From b74ffb04393f31de3aca5c514166c89c281b6c94 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 15:59:14 -0300 Subject: [PATCH 01/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 6 ++---- 1 file changed, 2 insertions(+), 4 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 5a41f91..7659bfb 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -1,7 +1,5 @@ # Single-cell transcriptomics -## Author: T Attenborough -## Affiliation: University of Glasgow - +### Author: Santiago Fontenla, addapted from tutorial by Teresa Attenborough ## Table of Contents 1. [Overview and Aims](#intro) @@ -654,4 +652,4 @@ plot1 <- DimPlot(day2somules, reduction = "umap", label = FALSE, repel = TRUE, l LabelClusters(plot1, id = "ident", color = 'black', size =4, repel = T, box.padding = 1.75, max.overlaps=Inf) ggsave(paste0("day2somules-labelledlclusters_40PC_0.5res_",st,".jpg"), width = 25, height = 15) -``` \ No newline at end of file +``` From efde2b2102d184d3b7dcefe84eb28b3ad2cbb0f0 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 16:06:13 -0300 Subject: [PATCH 02/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 7659bfb..c3b005b 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -23,7 +23,7 @@ In this module, we will cover some basic concepts in single-cell transcriptomics --- -[ **Back to top**](#top) +[↥ **Back to top**](#top) ## Introduction to single cell transcriptomics @@ -185,7 +185,7 @@ head(sample1@meta.data$orig.ident) ``` If you're not sure which metadata are available, or you want a summary of metadata in each category, that's an easy way to check. Anything that comes after the R object name and a dollar sign is a metadata value so you can look at it in a table as above, or in a UMAP, once you've generated one. -[ **Back to top**](#top) +[↥ **Back to top**](#top) ## Doublet ID @@ -266,7 +266,7 @@ table(day2somules@meta.data$batches) ``` -[ **Back to top**](#top) +[↥ **Back to top**](#top) ## QC removal @@ -400,7 +400,7 @@ day2somules <- readRDS(file = "day2somules_v10_firstfilt.rds") ``` -[ **Back to top**](#top) +[↥ **Back to top**](#top) ## Normalisation and scaling From 11addaeb235e3746808c9f269c6e38d353ffd372 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 17:28:44 -0300 Subject: [PATCH 03/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index c3b005b..ff25ad3 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -17,7 +17,7 @@ ## Overview and Aims -In this module, we will cover some basic concepts in single-cell transcriptomics. In the example we will use data of the somule stage from _Schistosoma mansoni_. We will start examining the data structure of counts per sample, filter low quality cells +In this module, you will learn basic concepts about the technology and data structure related to single-cell generated data. We will use data generated from 3 replicates of the 2 days somules of _Schistosoma mansoni_. We will start examining the raw data and the structure of the count matrices generated with Cell Ranger, we will transform these matrices to Seurat objects and we will generate some plots to visualize the data. Then we will remove doublet cells and do quality filter to retain good quality cells with the remianing cells we will low quality cells From 089d52c843265abc48de3734a2c69a1c3bdd2031 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 17:48:34 -0300 Subject: [PATCH 04/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index ff25ad3..3931afe 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -29,8 +29,7 @@ In this module, you will learn basic concepts about the technology and data stru Single-cell RNA sequencing (scRNA-seq) technology has become the state-of-the-art approach for unravelling the heterogeneity and complexity of RNA transcripts within individual cells, as well as revealing the composition of different cell types and functions within highly organized tissues/organs/organims. scRNA-seq allows the analysis of individual cells, revealing subtle differences in gene expression that might be masked in bulk sequencing, potentially identifying rare cell populations. However scRNA-seq can be expensive and complex to set up and performa, limiting its widespread adoption. -The scRNA-seq data used in the module was obtained from 2 day schistosomula of _S.mansoni_ after _in vitro_ transformation of cercariae. Single-cells were obtained after tissue dissociation with a digestion solution. Single-cell concentration was estimated by flow-cytometry and loaded according to the 10X Chromium Single Cell 3' Reagent Kits v2 protocol. Library construction was done using standar protocols and sequenced on an Illumina HiSeq4000 (PE reads 75 bp) [reference](https://www.nature.com/articles/s41467-020-20092-5). For these tutorial, scRNA-seq data were mapped to the reference genome V10 using Cell Ranger (version 7.0.1) - [here](https://www.10xgenomics.com/support/software/cell-ranger/latest) +The scRNA-seq data used in the module was obtained from 2 day schistosomula of _S.mansoni_ after _in vitro_ transformation of cercariae. Single-cells were obtained after tissue dissociation with a digestion solution. Single-cell concentration was estimated by flow-cytometry and loaded according to the 10X Chromium Single Cell 3' Reagent Kits v2 protocol. Library construction was done using standar protocols and sequenced on an Illumina HiSeq4000 (PE reads 75 bp) [ref](https://www.nature.com/articles/s41467-020-20092-5). For these tutorial, scRNA-seq data were mapped to the reference genome V10 using [Cell Ranger (version 7.0.1)](https://www.10xgenomics.com/support/software/cell-ranger/latest) --- @@ -50,6 +49,7 @@ _Three key metrics are presented at the top left of the Summary tab: *Median Genes per Cell: the median number of genes detected per cell-associated barcode. This metric is dependent on cell type and sequencing depth. ![](figures/SC_Figure_2.png) + **Figure 2.** Main metrics of Cell Ranger run We are not planning to go through the rest of the sections and metrics, for more details of each metrics and their expected range, please refer to the [10xGenomics_documentation](https://www.10xgenomics.com/analysis-guides/quality-assessment-using-the-cell-ranger-web-summary) From aa63def9f909b12f92fc7268c962b0fe5175eef3 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 18:01:38 -0300 Subject: [PATCH 05/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 16 ++++++++-------- 1 file changed, 8 insertions(+), 8 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 3931afe..798995c 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -42,11 +42,11 @@ After sequencing, reads are mapped to the reference genome with Cell Ranger, a s Navigate to the output folder of Cell Ranger, within each sample folder you will find a web_summary of the run and a feature-barcode folder containing the feature-barcode_matrix. Inspect each web_summary. -_Three key metrics are presented at the top left of the Summary tab: +Three key metrics are presented at the top left of the Summary tab: -*Estimated Number of Cells: the number of barcodes associated with cells. -*Mean Reads per Cell: the total number of sequenced reads divided by the number of cells. We recommend a minimum of 20,000 read pairs per cell. -*Median Genes per Cell: the median number of genes detected per cell-associated barcode. This metric is dependent on cell type and sequencing depth. +- **Estimated Number of Cells**: the number of barcodes associated with cells. +- **Mean Reads per Cell**: the total number of sequenced reads divided by the number of cells. A minimum of 20,000 read pairs per cell is recommended. +- **Median Genes per Cell**: the median number of genes detected per cell-associated barcode. This metric is dependent on cell type and sequencing depth. ![](figures/SC_Figure_2.png) @@ -101,11 +101,11 @@ Here we save an object that contains today's date, so we can save any files with st <- format(Sys.time(), "%Y-%m-%d") ``` -####Loading data +#### Importing the data to R -Next, we import the data files that contain the mapping outputs from Cellranger. There are three folders, from the three samples which were sequenced. Each folder contains the list of genes, the cell barcodes, and the count matrix showing the number of transcripts. +Next, we import the data files that contain the mapping outputs from Cell Ranger. There are three folders, from the three samples which were sequenced. Each folder contains the list of genes, the cell barcodes, and the count matrix showing the number of transcripts. -Since the we used 10X sequencing, we import this into R using the specific 10X import function. +Since the data was generated with 10X sequencing, we import this into R using the specific 10X import function. ```R @@ -382,7 +382,7 @@ dim(day2somules) #shows number of genes and number of cells How many cells were removed in total and by batch? -###Save and start next analysis +### Save and start next analysis Now that you have completed the QC filter, you can save your analysis in a R object. From 1ca7133eaca754924f0eda12b21813984b5c9edb Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Tue, 29 Apr 2025 18:37:58 -0300 Subject: [PATCH 06/51] Update Single_cell_module.md --- .../Single_cell_module.md | 55 ++++++++++++------- 1 file changed, 34 insertions(+), 21 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 3931afe..87dfd52 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -9,9 +9,9 @@ 5. [QC removal](#QCremoval) 6. [Normalizing and Scaling the data](#N&S) 7. [SCTransform of data](#SCTrasform) +8. [Plot UMAPs](#plotUMAPs) -8. [Exploring gene expression using gene plots and heatmaps](#plots) -9. [Functional analysis using GO term enrichment](#go) +10. [Functional analysis using GO term enrichment](#go) --- @@ -23,7 +23,7 @@ In this module, you will learn basic concepts about the technology and data stru --- -[↥ **Back to top**](#top) +[↥ **Back to top**](#top) ## Introduction to single cell transcriptomics @@ -42,11 +42,11 @@ After sequencing, reads are mapped to the reference genome with Cell Ranger, a s Navigate to the output folder of Cell Ranger, within each sample folder you will find a web_summary of the run and a feature-barcode folder containing the feature-barcode_matrix. Inspect each web_summary. -_Three key metrics are presented at the top left of the Summary tab: +Three key metrics are presented at the top left of the Summary tab: -*Estimated Number of Cells: the number of barcodes associated with cells. -*Mean Reads per Cell: the total number of sequenced reads divided by the number of cells. We recommend a minimum of 20,000 read pairs per cell. -*Median Genes per Cell: the median number of genes detected per cell-associated barcode. This metric is dependent on cell type and sequencing depth. +- **Estimated Number of Cells**: the number of barcodes associated