227 lines (148 with data), 4.6 kB

---
title: "Map 5 prime single cell RNA data"
author: Tobias Roider
date: "Last compiled on `r format(Sys.time(), '%d %B, %Y, %X')`"
output: 
  
  rmdformats::readthedown
editor_options: 
  chunk_output_type: inline
---

```{r options, include=FALSE, warning = FALSE}

library(knitr)
opts_chunk$set(echo=TRUE, tidy=FALSE, include=TRUE, message=FALSE,
               dpi = 100, cache = FALSE, warning = FALSE)
opts_knit$set(root.dir = "../")
options(bitmapType = "cairo")

```

# Load packages and functions
```{r Load packages and functions}

library(Seurat)
library(tidyverse)
library(readxl)

source("R/ReadPackages.R")
source("R/Functions.R")
source("R/ThemesColors.R")

mytheme <- 
  theme_bw()+
  theme(legend.position = "none",
        plot.title = element_text(hjust = 0.5),
        panel.grid = element_blank())
  
```

# Read data
```{r read data}

Combined_T <- readRDS("output/Tcells_Integrated.rds")

```

# Read and Process 5' scRNA data
```{r Read and Process}

files <- list.files(path = "countMatrices", pattern = "_Tcells_5'RNA.txt", full.names = T)
names(files) <- strsplit(files, split = "_") %>% sapply("[[", 1) %>% strsplit(split = "/") %>% sapply("[[", 2)

sobjs_T <- lapply(1:length(files), function(x) {
  
  # Read count tables
  rna <- read.delim(files[x], sep = " ")
  
  # Create Seurat Object
  sobj <- CreateSeuratObject(counts = rna)

  # Add Percentage of mitochondrial genes and PatientID
  sobj[["percent.mt"]] <- PercentageFeatureSet(sobj, pattern = "^MT-")
  sobj$PatientID <- names(files)[x]
  
  # Normalize data
  sobj <- NormalizeData(sobj, normalization.method = "LogNormalize", scale.factor = 10000)
  
  # Run Seurat Processing
  sobj <- SeuratProc_T(sobj, verbose=FALSE, 
                       dims.clustering=1:14, 
                       resolution.clustering = 0.4, 
                       dims.umap=1:13)
  
  return(sobj)
  
  })

names(sobjs_T) <- names(files) 

```

# Map individual samples to CITE-seq reference
```{r mapping}

sobjs_T_mapped <- lapply(sobjs_T, function(sobj) {
    
    anchors <- FindTransferAnchors(reference = Combined_T, query = sobj, 
                                   reference.reduction = "pcaRNA")
  
    sobj <- MapQuery(anchorset = anchors, reference = Combined_T, 
                     query = sobj, reduction.model = "wnn.umap",
                     refdata = list(celltype = "IdentI"),
                     reference.reduction = "pcaRNA")
    
    return(sobj)
    
    })

names(sobjs_T_mapped) <- names(sobjs_T)


```

# Plots
```{r generate plots}

p1 <- 
  DimPlot(Combined_T, group.by = "IdentI", raster = TRUE, raster.dpi = c(500,500),
          reduction = "wnn.umap", label = TRUE)+
  scale_color_manual(values = colors_umap_cl)+
  xlim(-10, 13)+ylim(-10, 10)+
  coord_fixed()+
  mytheme

p2 <- lapply(names(sobjs_T), function(sample){
  DimPlot(subset(Combined_T, subset=PatientID==sample), raster.dpi = c(500,500), raster = TRUE,
          reduction = "wnn.umap", group.by = "IdentI", label = TRUE)+
     scale_color_manual(values = colors_umap_cl)+
    xlim(-10, 13)+ylim(-10, 10)+
    coord_fixed()+
    mytheme
  }) %>% `names<-`(names(sobjs_T))

p3 <- lapply(sobjs_T_mapped, function(sample){
  DimPlot(sample, label = TRUE, raster.dpi = c(500,500), raster = TRUE,
          group.by = "predicted.celltype", reduction = "ref.umap")+
     scale_color_manual(values = colors_umap_cl)+
    xlim(-10, 13)+ylim(-10, 10)+
    coord_fixed()+
    mytheme
  }) %>% `names<-`(names(sobjs_T_mapped))

```

## LN0078
```{r LN0078, fig.height=4, fig.width=10}

p1+p2$LN0078+p3$LN0078

```

## LN0132
```{r LN0132, fig.height=4, fig.width=10}

p1+p2$LN0132+p3$LN0132

```

## LN0144
```{r LN0144, fig.height=4, fig.width=10}

p1+p2$LN0144+p3$LN0144

```

## LN0178
```{r LN0178, fig.height=4, fig.width=10}

p1+p2$LN0178+p3$LN0178

```

## LN0193
```{r LN0193, fig.height=4, fig.width=10}

p1+p2$LN0193+p3$LN0193

```

## LN0217
```{r LN0217, fig.height=4, fig.width=10}

p1+p2$LN0217+p3$LN0217

```

## LN0302
```{r LN0302, fig.height=4, fig.width=10}

p1+p2$LN0302+p3$LN0302

```

## LN0110
```{r LN0110, fig.height=4, fig.width=10}

p1+p2$LN0110+p3$LN0110

```

## LN0198
```{r LN0198, fig.height=4, fig.width=10}

p1+p2$LN0198+p3$LN0198

```

## LN0259
```{r LN0259, fig.height=4, fig.width=10}

p1+p2$LN0259+p3$LN0259

```

## LN0278
```{r LN0278, fig.height=4, fig.width=10}

p1+p2$LN0278+p3$LN0278

```

# Save object
```{r save, eval=FALSE}

saveRDS(sobjs_T_mapped, file = "output/List_SeuratObjects_T_5prime.rds")

```

# Session Info
```{r session info}

sessionInfo()

```