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Seurat Objects
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Ashley Richardson committed Feb 29, 2024
1 parent 1ecf810 commit 0401943
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Showing 6 changed files with 2,688 additions and 69 deletions.
9 changes: 6 additions & 3 deletions Fibril_Seurat.RMD
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Expand Up @@ -15,8 +15,9 @@ knitr::opts_chunk$set(echo = TRUE)
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(Seurat)
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library")
library(sctransform, lib.loc = "/hpc/users/richaa21/.Rlib")
library(Seurat, lib.loc = "/hpc/users/richaa21/.Rlib")
library(patchwork)
library(dplyr)
```

Expand Down Expand Up @@ -170,6 +171,8 @@ head([email protected])
# find markers for every cluster compared to all remaining cells, report only the positive
# ones
markers_f <- FindAllMarkers(pbmc.f, only.pos = TRUE)
cluster5.markers <- FindMarkers(pbmc.f, ident.1 = 5, logfc.threshold = 0.25, test.use = "roc", only.pos = TRUE)
```


Expand Down Expand Up @@ -197,7 +200,7 @@ FeaturePlot(pbmc.f, features = c("MS4A1", "GNLY", "CD3E", "CD14", "FCER1A", "FCG
```{r}
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.3.0/site-library")
library(patchwork)
library(tidyr)
f_demuxlet = read.delim("/sc/arion/projects/ad-omics/ashley/data/aF.best", header = T, stringsAsFactors = F, check.names = F)
head(f_demuxlet)
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886 changes: 886 additions & 0 deletions Fibril_Seurat.html
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34 changes: 2 additions & 32 deletions Monomer_Seurat.RMD
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Expand Up @@ -16,7 +16,7 @@ knitr::opts_chunk$set(echo = TRUE)
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(Seurat)
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library")
library(patchwork)
library(dplyr)
```

Expand Down Expand Up @@ -197,7 +197,7 @@ FeaturePlot(pbmc.m, features = c("MS4A1", "GNLY", "CD3E", "CD14", "FCER1A", "FCG
```{r}
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.3.0/site-library")
library(patchwork)
library(tidyr)
m_demuxlet = read.delim("/sc/arion/projects/ad-omics/ashley/data/aM.best", header = T, stringsAsFactors = F, check.names = F)
head(m_demuxlet)
Expand Down Expand Up @@ -271,36 +271,6 @@ names([email protected])[names([email protected]) == "m_demuxlet_edit.subset$BARCO
head([email protected])
```


## Remove douplets.
Note - It is a good idea to compare doublet determination by dumuxlet and another resource (i.e. DoupletFinder or scDblFinder). However, these packages do not work with SeuratV5, which is what I used to create my SeuratObject.
For now, I am just removing doublets defined by demuxlet.

```{r}
#Idents(pbmc.m) <- pbmc.m$DMX_classification.global
#pbmc.m_sing <- subset(x = pbmc.m, idents = c("SNG"), invert = F)
## confirm that there are no doublets left
#table(pbmc.m_sing$DMX_classification.global)
```

# Add metadata information.
```{r}
# Add patient data to Seurat object meta data.
#pt_data <- data.frame(DMX_maxID = c("NYUMD0289", "NYUMD0315", "NYUMD0327", "NYUMD0334"),
#DX = c("PD", "PD", "CO", "CO"),
# Sex = c("Male", "Male", "Male", "Male"),
#Age = c(68, 65, 65, 60))
#merged_df <- merge([email protected], pt_data, by = "DMX_maxID")
#[email protected] <- merged_df
#head([email protected])
```


```{r Save as .finalRDS file}
saveRDS(pbmc.m, file = "/sc/arion/projects/ad-omics/ashley/PD_Stim/pbmc.monomer.final.RDS")
```
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894 changes: 894 additions & 0 deletions Monomer_Seurat.html
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38 changes: 4 additions & 34 deletions Unstim_Seurat.RMD
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Expand Up @@ -15,8 +15,9 @@ knitr::opts_chunk$set(echo = TRUE)
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(Seurat)
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library")
library(sctransform, lib.loc = "/hpc/users/richaa21/.Rlib")
library(Seurat, lib.loc = "/hpc/users/richaa21/.Rlib")
library(patchwork)
library(dplyr)
```

Expand Down Expand Up @@ -107,7 +108,7 @@ VizDimLoadings(pbmc.unstim, dims = 1:2, reduction = "pca")
```

```{r PC2 vs PC1 scatter plot, warning=FALSE}
DimPlot(pbmc.unstim, reduction = "pca") + NoLegend()
DimPlot(pbmc.unstim, reduction = "pca")
```

```{r Heatmap of PC1, warning=FALSE}
Expand Down Expand Up @@ -196,7 +197,6 @@ FeaturePlot(pbmc.unstim, features = c("MS4A1", "GNLY", "CD3E", "CD14", "FCER1A",
```{r}
.libPaths(c("/hpc/packages/minerva-centos7/rpackages/4.2.0/site-library", "/hpc/packages/minerva-centos7/rpackages/bioconductor/3.15", .libPaths()))
library(patchwork, lib.loc = "/hpc/packages/minerva-centos7/rpackages/4.3.0/site-library")
library(tidyr)
u_demuxlet = read.delim("/sc/arion/projects/ad-omics/ashley/data/Unstim.best", header = T, stringsAsFactors = F, check.names = F)
head(u_demuxlet)
Expand Down Expand Up @@ -270,36 +270,6 @@ names([email protected])[names([email protected]) == "u_demuxlet_edit.su
head([email protected])
```


## Remove douplets.
Note - It is a good idea to compare doublet determination by dumuxlet and another resource (i.e. DoupletFinder or scDblFinder). However, these packages do not work with SeuratV5, which is what I used to create my SeuratObject.
For now, I am just removing doublets defined by demuxlet.

```{r}
#Idents(pbmc.unstim) <- pbmc.unstim$DMX_classification.global
#pbmc.unstim_sing <- subset(x = pbmc.unstim, idents = c("SNG"), invert = F)
## confirm that there are no doublets left
#table(pbmc.unstim_sing$DMX_classification.global)
```

# Add metadata information.
```{r}
# Add patient data to Seurat object meta data.
#pt_data <- data.frame(DMX_maxID = c("NYUMD0289", "NYUMD0315", "NYUMD0327", "NYUMD0334"),
#DX = c("PD", "PD", "CO", "CO"),
# Sex = c("Male", "Male", "Male", "Male"),
#Age = c(68, 65, 65, 60))
#merged_df <- merge([email protected], pt_data, by = "DMX_maxID")
#[email protected] <- merged_df
#head([email protected])
```


```{r Save as .finalRDS file}
saveRDS(pbmc.unstim, file = "/sc/arion/projects/ad-omics/ashley/PD_Stim/pbmc.unstim.final.RDS")
```
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896 changes: 896 additions & 0 deletions Unstim_Seurat.html
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