with cells. +- **Mean Reads per Cell**: the total number of sequenced reads divided by the number of cells. A minimum of 20,000 read pairs per cell is recommended. +- **Median Genes per Cell**: the median number of genes detected per cell-associated barcode. This metric is dependent on cell type and sequencing depth. ![](figures/SC_Figure_2.png) @@ -101,11 +101,11 @@ Here we save an object that contains today's date, so we can save any files with st <- format(Sys.time(), "%Y-%m-%d") ``` -####Loading data +#### Importing the data to R -Next, we import the data files that contain the mapping outputs from Cellranger. There are three folders, from the three samples which were sequenced. Each folder contains the list of genes, the cell barcodes, and the count matrix showing the number of transcripts. +Next, we import the data files that contain the mapping outputs from Cell Ranger. There are three folders, from the three samples which were sequenced. Each folder contains the list of genes, the cell barcodes, and the count matrix showing the number of transcripts. -Since the we used 10X sequencing, we import this into R using the specific 10X import function. +Since the data was generated with 10X sequencing, we import this into R using the specific 10X import function. ```R @@ -185,7 +185,7 @@ head(sample1@meta.data$orig.ident) ``` If you're not sure which metadata are available, or you want a summary of metadata in each category, that's an easy way to check. Anything that comes after the R object name and a dollar sign is a metadata value so you can look at it in a table as above, or in a UMAP, once you've generated one. -[↥ **Back to top**](#top) +[↥ **Back to top**](#top) ## Doublet ID @@ -266,7 +266,7 @@ table(day2somules@meta.data$batches) ``` -[↥ **Back to top**](#top) +[↥ **Back to top**](#top) ## QC removal @@ -382,7 +382,7 @@ dim(day2somules) #shows number of genes and number of cells How many cells were removed in total and by batch? -###Save and start next analysis +### Save and start next analysis Now that you have completed the QC filter, you can save your analysis in a R object. @@ -440,6 +440,7 @@ DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) ``` ![](figures/SC_Figure_8.png) + **Figure 8.** ### Jackstraw @@ -481,7 +482,8 @@ ggsave(paste0("day2somules_v10clust_40PC_0.4res_RNA_",st,".jpg")) ## SCTransform of data -Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat (and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()), though there are functions it's difficult to perform when the data is transformed with this method. +Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()). + ```R # run sctransform day2somules <- SCTransform(day2somules, verbose = TRUE) @@ -489,28 +491,37 @@ day2somules <- SCTransform(day2somules, verbose = TRUE) The results of this are saved in a different 'assay' of the R object - so you can still use both, and the data aren't overwritten. -#PCA on SCT +### PCA on SCT transformed data Now, perform dimensionality reduction by PCA and UMAP embedding Here, we perform PCA on the scaled data. The most variable features selected earlier are used. This follows the same approach as when using the RNA assay we used above. + ```R day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules), npcs=100) VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") #shows the weightings of top contributing features to PCs 1 and 2 DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) #plots heatmap of top 500 most variable cells for PC1, with relative gene expression ``` -#CLustering on SCT + +### Clustering on SCT ```R #here construct k-nearest neighbours graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) day2somules <- FindNeighbors(day2somules, dims = 1:40) #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. day2somules <- FindClusters(day2somules, resolution = 0.5) -#runs umap to visualize the clusters. Need to set the number of PCs +``` +## Plot UMAPs + +Runs umap to visualize the clusters. Need to set the number of PCs + +```R + day2somules <- RunUMAP(day2somules, dims = 1:40) #visualises the UMAP DimPlot(day2somules, reduction = "umap") ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) + ``` ![](figures/SC_Figure_11.png) @@ -541,7 +552,7 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_mt_SCT.jpg") What do you think about this figure? Is there any cluster with a higher proportion of MT RNA? -# Marker identification +## Markers identification We want to understand what the cell clusters might be. One approach to do that is find gene that are cluster markers - find differentially expressed genes that are descriptive of a cluster. @@ -554,8 +565,8 @@ day2somules<-JoinLayers(day2somules) day2somules.markers_roc_cluster0 <- FindMarkers(day2somules, ident.1 = 0, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") ``` - Import annotation information. I have collected this from the genome annotation file. + ```R v10_genelist <- read.csv("v10_genes_with_descriptions_2023-04-17.csv", stringsAsFactors = FALSE, header = TRUE) v10_genelist$X <- NULL @@ -585,7 +596,7 @@ A brainstorm question: how might you use this list to choose a gene to use for i How else might you classify cell cluster tissue types? -#Plot individual genes +### Plot individual genes Now we can look at gene expression in these data by gene. The example below shows you ago 2-1. As above, remove the hash symbol in front of the word ggsave and run this chunk of code if you want to save the plot. If you want to look at a different gene, simply type the gene ID where "Smp-179320" currently sits. Remember to use - rather than _ ! We can visualise genes that we already know something about in the organism, to see if that gives us some clues ```R @@ -594,6 +605,7 @@ FeaturePlot(day2somules, features = "Smp-179320") ``` We can also look at these genes with a violin plot - this visualisation can be helpful in many ways, including seeing which clusters a gene is most expressed in. + ```R VlnPlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320_",st, ".jpg"), width = 25, height = 15) @@ -602,8 +614,9 @@ VlnPlot(day2somules, features = "Smp-179320") Are there any genes you're particularly interested in? You can adapt the code below to see if, and where, it might be expressed in these data. If you're not sure, you can look up S mansoni on WBPS and choose a gene from there (https://parasite.wormbase.org/index.html). Remember to delete the # if you want to save the plot to your computer! ```R FeaturePlot(day2somules, features = "your_gene") -#ggsave(paste0("day2somules-Smp-179320_",st,".jpg"), width = 25, height = 15) +#ggsave(paste0("day2somules-your-gene_",st,".jpg"), width = 25, height = 15) ``` +### Plot co-expressed genes You can also look for co-expression - are two genes you're interested in expressed in the same cells? ```R From b3d452b2ba115305f2b6e0ea5d3aecc9b2942bc9 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Wed, 30 Apr 2025 13:41:39 -0300 Subject: [PATCH 07/51] Update Single_cell_module.md --- .../Single_cell_module.md | 25 +++++++++++++------ 1 file changed, 17 insertions(+), 8 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 87dfd52..a645db6 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -1,5 +1,5 @@ # Single-cell transcriptomics -### Author: Santiago Fontenla, addapted from tutorial by Teresa Attenborough +### Author: Santiago Fontenla, addapted from the tutorial by Teresa Attenborough ## Table of Contents 1. [Overview and Aims](#intro) @@ -10,8 +10,8 @@ 6. [Normalizing and Scaling the data](#N&S) 7. [SCTransform of data](#SCTrasform) 8. [Plot UMAPs](#plotUMAPs) - -10. [Functional analysis using GO term enrichment](#go) +9. [Plot the expression of individual genes in the UMAP](#plotGenes) +10. [Rename UMAP clusters IDs](#renameUMAP) --- @@ -477,7 +477,7 @@ DimPlot(day2somules, reduction = "umap") ggsave(paste0("day2somules_v10clust_40PC_0.4res_RNA_",st,".jpg")) ``` -![](figures/SC_Figure_10.jpg) +![](figures/SC_Figure_10.png) **Figure 10.** ## SCTransform of data @@ -546,7 +546,7 @@ FeaturePlot(day2somules, features="percent.mt", label=TRUE) ggsave("day2somules_v10_40PC_0.5res_after_one_filt_mt_SCT.jpg") ``` -![](figures/SC_Figure_13.png) +![](figures/SC_Figure_13.jpg) **Figure 13.** What do you think about this figure? Is there any cluster with a higher proportion of MT RNA? @@ -596,13 +596,15 @@ A brainstorm question: how might you use this list to choose a gene to use for i How else might you classify cell cluster tissue types? -### Plot individual genes +## Plot the expression of individual genes in the UMAP -Now we can look at gene expression in these data by gene. The example below shows you ago 2-1. As above, remove the hash symbol in front of the word ggsave and run this chunk of code if you want to save the plot. If you want to look at a different gene, simply type the gene ID where "Smp-179320" currently sits. Remember to use - rather than _ ! We can visualise genes that we already know something about in the organism, to see if that gives us some clues +Now we can look at gene expression in these data by gene, which could be useful to identify cluster marker genes. The example below shows you ago 2-1. If you want to look at a different gene, simply type the gene ID where "Smp-179320" currently sits. Remember to use - rather than _ ! We can visualise genes that we already know something about in the organism, to see if that gives us some clues ```R FeaturePlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320-",st, ".jpg"), width = 25, height = 15) ``` +![](figures/SC_Figure_14.png) +**Figure 14.** We can also look at these genes with a violin plot - this visualisation can be helpful in many ways, including seeing which clusters a gene is most expressed in. @@ -610,8 +612,11 @@ We can also look at these genes with a violin plot - this visualisation can be h VlnPlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320_",st, ".jpg"), width = 25, height = 15) ``` +![](figures/SC_Figure_15.png) +**Figure 15.** Are there any genes you're particularly interested in? You can adapt the code below to see if, and where, it might be expressed in these data. If you're not sure, you can look up S mansoni on WBPS and choose a gene from there (https://parasite.wormbase.org/index.html). Remember to delete the # if you want to save the plot to your computer! + ```R FeaturePlot(day2somules, features = "your_gene") #ggsave(paste0("day2somules-your-gene_",st,".jpg"), width = 25, height = 15) @@ -624,6 +629,8 @@ coexpression.plot <-FeaturePlot(day2somules, features = c("Smp-179320", "Smp-086 coexpression.plot #ggsave(paste0("day2somules-coexpressed-Smp-179320-Smp-086860-",st, ".jpg"), width = 45, height = 25) ``` +![](figures/SC_Figure_16.png) +**Figure 16.