fig.width = 15,
prompt = FALSE,
tidy = FALSE,
message = FALSE,
warning = TRUE
)
knitr::opts_knit$set(width = 75)
```
This is a documentation for analyses of scATAC-seq data, generated from rat metrial gland tissues on gestational day (GD) 15.5 and 19.5. <br>
## GD 15.5:
```{r}
library(Signac)
library(Seurat)
library(ggplot2)
library(GenomicRanges)
library(patchwork)
set.seed(1234)
load("/work/LAS/geetu-lab/hhvu/project3_scATAC/rnor6.rda")
load("/work/LAS/geetu-lab/hhvu/project3_scATAC/rnor6.ranges.rda")
load("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-ATACwithRNAlables.rda")
Idents(rats) <- [email protected]$predicted.id # change from old identities to those predicted by scRNA-seq
DefaultAssay(rats) <- 'MACSpeaks'
Annotation(rats) <- rnor6.ranges
rats1.markers <- read.table("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-unfilteredMarkers.txt", header = T, sep = "\t")
```
Natural killer cells:
```{r}
nk15.5 <- rats1.markers[rats1.markers$cluster == "Natural killer cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(nk15.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-nk.txt", quote = F, sep = '\t')
dim(nk15.5)
closest_genes <- ClosestFeature(rats, regions = rownames(nk15.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-nk-toGenes.txt", quote = F, sep = '\t')
```
Smooth muscle cells:
```{r}
sm15.5 <- rats1.markers[rats1.markers$cluster == "Smooth muscle cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(sm15.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-sm.txt", quote = F, sep = '\t')
dim(sm15.5)
closest_genes <- ClosestFeature(rats, regions = rownames(sm15.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-sm-toGenes.txt", quote = F, sep = '\t')
```
Endothelial cells:
```{r}
endo15.5 <- rats1.markers[rats1.markers$cluster == "Endothelial cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(endo15.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-endo.txt", quote = F, sep = '\t')
dim(endo15.5)
closest_genes <- ClosestFeature(rats, regions = rownames(endo15.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-endo-toGenes.txt", quote = F, sep = '\t')
```
Macrophage cells:
```{r}
macro15.5 <- rats1.markers[rats1.markers$cluster == "Macrophage cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(macro15.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-macro.txt", quote = F, sep = '\t')
dim(macro15.5)
closest_genes <- ClosestFeature(rats, regions = rownames(macro15.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-macro-toGenes.txt", quote = F, sep = '\t')
```
In BASH, run the following command to obtain a bed file of the peaks:
```{BASH, eval = F}
cd /work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/
for i in gd15.5-endo.txt gd15.5-macro.txt gd15.5-nk.txt gd15.5-sm.txt; do e=`echo $i | sed 's/txt/bed/g'`; cut -f 6 $i | tail -n +2 | sed 's/-/\t/g' | awk '{print $1"\t"$2"\t"$3"\t"$1"-"$2"-"$3}' > $e; done
```
We can look at the distribution of the cell type-enriched peaks. For natural killer cells (NKs):
```{r}
library(ChIPseeker)
library(GenomicFeatures)
library(RMariaDB)
txdb <- makeTxDbFromEnsembl(organism= "Rattus norvegicus", release = 98)
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-nk.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-15.5-NKs.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - natural killer cells GD15.5")
#dev.off()
```
Smooth muscle cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-sm.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-15.5-sm.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - smooth muscle cells GD15.5")
#dev.off()
```
Endothelial cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-endo.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-15.5-endo.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - Endothelial cells GD15.5")
#dev.off()
```
Macrophage cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd15.5-macro.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-15.5-macro.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - Macrophage cells GD15.5")
#dev.off()
```
## GD 19.5:
```{r}
set.seed(1234)
load("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-ATACwithRNAlables.rda")
Idents(rats) <- [email protected]$predicted.id # change from old identities to those predicted by scRNA-seq
DefaultAssay(rats) <- 'MACSpeaks'
Annotation(rats) <- rnor6.ranges
rats1.markers <- read.table("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-unfilteredMarkers.txt", header = T, sep = "\t")
```
Natural killer cells:
```{r}
nk19.5 <- rats1.markers[rats1.markers$cluster == "Natural killer cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(nk19.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-nk.txt", quote = F, sep = '\t')
dim(nk19.5)
closest_genes <- ClosestFeature(rats, regions = rownames(nk19.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-nk-toGenes.txt", quote = F, sep = '\t')
```
Smooth muscle cells:
```{r}
sm19.5 <- rats1.markers[rats1.markers$cluster == "Smooth muscle cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(sm19.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-sm.txt", quote = F, sep = '\t')
dim(sm19.5)
closest_genes <- ClosestFeature(rats, regions = rownames(sm19.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-sm-toGenes.txt", quote = F, sep = '\t')
```
Endothelial cells:
```{r}
endo19.5 <- rats1.markers[rats1.markers$cluster == "Endothelial cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(endo19.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-endo.txt", quote = F, sep = '\t')
dim(endo19.5)
closest_genes <- ClosestFeature(rats, regions = rownames(endo19.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-endo-toGenes.txt", quote = F, sep = '\t')
```
Macrophage cells:
```{r}
macro19.5 <- rats1.markers[rats1.markers$cluster == "Macrophage cells" & rats1.markers$avg_log2FC >= log2(1.5) & rats1.markers$p_val_adj <= 0.05,]
#write.table(macro19.5, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-macro.txt", quote = F, sep = '\t')
dim(macro19.5)
closest_genes <- ClosestFeature(rats, regions = rownames(macro19.5))
length(unique(closest_genes$gene_name))
#write.table(closest_genes, "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-macro-toGenes.txt", quote = F, sep = '\t')
```
In BASH, run the following command to obtain a bed file of the TBC peaks:
```{BASH, eval = F}
cd /work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/
for i in gd19.5-endo.txt gd19.5-macro.txt gd19.5-nk.txt gd19.5-sm.txt; do e=`echo $i | sed 's/txt/bed/g'`; cut -f 8 $i | tail -n +2 | sed 's/-/\t/g' | awk '{print $1"\t"$2"\t"$3"\t"$1"-"$2"-"$3}' > $e; done
```
We can look at the distribution of the cell type-enriched peaks. For natural killer cells (NKs):
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-nk.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-19.5-NKs.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - natural killer cells GD19.5")
#dev.off()
```
Smooth muscle cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-sm.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-19.5-sm.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - smooth muscle cells GD19.5")
#dev.off()
```
Endothelial cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-endo.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-19.5-endo.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - Endothelial cells GD19.5")
#dev.off()
```
Macrophage cells:
```{r}
file <- "/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/4_RNAintegration/2_MACS2Peaks/annotation_scRNAseq/gd19.5-macro.bed"
peakAnno <- annotatePeak(file, tssRegion=c(-3000, 3000), TxDb=txdb)
#pdf("/work/LAS/geetu-lab/hhvu/project3_scATAC/scATAC-seq-analysis/FIGURES/20220829_draftV3/figureS3-19.5-macro.#pdf", height = 5, width = 6)
plotDistToTSS(peakAnno,
title="Distribution of transcription factor-binding loci relative to TSS - Macrophage cells GD19.5")
#dev.off()
```
```{r}
sessionInfo()
```