** Can you find a pair genes whose products you might expect to interact, and see if they are co-expressed? Again, you can use WBPS to learn more about genes. @@ -631,7 +638,7 @@ Can you find a pair genes whose products you might expect to interact, and see i #put your code in here! ``` -#Cluster IDs +## Rename UMAP cluster IDs You want all the cluster names to be unique - why might that be? ```R @@ -666,3 +673,5 @@ LabelClusters(plot1, id = "ident", color = 'black', size =4, repel = T, box.pad ggsave(paste0("day2somules-labelledlclusters_40PC_0.5res_",st,".jpg"), width = 25, height = 15) ``` +![](figures/SC_Figure_17.png) +**Figure 17.** From 4272cf0541e6c0bff42e62034e23664b44b13695 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Wed, 30 Apr 2025 13:46:10 -0300 Subject: [PATCH 08/51] Create SC_Figure_14.png --- .../module_transcriptomics/figures/SC_Figure_14.png | 1 + 1 file changed, 1 insertion(+) create mode 100644 course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png new file mode 100644 index 0000000..8b13789 --- /dev/null +++ b/course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png @@ -0,0 +1 @@ + From e9a617b87a3f9b23b6a1d63510558a8986ac4e0f Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Wed, 30 Apr 2025 13:49:02 -0300 Subject: [PATCH 09/51] Add files via upload --- .../day2somules-Smp-179320-2025-04-30.png | Bin 0 -> 201712 bytes 1 file changed, 0 insertions(+), 0 deletions(-) create mode 100644 course_modules_2025/module_transcriptomics/figures/day2somules-Smp-179320-2025-04-30.png diff --git a/course_modules_2025/module_transcriptomics/figures/day2somules-Smp-179320-2025-04-30.png b/course_modules_2025/module_transcriptomics/figures/day2somules-Smp-179320-2025-04-30.png new file mode 100644 index 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a/course_modules_2025/module_transcriptomics/figures/day2somules-coexpressed-Smp-179320-Smp-086860-2025-04-30.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_16.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/day2somules-coexpressed-Smp-179320-Smp-086860-2025-04-30.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_16.png From 32362ed7d81f5b899ce156554932e4393ec101b1 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Wed, 30 Apr 2025 14:28:06 -0300 Subject: [PATCH 17/51] Rename day2somules-labelledlclusters_40PC_0.5res_2025-04-30.png to SC_Figure_17.png --- ..._40PC_0.5res_2025-04-30.png => SC_Figure_17.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{day2somules-labelledlclusters_40PC_0.5res_2025-04-30.png => SC_Figure_17.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/day2somules-labelledlclusters_40PC_0.5res_2025-04-30.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_17.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/day2somules-labelledlclusters_40PC_0.5res_2025-04-30.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_17.png From 87c13b6fa1f43d576c4dac2ae55172258e3fc9ca Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:24:03 -0300 Subject: [PATCH 18/51] Update Single_cell_module.md --- .../Single_cell_module.md | 22 ++++++++++++++----- 1 file changed, 16 insertions(+), 6 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index a645db6..9e31e59 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -364,7 +364,7 @@ ggsave(paste0("raw_cgenesVumis_mito_somules_v10_bybatch_",st,".png"), width = 10 ``` ![](figures/SC_Figure_6.png) -**Figure 6.** +**Figure 6.** Correlation between feature (genes) and UMIs (transcript) counts per cell. Gray scale indicates the percentage of mito RNA. ### QC filtering @@ -404,16 +404,26 @@ day2somules <- readRDS(file = "day2somules_v10_firstfilt.rds") ## Normalisation and scaling -On to the next stage of analysis: initial normalization and clustering +On to the next stage of analysis we will do an initial normalization, scalinig and clustering -```R +We do an initial noramlization using default parameters, that is divide the feature (gene) expression for each cell by the total expression, multiplie this by a 10,000 factor and log-transform the result. +```R day2somules <- NormalizeData(day2somules) +``` +We next calculate a subset of features that exhibit high cell-to-cell variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others). + +```R day2somules <- FindVariableFeatures(day2somules, selection.method = "vst", nfeatures = 2000) # Identify the 10 most highly variable genes top10 <- head(VariableFeatures(day2somules), 10) -top10 +plot<-VariableFeaturePlot(day2somules) +LabelPoints((plot=plot),points=top10, repel=TRUE) +``` +![](figures/SC_Figure_6.png) +**Figure 6.** Correlation between feature (genes) and UMIs (transcript) counts per cell. Gray scale indicates the percentage of mito RNA. +```R all.genes <- rownames(day2somules) day2somules <- ScaleData(day2somules, features = all.genes) @@ -432,7 +442,7 @@ VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") ``` ![](figures/SC_Figure_7.png) -**Figure 7.** +**Figure 7.** Top most variable genes for 1st and 2nd PCs after the initial normalization and scaling. ```R #plots heatmap of top 500 most variable cells for PC1, with relative gene expression @@ -441,7 +451,7 @@ DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) ![](figures/SC_Figure_8.png) -**Figure 8.** +**Figure 8.** heatmap of top 500 most variable cells for PC1 ### Jackstraw From 1167f7df7a99211fcd5326ca2b32cb4abf5008ec Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:24:42 -0300 Subject: [PATCH 19/51] Add files via upload --- .../figures/top10VariableFeatures.png | Bin 0 -> 21978 bytes 1 file changed, 0 insertions(+), 0 deletions(-) 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to SC_Figure_18.png --- .../figures/{SC_Figure_17.png => SC_Figure_18.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_17.png => SC_Figure_18.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_17.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_18.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_17.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_18.png From d8cd70c10e82523d8bfe33ceabe65d922983f88f Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:25:57 -0300 Subject: [PATCH 21/51] Rename SC_Figure_16.png to SC_Figure_17.png --- .../figures/{SC_Figure_16.png => SC_Figure_17.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_16.png => 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course_modules_2025/module_transcriptomics/figures/SC_Figure_15.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_16.png From abd9413c1a824033bd79784d68296a479a027ed0 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:26:41 -0300 Subject: [PATCH 23/51] Rename SC_Figure_14.png to SC_Figure_15.png --- .../figures/{SC_Figure_14.png => SC_Figure_15.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_14.png => SC_Figure_15.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_15.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_14.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_15.png From 216fdc169374b23bf515805c8eb4fd9b1da4b374 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:27:36 -0300 Subject: [PATCH 24/51] Rename SC_Figure_13.jpg to SC_Figure_14.jpg --- .../figures/{SC_Figure_13.jpg => SC_Figure_14.jpg} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_13.jpg => SC_Figure_14.jpg} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_13.jpg b/course_modules_2025/module_transcriptomics/figures/SC_Figure_14.jpg similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_13.jpg rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_14.jpg From be1d1db51c4666daf31315af7623ec2315fd1913 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:27:53 -0300 Subject: [PATCH 25/51] Rename SC_Figure_12.png to SC_Figure_13.png --- .../figures/{SC_Figure_12.png => SC_Figure_13.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_12.png => SC_Figure_13.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_12.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_13.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_12.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_13.png From 5a4fda551d5c76eca2ed212d009c17efb478049b Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:28:12 -0300 Subject: [PATCH 26/51] Rename SC_Figure_11.png to SC_Figure_12.png --- .../figures/{SC_Figure_11.png => SC_Figure_12.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_11.png => SC_Figure_12.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_11.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_12.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_11.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_12.png From 4269bae807b452debc900d985e330471edf5a03c Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:28:28 -0300 Subject: [PATCH 27/51] Rename SC_Figure_10.png to SC_Figure_11.png --- .../figures/{SC_Figure_10.png => SC_Figure_11.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_10.png => SC_Figure_11.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_10.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_11.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_10.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_11.png From 4ba78e1dea2dbaecf94cf730bb662535a84be4c1 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:28:46 -0300 Subject: [PATCH 28/51] Rename SC_Figure_9.jpg to SC_Figure_10.jpg --- .../figures/{SC_Figure_9.jpg => SC_Figure_10.jpg} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_9.jpg => SC_Figure_10.jpg} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_9.jpg b/course_modules_2025/module_transcriptomics/figures/SC_Figure_10.jpg similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_9.jpg rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_10.jpg From 02faa4df3f5810ffeba9b7ad30741f19aaab65f0 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:29:07 -0300 Subject: [PATCH 29/51] Rename SC_Figure_8.png to SC_Figure_9.png --- .../figures/{SC_Figure_8.png => SC_Figure_9.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_8.png => SC_Figure_9.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_8.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_9.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_8.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_9.png From cbae6b5fd6b1211505e4597d31eae426858e3e71 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:29:25 -0300 Subject: [PATCH 30/51] Rename SC_Figure_7.png to SC_Figure_8.png --- .../figures/{SC_Figure_7.png => SC_Figure_8.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{SC_Figure_7.png => SC_Figure_8.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/SC_Figure_7.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_8.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/SC_Figure_7.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_8.png From 2f940324cfe70e88514530f06b07643b51a92022 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 12:29:56 -0300 Subject: [PATCH 31/51] Rename top10VariableFeatures.png to SC_Figure_7.png --- .../{top10VariableFeatures.png => SC_Figure_7.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{top10VariableFeatures.png => SC_Figure_7.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/top10VariableFeatures.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_7.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/top10VariableFeatures.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_7.png From ae515264b39a0b7cd2469d377367fc7de212748e Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 14:38:44 -0300 Subject: [PATCH 32/51] Update Single_cell_module.md --- .../Single_cell_module.md | 23 +++++++++++-------- 1 file changed, 13 insertions(+), 10 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 9e31e59..f0279bc 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -420,10 +420,12 @@ top10 <- head(VariableFeatures(day2somules), 10) plot<-VariableFeaturePlot(day2somules) LabelPoints((plot=plot),points=top10, repel=TRUE) ``` -![](figures/SC_Figure_6.png) -**Figure 6.** Correlation between feature (genes) and UMIs (transcript) counts per cell. Gray scale indicates the percentage of mito RNA. +![](figures/SC_Figure_7.png) +**Figure 7.** Labeled are the top 10 most variable fetures in the data. Red dots represent the 2000 most variable features. +We apply a linear transformation that is a standard pre-processing step prior to dimensional reduction techniques. We scale the data considering all genes (by default only variable features are scaled) ```R + all.genes <- rownames(day2somules) day2somules <- ScaleData(day2somules, features = all.genes) @@ -431,9 +433,10 @@ day2somules <- ScaleData(day2somules, features = all.genes) ### PCA -Now, we can look at look at gene expression scaled across the cells, and find variable features: genes. +Here, we perform PCA on the scaled data. + +Now, we can look at gene expression scaled across the cells, and find variable features (genes). -Here, we perform PCA on the scaled data. The most variable features selected earlier are used. ```R #shows top contributing features for the PCs day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules),npcs = 100) @@ -442,20 +445,20 @@ VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") ``` ![](figures/SC_Figure_7.png) -**Figure 7.** Top most variable genes for 1st and 2nd PCs after the initial normalization and scaling. +**Figure 7.** Top variable genes for 1st and 2nd PCs after the initial normalization and scaling. +We can also plot a heatmap to visualize the top features contributing to heterogenity in each PC. We will plot the expression for each feature in the top 500 cells in the exptremes of the spectrum. ```R -#plots heatmap of top 500 most variable cells for PC1, with relative gene expression DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) ``` ![](figures/SC_Figure_8.png) -**Figure 8.** heatmap of top 500 most variable cells for PC1 +**Figure 8.** Heatmap showing the gene expression of top 500 most variable cells for PC1 -### Jackstraw +### Plotting the variation in each PC: Jackstraw and Elbowplot -It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, for these data it's too slow for this tutorial - on my computer it took approximately 28 minutes. +It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, for these data it's too slow for this tutorial. An elbow plot is a quick way to assess the contribution of the principal components by percentage of variation. ```R @@ -471,7 +474,7 @@ ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ``` ![](figures/SC_Figure_9.jpg) -**Figure 9.** +**Figure 9.** ### Find clusters From ce0ff9863842677239957727756253ff1acd2b96 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 14:50:58 -0300 Subject: [PATCH 33/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index f0279bc..aa41201 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -4,7 +4,7 @@ ## Table of Contents 1. [Overview and Aims](#intro) 2. [Introduction to single cell transcriptomics](#basic) -3. [Working with Seurat objects](#seurat) +3. [Setup the Seurat Object](#seurat) 4. [Doublet cells removal](#doubletremoval) 5. [QC removal](#QCremoval) 6. [Normalizing and Scaling the data](#N&S) @@ -33,7 +33,7 @@ The scRNA-seq data used in the module was obtained from 2 day schistosomula of _ --- -## Working with Seurat objects +## Setup the Seurat Object After sequencing, reads are mapped to the reference genome with Cell Ranger, a set of analysis pipelines that process Chromium Next GEM single cell data to align reads and generate feature-barcode matrices. To do this, transcripts from different cells are identified through a 10X barcode, while different transcripts of each cell are identified through a unique molecular identifier or UMI. 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zdLnxF-mJE|ugyce_UnSndcS(5chGq=V>TM-Oeq#?cVP;zkhB^Y4iT9Zl3WGk&Ci1& zdI%^3c@wwiiZN3Yu8k$zP^BW`PAyp}rMBG$%P1>7L?f6>Ko9IGbBY_YVGWA5Jf;Gw znG^S4%bVCs2;^w$k)t3K3x#A56l_osG%*E< zG?05HB7U#AY38XE&58B+WM<;SIJ-_iOOA_1oOz%pgn_h7<9s)t<&lf;NnZpI3#j-x zl~V`WaF{;nJ;(KN}-RQ~l5HYS#IQmTQY_bJ-l~CKnRbBWeikeH%Y$co>e7 zlDi^su(|M}P}5!|d{sBZ%A9s6QdnN1_s1coYTG}9X@tB~<$W literal 0 HcmV?d00001 From 724e7cf23984a1fc7af685e79b26918deb7edc4d Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 14:58:53 -0300 Subject: [PATCH 39/51] Rename NewSC_Figure_9.png to SC_Figure_9.png --- .../figures/{NewSC_Figure_9.png => SC_Figure_9.png} | Bin 1 file changed, 0 insertions(+), 0 deletions(-) rename course_modules_2025/module_transcriptomics/figures/{NewSC_Figure_9.png => SC_Figure_9.png} (100%) diff --git a/course_modules_2025/module_transcriptomics/figures/NewSC_Figure_9.png b/course_modules_2025/module_transcriptomics/figures/SC_Figure_9.png similarity index 100% rename from course_modules_2025/module_transcriptomics/figures/NewSC_Figure_9.png rename to course_modules_2025/module_transcriptomics/figures/SC_Figure_9.png From d82bfba8c5fa6c0a7c4af62263371fc78010f5ec Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 15:03:58 -0300 Subject: [PATCH 40/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index aa41201..cb41075 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -444,17 +444,17 @@ day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somu VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") ``` -![](figures/SC_Figure_7.png) -**Figure 7.** Top variable genes for 1st and 2nd PCs after the initial normalization and scaling. +![](figures/SC_Figure_8.png) +**Figure 8.** Top variable genes for 1st and 2nd PCs after the initial normalization and scaling. We can also plot a heatmap to visualize the top features contributing to heterogenity in each PC. We will plot the expression for each feature in the top 500 cells in the exptremes of the spectrum. ```R DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) ``` -![](figures/SC_Figure_8.png) +![](figures/SC_Figure_9.png) -**Figure 8.** Heatmap showing the gene expression of top 500 most variable cells for PC1 +**Figure 9.** Heatmap showing the gene expression of top 500 most variable cells for PC1 ### Plotting the variation in each PC: Jackstraw and Elbowplot From 64999f1aa1931a4009bbad442823c6336762e0c3 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 15:09:12 -0300 Subject: [PATCH 41/51] Update Single_cell_module.md --- .../Single_cell_module.md | 36 +++++++++---------- 1 file changed, 18 insertions(+), 18 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index cb41075..f169f9d 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -473,8 +473,8 @@ ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A cl ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ``` -![](figures/SC_Figure_9.jpg) -**Figure 9.** +![](figures/SC_Figure_10.jpg) +**Figure 10.** ### Find clusters @@ -490,8 +490,8 @@ DimPlot(day2somules, reduction = "umap") ggsave(paste0("day2somules_v10clust_40PC_0.4res_RNA_",st,".jpg")) ``` -![](figures/SC_Figure_10.png) -**Figure 10.** +![](figures/SC_Figure_11.png) +**Figure 11.** ## SCTransform of data @@ -537,8 +537,8 @@ ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) ``` -![](figures/SC_Figure_11.png) -**Figure 11.** +![](figures/SC_Figure_12.png) +**Figure 12.** #Plot metadata @@ -549,8 +549,8 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_shuffled_batch_SCT.jpg") ``` -![](figures/SC_Figure_12.png) -**Figure 12.** +![](figures/SC_Figure_13.png) +**Figure 13.** What do you notice once the UMAP is coloured by sample? Is there anything you might do about this? @@ -559,8 +559,8 @@ FeaturePlot(day2somules, features="percent.mt", label=TRUE) ggsave("day2somules_v10_40PC_0.5res_after_one_filt_mt_SCT.jpg") ``` -![](figures/SC_Figure_13.jpg) -**Figure 13.** +![](figures/SC_Figure_14.jpg) +**Figure 14.** What do you think about this figure? Is there any cluster with a higher proportion of MT RNA? @@ -616,8 +616,8 @@ Now we can look at gene expression in these data by gene, which could be useful FeaturePlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320-",st, ".jpg"), width = 25, height = 15) ``` -![](figures/SC_Figure_14.png) -**Figure 14.** +![](figures/SC_Figure_15.png) +**Figure 15.** We can also look at these genes with a violin plot - this visualisation can be helpful in many ways, including seeing which clusters a gene is most expressed in. @@ -625,8 +625,8 @@ We can also look at these genes with a violin plot - this visualisation can be h VlnPlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320_",st, ".jpg"), width = 25, height = 15) ``` -![](figures/SC_Figure_15.png) -**Figure 15.** +![](figures/SC_Figure_16.png) +**Figure 16.** Are there any genes you're particularly interested in? You can adapt the code below to see if, and where, it might be expressed in these data. If you're not sure, you can look up S mansoni on WBPS and choose a gene from there (https://parasite.wormbase.org/index.html). Remember to delete the # if you want to save the plot to your computer! @@ -642,8 +642,8 @@ coexpression.plot <-FeaturePlot(day2somules, features = c("Smp-179320", "Smp-086 coexpression.plot #ggsave(paste0("day2somules-coexpressed-Smp-179320-Smp-086860-",st, ".jpg"), width = 45, height = 25) ``` -![](figures/SC_Figure_16.png) -**Figure 16.** +![](figures/SC_Figure_17.png) +**Figure 17.** Can you find a pair genes whose products you might expect to interact, and see if they are co-expressed? Again, you can use WBPS to learn more about genes. @@ -686,5 +686,5 @@ LabelClusters(plot1, id = "ident", color = 'black', size =4, repel = T, box.pad ggsave(paste0("day2somules-labelledlclusters_40PC_0.5res_",st,".jpg"), width = 25, height = 15) ``` -![](figures/SC_Figure_17.png) -**Figure 17.** +![](figures/SC_Figure_18.png) +**Figure 18.** From 63e5e8d56d9514d4622ed255fe41bcb73acd74d3 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 16:06:30 -0300 Subject: [PATCH 42/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 13 ++++++++----- 1 file changed, 8 insertions(+), 5 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index f169f9d..8ba4092 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -449,18 +449,19 @@ VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") We can also plot a heatmap to visualize the top features contributing to heterogenity in each PC. We will plot the expression for each feature in the top 500 cells in the exptremes of the spectrum. ```R -DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) +DimHeatmap(day2somules, dims = 1:3, cells = 500, balanced = TRUE) ``` ![](figures/SC_Figure_9.png) -**Figure 9.** Heatmap showing the gene expression of top 500 most variable cells for PC1 +**Figure 9.** Heatmap showing the most contributing features in PCs 1 to 3 and the gene expression in the top 500 most variable cells (positive and negative) -### Plotting the variation in each PC: Jackstraw and Elbowplot +### Plotting the variation in each PCA: Jackstraw and Elbowplot + +Principal component analysis (PCA) is a fundamental dimension reduction technique in analyzing single-cell genomic data. It maps the cells with high-dimensional and noisy genomic information to a low-dimensional and denoised principal component space. The principal components (PCs) are then used to group cells into clusters. The number of PCs plays a critical role in downstream analyses. With too many PCs, PCs with the smallest variations may represent the noise in the data and dilute the signal. With too few PCs, the essential information in the data may not be captured. An optimal number of PCs should keep the essential information in the data while filtering out as much noise as possible. It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, for these data it's too slow for this tutorial. -An elbow plot is a quick way to assess the contribution of the principal components by percentage of variation. ```R DefaultAssay(day2somules) <- "RNA" @@ -468,7 +469,9 @@ DefaultAssay(day2somules) <- "RNA" #day2somules <- ScoreJackStraw(day2somules, dims = 1:100) #extract the scores #JackStrawPlot(day2somules, dims = 1:100) #plot the p-vals for PCs. Dashed line giives null distribution #ggsave("day2somules_v10_jackstrawplot100.pdf", width = 30, height = 15, units = c('cm')) - +``` +An elbow plot is a quick way to assess the contribution of the principal components by standard deviation. +```R ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A clear dropoff is sometimes seen, though not really here. ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ``` From f40a564931e4382b3c5a719aa00b05dbc2de58b2 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 16:33:20 -0300 Subject: [PATCH 43/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 4 +++- 1 file changed, 3 insertions(+), 1 deletion(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 8ba4092..4e55347 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -448,6 +448,7 @@ VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") **Figure 8.** Top variable genes for 1st and 2nd PCs after the initial normalization and scaling. We can also plot a heatmap to visualize the top features contributing to heterogenity in each PC. We will plot the expression for each feature in the top 500 cells in the exptremes of the spectrum. + ```R DimHeatmap(day2somules, dims = 1:3, cells = 500, balanced = TRUE) ``` @@ -470,7 +471,8 @@ DefaultAssay(day2somules) <- "RNA" #JackStrawPlot(day2somules, dims = 1:100) #plot the p-vals for PCs. Dashed line giives null distribution #ggsave("day2somules_v10_jackstrawplot100.pdf", width = 30, height = 15, units = c('cm')) ``` -An elbow plot is a quick way to assess the contribution of the principal components by standard deviation. +An elbow plot is a quick way to assess the contribution of the principal components to the variation. It starts from a scatterplot where the y-axis shows the standard deviations of PCs and the x-axis shows the numbers of PCs. Since the PCs are ordered decreasingly by their standard deviations, the scatterplot usually presents a monotonically decreasing curve that sharply descends for the first several PCs and gradually flattens out for the subsequent PCs. + ```R ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A clear dropoff is sometimes seen, though not really here. ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) From db622ed516a91e4bec9bec0fc82ae08f775bdcbb Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 18:10:46 -0300 Subject: [PATCH 44/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 12 ++++++++++-- 1 file changed, 10 insertions(+), 2 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 8ba4092..2dc67fc 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -448,6 +448,7 @@ VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") **Figure 8.** Top variable genes for 1st and 2nd PCs after the initial normalization and scaling. We can also plot a heatmap to visualize the top features contributing to heterogenity in each PC. We will plot the expression for each feature in the top 500 cells in the exptremes of the spectrum. + ```R DimHeatmap(day2somules, dims = 1:3, cells = 500, balanced = TRUE) ``` @@ -470,7 +471,8 @@ DefaultAssay(day2somules) <- "RNA" #JackStrawPlot(day2somules, dims = 1:100) #plot the p-vals for PCs. Dashed line giives null distribution #ggsave("day2somules_v10_jackstrawplot100.pdf", width = 30, height = 15, units = c('cm')) ``` -An elbow plot is a quick way to assess the contribution of the principal components by standard deviation. +An elbow plot is a quick way to assess the contribution of the principal components to the variation. It starts from a scatterplot where the y-axis shows the standard deviations of PCs and the x-axis shows the numbers of PCs. Since the PCs are ordered decreasingly by their standard deviations, the scatterplot usually presents a monotonically decreasing curve that sharply descends for the first several PCs and gradually flattens out for the subsequent PCs. + ```R ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A clear dropoff is sometimes seen, though not really here. ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) @@ -481,9 +483,15 @@ ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ### Find clusters +Seurat applies a graph-based clustering approach. Constructs a K-nearest neighbor graph based on the euclidean distance in PCA space, and refine the edge weights between any two cells based on the shared overlap in their local neighborhoods (Jaccard similarity). This step is performed using the FindNeighbors() function, and takes as input the previously defined dimensionality of the dataset (PCs). + ```R #here construct k-nearest neighbors graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) -day2somules <- FindNeighbors(day2somules, dims = 1:40) +day2somules <- FindNeighbors(day2somules, dims = 1:40) + +``` +To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. +```R #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. day2somules <- FindClusters(day2somules, resolution = 0.5) #runs umap to visualise the clusters. Need to set the number of PCs From 652e3c9760fe124f219e9929f6f44158202ff8b7 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Fri, 2 May 2025 18:31:41 -0300 Subject: [PATCH 45/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 8 +++++++- 1 file changed, 7 insertions(+), 1 deletion(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 2dc67fc..9a2cd0b 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -493,7 +493,13 @@ day2somules <- FindNeighbors(day2somules, dims = 1:40) To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. ```R #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. -day2somules <- FindClusters(day2somules, resolution = 0.5) +day2somules <- FindClusters(day2somules, resolution = 0.5) +``` +### Run non-linear dimensional reduction + +Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. + +```R #runs umap to visualise the clusters. Need to set the number of PCs day2somules <- RunUMAP(day2somules, dims = 1:40) #visulaises the UMAP From 85faa8660583f5465caffdfe7c16c04941312e3a Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Sat, 3 May 2025 12:35:48 -0300 Subject: [PATCH 46/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 9a2cd0b..8239c03 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -495,7 +495,7 @@ To cluster the cells, we next apply modularity optimization techniques, to itera #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. day2somules <- FindClusters(day2somules, resolution = 0.5) ``` -### Run non-linear dimensional reduction +### Run non-linear dimensional reduction5 Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. From c494cf8b5715380ff8b510b3fd8f6b58a0534fa2 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Mon, 5 May 2025 12:15:04 -0300 Subject: [PATCH 47/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 15 ++++++++------- 1 file changed, 8 insertions(+), 7 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 8239c03..dfda169 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -5,13 +5,14 @@ 1. [Overview and Aims](#intro) 2. [Introduction to single cell transcriptomics](#basic) 3. [Setup the Seurat Object](#seurat) -4. [Doublet cells removal](#doubletremoval) -5. [QC removal](#QCremoval) -6. [Normalizing and Scaling the data](#N&S) -7. [SCTransform of data](#SCTrasform) -8. [Plot UMAPs](#plotUMAPs) -9. [Plot the expression of individual genes in the UMAP](#plotGenes) -10. [Rename UMAP clusters IDs](#renameUMAP) +4. [Standard preprocessing workflow] +5. *[Doublet cells removal](#doubletremoval) +7. *[QC removal](#QCremoval) +8. [Normalizing and Scaling the data](#N&S) +9. [SCTransform of data](#SCTrasform) +10. [Plot UMAPs](#plotUMAPs) +11. [Plot the expression of individual genes in the UMAP](#plotGenes) +12. [Rename UMAP clusters IDs](#renameUMAP) --- From f7a5e0e71e0f91b9a9be89e4b8acae25c9dfbe80 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Mon, 5 May 2025 12:22:28 -0300 Subject: [PATCH 48/51] Update Single_cell_module.md --- .../module_transcriptomics/Single_cell_module.md | 16 +++++++++------- 1 file changed, 9 insertions(+), 7 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index dfda169..998a48d 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -6,13 +6,15 @@ 2. [Introduction to single cell transcriptomics](#basic) 3. [Setup the Seurat Object](#seurat) 4. [Standard preprocessing workflow] -5. *[Doublet cells removal](#doubletremoval) -7. *[QC removal](#QCremoval) -8. [Normalizing and Scaling the data](#N&S) -9. [SCTransform of data](#SCTrasform) -10. [Plot UMAPs](#plotUMAPs) -11. [Plot the expression of individual genes in the UMAP](#plotGenes) -12. [Rename UMAP clusters IDs](#renameUMAP) + * [Doublet cells removal](#doubletremoval) + * [QC removal](#QCremoval) +5. [SCTransform the data](#SCTrasform) +6. [Perform dimentional reduction](#plotUMAPs) +7. [Determine the 'dimentionality' of the dataset] +8. [Cluster the cells] +9. [Run non-linear dimensional reduction (UMAP)] +10. [Finding differentially expressed features](#plotGenes) +11. [Assigning cell type identity to clusters](#renameUMAP) --- From 035109064a3d93fccbd89f4f10ebfca2533ee066 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Mon, 5 May 2025 16:20:52 -0300 Subject: [PATCH 49/51] Update Single_cell_module.md --- .../Single_cell_module.md | 144 ++++++------------ 1 file changed, 45 insertions(+), 99 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 998a48d..b69a891 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -5,15 +5,16 @@ 1. [Overview and Aims](#intro) 2. [Introduction to single cell transcriptomics](#basic) 3. [Setup the Seurat Object](#seurat) -4. [Standard preprocessing workflow] +4. [Standard preprocessing workflow](#preprocessing) * [Doublet cells removal](#doubletremoval) * [QC removal](#QCremoval) 5. [SCTransform the data](#SCTrasform) -6. [Perform dimentional reduction](#plotUMAPs) -7. [Determine the 'dimentionality' of the dataset] -8. [Cluster the cells] -9. [Run non-linear dimensional reduction (UMAP)] -10. [Finding differentially expressed features](#plotGenes) +6. [Perform dimentional reduction](#PCA) +7. [Determine the 'dimentionality' of the dataset](#dimentionality) +8. [Cluster the cells](#cluster) +9. [Run non-linear dimensional reduction (UMAP)](#umap) +10. [Finding differentially expressed features (cluster biomarkers)](#biomarkers) + * [Explote the expression of specific genes in the UMAP](#plotGenes) 11. [Assigning cell type identity to clusters](#renameUMAP) --- @@ -190,7 +191,9 @@ If you're not sure which metadata are available, or you want a summary of metada [↥ **Back to top**](#top) -## Doublet ID +## Standard preprocessing workflow + +### Doublet ID Doublets, or multiplets, form when two or more cells are associated with one cell barcode - for example you might have a 'cell' in your data that looks like a hybrid of a muscle and a neural cell. Since these don't describe a cell type found in the sample, we want to exclude these cells as much as possible. There are many tools/strategies, the below tool assigns each cell a doublet score, and assigns cells as doublets or singlets based on these scores and the expected percentage of doublets. Run the following lines and set the function. @@ -271,7 +274,7 @@ table(day2somules@meta.data$batches) [↥ **Back to top**](#top) -## QC removal +### QC removal ### Mito % @@ -323,7 +326,7 @@ day2somules_metadata %>% geom_vline(xintercept = 500) + geom_vline(xintercept = 1000)+ ggtitle("UMIs per cell") -ggsave(paste0("raw_umisPerCell_somules_v10_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_umisPerCell_somules_v10_",st,".png"), width = 10, height = 5) ``` @@ -342,7 +345,7 @@ day2somules_metadata %>% scale_x_log10() + geom_vline(xintercept = 300)+ ggtitle("Genes per cell") -ggsave(paste0("raw_genesPerCell_somules_v10_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_genesPerCell_somules_v10_",st,".png"), width = 10, height = 5) ``` ![](figures/SC_Figure_5.jpg) @@ -363,7 +366,7 @@ day2somules_metadata %>% geom_vline(xintercept = 1000) + geom_hline(yintercept = 500) + facet_wrap(~batches) -ggsave(paste0("raw_cgenesVumis_mito_somules_v10_bybatch_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_cgenesVumis_mito_somules_v10_bybatch_",st,".png"), width = 10, height = 5) ``` ![](figures/SC_Figure_6.png) @@ -405,20 +408,16 @@ day2somules <- readRDS(file = "day2somules_v10_firstfilt.rds") [↥ **Back to top**](#top) -## Normalisation and scaling - -On to the next stage of analysis we will do an initial normalization, scalinig and clustering +## SCTransform of data -We do an initial noramlization using default parameters, that is divide the feature (gene) expression for each cell by the total expression, multiplie this by a 10,000 factor and log-transform the result. +Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()). ```R -day2somules <- NormalizeData(day2somules) +# run sctransform +day2somules <- SCTransform(day2somules, verbose = TRUE) ``` -We next calculate a subset of features that exhibit high cell-to-cell variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others). - +We can visualize the features (genes) that exhibit high cell-to-cell variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others). ```R -day2somules <- FindVariableFeatures(day2somules, selection.method = "vst", nfeatures = 2000) -# Identify the 10 most highly variable genes top10 <- head(VariableFeatures(day2somules), 10) plot<-VariableFeaturePlot(day2somules) LabelPoints((plot=plot),points=top10, repel=TRUE) @@ -426,25 +425,16 @@ LabelPoints((plot=plot),points=top10, repel=TRUE) ![](figures/SC_Figure_7.png) **Figure 7.** Labeled are the top 10 most variable fetures in the data. Red dots represent the 2000 most variable features. -We apply a linear transformation that is a standard pre-processing step prior to dimensional reduction techniques. We scale the data considering all genes (by default only variable features are scaled) -```R - -all.genes <- rownames(day2somules) -day2somules <- ScaleData(day2somules, features = all.genes) - -``` - -### PCA +The results of this are saved in a different 'assay' of the R object - so you can still use both, and the data aren't overwritten. -Here, we perform PCA on the scaled data. +## Perform dimentional reduction -Now, we can look at gene expression scaled across the cells, and find variable features (genes). +Principal component analysis (PCA) is a fundamental dimension reduction technique in analyzing single-cell genomic data. It maps the cells with high-dimensional and noisy genomic information to a low-dimensional and denoised principal component space. ```R -#shows top contributing features for the PCs -day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules),npcs = 100) +day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules), npcs=100) #shows the weightings of top contributing features to pcs 1 and 2 -VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") +VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") #shows the weightings of top contributing features to PCs 1 and 2 ``` ![](figures/SC_Figure_8.png) @@ -460,11 +450,11 @@ DimHeatmap(day2somules, dims = 1:3, cells = 500, balanced = TRUE) **Figure 9.** Heatmap showing the most contributing features in PCs 1 to 3 and the gene expression in the top 500 most variable cells (positive and negative) -### Plotting the variation in each PCA: Jackstraw and Elbowplot +## Determine the 'dimentionality' of the dataset -Principal component analysis (PCA) is a fundamental dimension reduction technique in analyzing single-cell genomic data. It maps the cells with high-dimensional and noisy genomic information to a low-dimensional and denoised principal component space. The principal components (PCs) are then used to group cells into clusters. The number of PCs plays a critical role in downstream analyses. With too many PCs, PCs with the smallest variations may represent the noise in the data and dilute the signal. With too few PCs, the essential information in the data may not be captured. An optimal number of PCs should keep the essential information in the data while filtering out as much noise as possible. +The principal components (PCs) are then used to group cells into clusters. The number of PCs plays a critical role in downstream analyses. With too many PCs, PCs with the smallest variations may represent the noise in the data and dilute the signal. With too few PCs, the essential information in the data may not be captured. An optimal number of PCs should keep the essential information in the data while filtering out as much noise as possible. -It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, for these data it's too slow for this tutorial. +It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, it's too slow for this tutorial. ```R DefaultAssay(day2somules) <- "RNA" @@ -478,89 +468,45 @@ An elbow plot is a quick way to assess the contribution of the principal compone ```R ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A clear dropoff is sometimes seen, though not really here. -ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) +#ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ``` ![](figures/SC_Figure_10.jpg) -**Figure 10.** +**Figure 10.** Elbowplot showing the proportion of variation captured by each PC -### Find clusters +## Cluster the cells Seurat applies a graph-based clustering approach. Constructs a K-nearest neighbor graph based on the euclidean distance in PCA space, and refine the edge weights between any two cells based on the shared overlap in their local neighborhoods (Jaccard similarity). This step is performed using the FindNeighbors() function, and takes as input the previously defined dimensionality of the dataset (PCs). ```R -#here construct k-nearest neighbors graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) +#here construct k-nearest neighbours graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) day2somules <- FindNeighbors(day2somules, dims = 1:40) - -``` -To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. -```R -#this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. -day2somules <- FindClusters(day2somules, resolution = 0.5) -``` -### Run non-linear dimensional reduction5 - -Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. - -```R -#runs umap to visualise the clusters. Need to set the number of PCs -day2somules <- RunUMAP(day2somules, dims = 1:40) -#visulaises the UMAP -DimPlot(day2somules, reduction = "umap") -ggsave(paste0("day2somules_v10clust_40PC_0.4res_RNA_",st,".jpg")) -``` - -![](figures/SC_Figure_11.png) -**Figure 11.** - -## SCTransform of data - -Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()). - -```R -# run sctransform -day2somules <- SCTransform(day2somules, verbose = TRUE) ``` -The results of this are saved in a different 'assay' of the R object - so you can still use both, and the data aren't overwritten. - -### PCA on SCT transformed data - -Now, perform dimensionality reduction by PCA and UMAP embedding - -Here, we perform PCA on the scaled data. The most variable features selected earlier are used. This follows the same approach as when using the RNA assay we used above. - -```R -day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules), npcs=100) -VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") #shows the weightings of top contributing features to PCs 1 and 2 -DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) #plots heatmap of top 500 most variable cells for PC1, with relative gene expression -``` - -### Clustering on SCT +To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. -```R -#here construct k-nearest neighbours graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) -day2somules <- FindNeighbors(day2somules, dims = 1:40) +```R #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. day2somules <- FindClusters(day2somules, resolution = 0.5) ``` -## Plot UMAPs +## Run non-linear dimensional reduction -Runs umap to visualize the clusters. Need to set the number of PCs +Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. ```R +#runs umap to visualise the clusters. Need to set the number of PCs day2somules <- RunUMAP(day2somules, dims = 1:40) #visualises the UMAP DimPlot(day2somules, reduction = "umap") -ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) +#ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) ``` ![](figures/SC_Figure_12.png) -**Figure 12.** +**Figure 12.** UMAP plot, cells are colored by cluster -#Plot metadata +### Plot metadata Now we have a UMAP representation of our cells, we can also use that to visualise the metadata. ```R @@ -570,7 +516,7 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_shuffled_batch_SCT.jpg") ``` ![](figures/SC_Figure_13.png) -**Figure 13.** +**Figure 13.** UMAP plot, cells are colored by batch What do you notice once the UMAP is coloured by sample? Is there anything you might do about this? @@ -580,12 +526,12 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_mt_SCT.jpg") ``` ![](figures/SC_Figure_14.jpg) -**Figure 14.** +**Figure 14.** UMAP plot, cells are colored by percentage of mtRNA What do you think about this figure? Is there any cluster with a higher proportion of MT RNA? -## Markers identification +## Finding differentially expressed features (cluster biomarkers) We want to understand what the cell clusters might be. One approach to do that is find gene that are cluster markers - find differentially expressed genes that are descriptive of a cluster. @@ -594,7 +540,7 @@ DefaultAssay(day2somules) <- "RNA" day2somules<-JoinLayers(day2somules) -#day2somules.all.markers_roc <- FindAllMarkers(day2somules, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") +#day2somules.all.markers_roc <- FindAllMarkers(day2somules, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") #We could search the markers for all clusters, however that would take too long. day2somules.markers_roc_cluster0 <- FindMarkers(day2somules, ident.1 = 0, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") ``` @@ -629,7 +575,7 @@ A brainstorm question: how might you use this list to choose a gene to use for i How else might you classify cell cluster tissue types? -## Plot the expression of individual genes in the UMAP +### Explore the expression of specific genes in the UMAP Now we can look at gene expression in these data by gene, which could be useful to identify cluster marker genes. The example below shows you ago 2-1. If you want to look at a different gene, simply type the gene ID where "Smp-179320" currently sits. Remember to use - rather than _ ! We can visualise genes that we already know something about in the organism, to see if that gives us some clues ```R @@ -671,7 +617,7 @@ Can you find a pair genes whose products you might expect to interact, and see i #put your code in here! ``` -## Rename UMAP cluster IDs +## Assigning cell type identity to clusters You want all the cluster names to be unique - why might that be? ```R From e5fe3ccff81135defd2fbacb3b2df7b2f27ebd61 Mon Sep 17 00:00:00 2001 From: santifo <39537895+santifo@users.noreply.github.com> Date: Mon, 5 May 2025 16:52:41 -0300 Subject: [PATCH 50/51] Update Single_cell_module.md --- .../Single_cell_module.md | 154 ++++++------------ 1 file changed, 50 insertions(+), 104 deletions(-) diff --git a/course_modules_2025/module_transcriptomics/Single_cell_module.md b/course_modules_2025/module_transcriptomics/Single_cell_module.md index 998a48d..0520b07 100644 --- a/course_modules_2025/module_transcriptomics/Single_cell_module.md +++ b/course_modules_2025/module_transcriptomics/Single_cell_module.md @@ -5,15 +5,16 @@ 1. [Overview and Aims](#intro) 2. [Introduction to single cell transcriptomics](#basic) 3. [Setup the Seurat Object](#seurat) -4. [Standard preprocessing workflow] +4. [Standard preprocessing workflow](#preprocessing) * [Doublet cells removal](#doubletremoval) * [QC removal](#QCremoval) 5. [SCTransform the data](#SCTrasform) -6. [Perform dimentional reduction](#plotUMAPs) -7. [Determine the 'dimentionality' of the dataset] -8. [Cluster the cells] -9. [Run non-linear dimensional reduction (UMAP)] -10. [Finding differentially expressed features](#plotGenes) +6. [Perform dimentional reduction](#PCA) +7. [Determine the 'dimentionality' of the dataset](#dimentionality) +8. [Cluster the cells](#cluster) +9. [Run non-linear dimensional reduction (UMAP)](#umap) +10. [Finding differentially expressed features (cluster biomarkers)](#biomarkers) + * [Explote the expression of specific genes in the UMAP](#plotGenes) 11. [Assigning cell type identity to clusters](#renameUMAP) --- @@ -190,7 +191,9 @@ If you're not sure which metadata are available, or you want a summary of metada [↥ **Back to top**](#top) -## Doublet ID +## Standard preprocessing workflow + +### Doublet ID Doublets, or multiplets, form when two or more cells are associated with one cell barcode - for example you might have a 'cell' in your data that looks like a hybrid of a muscle and a neural cell. Since these don't describe a cell type found in the sample, we want to exclude these cells as much as possible. There are many tools/strategies, the below tool assigns each cell a doublet score, and assigns cells as doublets or singlets based on these scores and the expected percentage of doublets. Run the following lines and set the function. @@ -271,7 +274,7 @@ table(day2somules@meta.data$batches) [↥ **Back to top**](#top) -## QC removal +### QC removal ### Mito % @@ -323,7 +326,7 @@ day2somules_metadata %>% geom_vline(xintercept = 500) + geom_vline(xintercept = 1000)+ ggtitle("UMIs per cell") -ggsave(paste0("raw_umisPerCell_somules_v10_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_umisPerCell_somules_v10_",st,".png"), width = 10, height = 5) ``` @@ -342,7 +345,7 @@ day2somules_metadata %>% scale_x_log10() + geom_vline(xintercept = 300)+ ggtitle("Genes per cell") -ggsave(paste0("raw_genesPerCell_somules_v10_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_genesPerCell_somules_v10_",st,".png"), width = 10, height = 5) ``` ![](figures/SC_Figure_5.jpg) @@ -363,7 +366,7 @@ day2somules_metadata %>% geom_vline(xintercept = 1000) + geom_hline(yintercept = 500) + facet_wrap(~batches) -ggsave(paste0("raw_cgenesVumis_mito_somules_v10_bybatch_",st,".png"), width = 10, height = 5) +#ggsave(paste0("raw_cgenesVumis_mito_somules_v10_bybatch_",st,".png"), width = 10, height = 5) ``` ![](figures/SC_Figure_6.png) @@ -405,20 +408,16 @@ day2somules <- readRDS(file = "day2somules_v10_firstfilt.rds") [↥ **Back to top**](#top) -## Normalisation and scaling - -On to the next stage of analysis we will do an initial normalization, scalinig and clustering +## SCTransform of data -We do an initial noramlization using default parameters, that is divide the feature (gene) expression for each cell by the total expression, multiplie this by a 10,000 factor and log-transform the result. +Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()). ```R -day2somules <- NormalizeData(day2somules) +# run sctransform +day2somules <- SCTransform(day2somules, verbose = TRUE) ``` -We next calculate a subset of features that exhibit high cell-to-cell variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others). - +We can visualize the features (genes) that exhibit high cell-to-cell variation in the dataset (i.e, they are highly expressed in some cells, and lowly expressed in others). ```R -day2somules <- FindVariableFeatures(day2somules, selection.method = "vst", nfeatures = 2000) -# Identify the 10 most highly variable genes top10 <- head(VariableFeatures(day2somules), 10) plot<-VariableFeaturePlot(day2somules) LabelPoints((plot=plot),points=top10, repel=TRUE) @@ -426,25 +425,16 @@ LabelPoints((plot=plot),points=top10, repel=TRUE) ![](figures/SC_Figure_7.png) **Figure 7.** Labeled are the top 10 most variable fetures in the data. Red dots represent the 2000 most variable features. -We apply a linear transformation that is a standard pre-processing step prior to dimensional reduction techniques. We scale the data considering all genes (by default only variable features are scaled) -```R - -all.genes <- rownames(day2somules) -day2somules <- ScaleData(day2somules, features = all.genes) - -``` - -### PCA +The results of this are saved in a different 'assay' of the R object - so you can still use both, and the data aren't overwritten. -Here, we perform PCA on the scaled data. +## Perform dimentional reduction -Now, we can look at gene expression scaled across the cells, and find variable features (genes). +Principal component analysis (PCA) is a fundamental dimension reduction technique in analyzing single-cell genomic data. It maps the cells with high-dimensional and noisy genomic information to a low-dimensional and denoised principal component space. ```R -#shows top contributing features for the PCs -day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules),npcs = 100) +day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules), npcs=100) #shows the weightings of top contributing features to pcs 1 and 2 -VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") +VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") #shows the weightings of top contributing features to PCs 1 and 2 ``` ![](figures/SC_Figure_8.png) @@ -460,11 +450,11 @@ DimHeatmap(day2somules, dims = 1:3, cells = 500, balanced = TRUE) **Figure 9.** Heatmap showing the most contributing features in PCs 1 to 3 and the gene expression in the top 500 most variable cells (positive and negative) -### Plotting the variation in each PCA: Jackstraw and Elbowplot +## Determine the 'dimentionality' of the dataset -Principal component analysis (PCA) is a fundamental dimension reduction technique in analyzing single-cell genomic data. It maps the cells with high-dimensional and noisy genomic information to a low-dimensional and denoised principal component space. The principal components (PCs) are then used to group cells into clusters. The number of PCs plays a critical role in downstream analyses. With too many PCs, PCs with the smallest variations may represent the noise in the data and dilute the signal. With too few PCs, the essential information in the data may not be captured. An optimal number of PCs should keep the essential information in the data while filtering out as much noise as possible. +The principal components (PCs) are then used to group cells into clusters. The number of PCs plays a critical role in downstream analyses. With too many PCs, PCs with the smallest variations may represent the noise in the data and dilute the signal. With too few PCs, the essential information in the data may not be captured. An optimal number of PCs should keep the essential information in the data while filtering out as much noise as possible. -It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, for these data it's too slow for this tutorial. +It's possible to use JackStraw to randomly permute data in 1% chunks. Here with 100 replicates and for 100 PCs. However, it's too slow for this tutorial. ```R DefaultAssay(day2somules) <- "RNA" @@ -478,89 +468,45 @@ An elbow plot is a quick way to assess the contribution of the principal compone ```R ElbowPlot(day2somules, ndims = 100) #ranks PCs by percentage of variation. A clear dropoff is sometimes seen, though not really here. -ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) +#ggsave(paste0("day2somules_v10_elbowplot100_",st,".jpg")) ``` ![](figures/SC_Figure_10.jpg) -**Figure 10.** +**Figure 10.** Elbowplot showing the proportion of variation captured by each PC -### Find clusters +## Cluster the cells Seurat applies a graph-based clustering approach. Constructs a K-nearest neighbor graph based on the euclidean distance in PCA space, and refine the edge weights between any two cells based on the shared overlap in their local neighborhoods (Jaccard similarity). This step is performed using the FindNeighbors() function, and takes as input the previously defined dimensionality of the dataset (PCs). ```R -#here construct k-nearest neighbors graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) +#here construct k-nearest neighbours graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) day2somules <- FindNeighbors(day2somules, dims = 1:40) - -``` -To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. -```R -#this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. -day2somules <- FindClusters(day2somules, resolution = 0.5) -``` -### Run non-linear dimensional reduction5 - -Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. - -```R -#runs umap to visualise the clusters. Need to set the number of PCs -day2somules <- RunUMAP(day2somules, dims = 1:40) -#visulaises the UMAP -DimPlot(day2somules, reduction = "umap") -ggsave(paste0("day2somules_v10clust_40PC_0.4res_RNA_",st,".jpg")) -``` - -![](figures/SC_Figure_11.png) -**Figure 11.** - -## SCTransform of data - -Do a basic analysis to start with, using SCTransform. This function normalises and scales the data, and finds variable features. It has some improvements from earlier versions of Seurat and replaces NormalizeData(), ScaleData(), and FindVariableFeatures()). - -```R -# run sctransform -day2somules <- SCTransform(day2somules, verbose = TRUE) ``` -The results of this are saved in a different 'assay' of the R object - so you can still use both, and the data aren't overwritten. - -### PCA on SCT transformed data - -Now, perform dimensionality reduction by PCA and UMAP embedding - -Here, we perform PCA on the scaled data. The most variable features selected earlier are used. This follows the same approach as when using the RNA assay we used above. - -```R -day2somules <- RunPCA(day2somules, features = VariableFeatures(object = day2somules), npcs=100) -VizDimLoadings(day2somules, dims = 1:2, reduction = "pca") #shows the weightings of top contributing features to PCs 1 and 2 -DimHeatmap(day2somules, dims = 1, cells = 500, balanced = TRUE) #plots heatmap of top 500 most variable cells for PC1, with relative gene expression -``` - -### Clustering on SCT +To cluster the cells, we next apply modularity optimization techniques, to iteratively group cells together, with the goal of optimizing the standard modularity function. The FindClusters() function implements this procedure, and contains a resolution parameter that sets the ‘granularity’ of the downstream clustering, with increased values leading to a greater number of clusters. -```R -#here construct k-nearest neighbours graph based on euclidean distance in PCA space, then refine edge weights based on Jaccard similarity. this takes the number of PCs previously determined as important (here 40 PCs_) -day2somules <- FindNeighbors(day2somules, dims = 1:40) +```R #this iteratively groups cells using Louvain algorithm (default). Resolution sets the granularity. 0.4-1.2 gives good results for ~3K cells, with larger number suiting larger datasets. day2somules <- FindClusters(day2somules, resolution = 0.5) ``` -## Plot UMAPs +## Run non-linear dimensional reduction -Runs umap to visualize the clusters. Need to set the number of PCs +Seurat offers several non-linear dimensional reduction techniques, such as tSNE and UMAP, to visualize and explore these datasets. The goal of these algorithms is to learn underlying structure in the dataset, in order to place similar cells together in low-dimensional space. Therefore, cells that are grouped together within graph-based clusters determined above should co-localize on these dimension reduction plots. ```R +#runs umap to visualise the clusters. Need to set the number of PCs day2somules <- RunUMAP(day2somules, dims = 1:40) #visualises the UMAP DimPlot(day2somules, reduction = "umap") -ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) +#ggsave(paste0("day2somules_v10clust_40PC_0.4res_SCT",st,".jpg")) ``` ![](figures/SC_Figure_12.png) -**Figure 12.** +**Figure 12.** UMAP plot, cells are colored by cluster -#Plot metadata +### Plot metadata Now we have a UMAP representation of our cells, we can also use that to visualise the metadata. ```R @@ -570,7 +516,7 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_shuffled_batch_SCT.jpg") ``` ![](figures/SC_Figure_13.png) -**Figure 13.** +**Figure 13.** UMAP plot, cells are colored by batch What do you notice once the UMAP is coloured by sample? Is there anything you might do about this? @@ -580,12 +526,12 @@ ggsave("day2somules_v10_40PC_0.5res_after_one_filt_mt_SCT.jpg") ``` ![](figures/SC_Figure_14.jpg) -**Figure 14.** +**Figure 14.** UMAP plot, cells are colored by percentage of mtRNA What do you think about this figure? Is there any cluster with a higher proportion of MT RNA? -## Markers identification +## Finding differentially expressed features (cluster biomarkers) We want to understand what the cell clusters might be. One approach to do that is find gene that are cluster markers - find differentially expressed genes that are descriptive of a cluster. @@ -594,7 +540,7 @@ DefaultAssay(day2somules) <- "RNA" day2somules<-JoinLayers(day2somules) -#day2somules.all.markers_roc <- FindAllMarkers(day2somules, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") +#day2somules.all.markers_roc <- FindAllMarkers(day2somules, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") #We could search the markers for all clusters, however that would take too long. day2somules.markers_roc_cluster0 <- FindMarkers(day2somules, ident.1 = 0, only.pos = TRUE, min.pct = 0.0, logfc.threshold = 0.0, test.use = "roc") ``` @@ -629,7 +575,7 @@ A brainstorm question: how might you use this list to choose a gene to use for i How else might you classify cell cluster tissue types? -## Plot the expression of individual genes in the UMAP +### Explore the expression of specific genes in the UMAP Now we can look at gene expression in these data by gene, which could be useful to identify cluster marker genes. The example below shows you ago 2-1. If you want to look at a different gene, simply type the gene ID where "Smp-179320" currently sits. Remember to use - rather than _ ! We can visualise genes that we already know something about in the organism, to see if that gives us some clues ```R @@ -637,7 +583,7 @@ FeaturePlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320-",st, ".jpg"), width = 25, height = 15) ``` ![](figures/SC_Figure_15.png) -**Figure 15.** +**Figure 15.** UMAP plot showing the expression of Smp_179320 (ago2-1) per cell We can also look at these genes with a violin plot - this visualisation can be helpful in many ways, including seeing which clusters a gene is most expressed in. @@ -646,9 +592,9 @@ VlnPlot(day2somules, features = "Smp-179320") #ggsave(paste0("day2somules-Smp-179320_",st, ".jpg"), width = 25, height = 15) ``` ![](figures/SC_Figure_16.png) -**Figure 16.** +**Figure 16.** Violin plot showing the expression of Smp_179320 (ago2-1) per cluster -Are there any genes you're particularly interested in? You can adapt the code below to see if, and where, it might be expressed in these data. If you're not sure, you can look up S mansoni on WBPS and choose a gene from there (https://parasite.wormbase.org/index.html). Remember to delete the # if you want to save the plot to your computer! +Are there any genes you're particularly interested in? You can adapt the code below to see if, and where, it might be expressed in these data. If you're not sure, you can look up _S.mansoni_ on WBPS and choose a gene from there (https://parasite.wormbase.org/index.html). Remember to delete the # if you want to save the plot to your computer! ```R FeaturePlot(day2somules, features = "your_gene") @@ -663,7 +609,7 @@ coexpression.plot #ggsave(paste0("day2somules-coexpressed-Smp-179320-Smp-086860-",st, ".jpg"), width = 45, height = 25) ``` ![](figures/SC_Figure_17.png) -**Figure 17.** +**Figure 17.** UMAPs showing the co-expression of Smp_179320 (ago2-1) and Smp_086860 (histone H2A) per cell Can you find a pair genes whose products you might expect to interact, and see if they are co-expressed? Again, you can use WBPS to learn more about genes. @@ -671,7 +617,7 @@ Can you find a pair genes whose products you might expect to interact, and see i #put your code in here! ``` -## Rename UMAP cluster IDs +## Assigning cell type identity to clusters You want all the cluster names to be unique - why might that be? ```R @@ -707,4 +653,4 @@ LabelClusters(plot1, id = "ident", color = 'black', size =4, repel = T, box.pad ggsave(paste0("day2somules-labelledlclusters_40PC_0.5res_",st,".jpg"), width = 25, height = 15) ``` ![](figures/SC_Figure_18.png) -**Figure 18.** +**Figure 18.** Once you have identified the cell types you can rename the clusters From 988fd76d38ba207334eb2ee48a2adf982e98d1cb Mon Sep 17 00:00:00 2001 From: santifo-creator Date: Wed, 7 May 2025 18:46:39 -0300 Subject: [PATCH 51/51] Delete course_modules_2025/module_transcriptomics/figures/SC_Figure_2.png --- .../figures/SC_Figure_2.png | Bin 23352 -> 0 bytes 1 file changed, 0 insertions(+), 0 deletions(-) delete mode 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