Questions tagged [scrnaseq]

Use this tag for questions related to single-cell RNA-seq.

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10 views

umap highlighting two different models

I'm trying to create a umap for single cell data from human samples and ptx samples. I can get the umap to where it shows the umap with the different clusters but I want to show where the ptx samples ...
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1answer
37 views

How to identify latent variables in single-cell RNA-Seq data

I have a single-cell RNASeq sample, in which I'd like to identify latent variables (e.g. response to stress) that I think might be affecting the clustering. The approach I was planning to use is to ...
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1answer
61 views

Reading multiple raw files in Seurat

I have multiple single cell samples to analyze and I'm following the instructions in Satija Lab's website. I want to merge all the count files from all the samples at once, and associate the metadata ...
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1answer
117 views

Identifying mutually **exclusive** gene sets

I am interested in identifying gene pairs (or better: sets of genes) whose expression is mutually exclusive. Ideally, both genes (or gene sets) would be widely expressed but I am also interested in ...
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1answer
44 views

Seurat clusters

I'm trying to show cell cycle regression and input sample metadata to my umap. I've been following Satija Lab's tutorials and have generated my umap but now want to specify which sample corresponds ...
3
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1answer
76 views

Differential gene expression bias due to effect of an individual sample

I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various ...
1
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1answer
28 views

Most scalable pseudotime ordering algorithm

What algorithms for linear Pseudotime trajectory construction (diffusion-based) are the most scalable to large datasets? I'm currently using Slingshot based on the recommendation in this manuscript: ...
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1answer
38 views

Percentage of each cluster in Seurat

I am using Seurat to analyze my single cell data. I have 2 conditions, treated and untreated. I am trying to create a stacked bar graph in order to show the differences in cell types for each ...
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1answer
31 views

Processed spliced and unspliced count matrices for existing scRNA-seq atlases

Before I do this myself... Is there any compiled batch-corrected dataset of the major scRNA-seq atlases (Mouse Cell Atlas, Mouse Organogenesis Atlas, Mouse Gastrulation Atlas, Tabula Muris, Tabula ...
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1answer
67 views

Low Fraction of usable antibody reads in CiteSeq

we performed a combined gene expression and CiteSeq experiment with the 10x VDJ kit and 20 conjugated antibodies and sequenced on hiseq. I used cellranger to process the sequencing output. The ...
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1answer
16 views

Calculating celltype proportion changes between conditions in single-cell data

I want to test for significance between differences in cell count between conditions. I have 2 conditions (N, T), 3 donors (samples) each, and four cell types (A-D) as shown in the example below. I ...
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1answer
339 views

Seurat VlnPlot presenting expression of multiple genes in a single cluster

Seurat VlnPlots are most commonly used to visualize differences in any given gene expression across multiple clusters or cell types. For example: ...
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2answers
223 views

Trim Seurat object to contain expression info only for selected genes

I'm working with some large Seurat objects (MOCA, MCA, Tabula Muris) studying gene coexpression, and I'm running into memory issues. Is it possible to remove all genes in a Seurat object that are ...
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0answers
36 views

How to set equal distances between axes ticks irrespective of number of points I plot in a ggplot?

how can I set an equal distance between the ticks of axes in a ggplot irrespective of the number of items I plot? For example I want to have the kind of plot below from Seurat's ...
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1answer
97 views

How to calculate the number of cells in different cell clusters with Seurat v3?

I was supposed to use the code below to get the number of cells per cluster. The code works with Seurat version 2, but while using version 3 I got the error ...
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3answers
193 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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0answers
26 views

Human Cell Atlas - cell annotations

Human Cell Atlas Preview Datasets have been available for a while now (there was some discussion about that earlier). However, although the raw data is available, cell labels are not (for example, ...
2
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1answer
189 views

How to load and split my two individual datasets when integrating datasets in Seurat?

I want to do comparative analyses of my two individual single cell RNA-Seq datasets in Seurat. And I load and list my data as follows: ...
3
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1answer
31 views

public multi-modal single-cell data

There is a scRNAseq Bioconductor package with a few different example scRNA-seq datasets. Are there any R packages that offer multiple modalities of single-cell data? For example, hashtags or ADTs or ...
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43 views

Want to make a tsne plot show subset

I am new to R. I make a tsne plot for my data "yfp". And there are two subsets in my data (yfppt and yfpng). I want to show their distribution or location in yfp tsne plot but failed. And I even did ...
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2answers
68 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
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3answers
142 views

Understanding Single Cell RNAseq Plots

I have just started some single cell RNAseq analysis with 500 cells. I have used Seurat for analysis. Following the guideline I have generated few plots. Now I am trying to understand how to read ...
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2answers
190 views

how to generate an scRNA counts file

I have barcodes.tsv, genes.tsv and matrix.mtx file from my RNA seq data and I want to generate a scRNA counts files using R. When I tried using the readMM function, ...
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0answers
70 views

High percentage of poly A sequences in 10X chromium R2 read

I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality. According to the Cell Ranger summary 56% of the reads can be mapped to ...
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0answers
114 views

PCA on large sparse matrix of single cell RNA-seq

I received a large sc-RNA-seq data matrix, as of the nature of sc-RNA-seq compared to bulk-RNA-seq the data matrix is very sparse. Moreover, due to the fact that this is single cell data the cell ...
2
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1answer
56 views

Single Cell RNA seq Analysis for low input Cells?

I am having a single cell RNA seq data from ~500 Cells. I do understand this number is very low for present day analysis tools like Seurat. So, I want to know what if one have a such a small number of ...
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1answer
178 views

Hierarchical clustering for outlier detection - single cell RNASeq & WGCNA

I wish to conduct WGCNA on a single cell RNASeq dataset and, when choosing the optimal beta parameter after running pickSoftThreshold am presented with a rather ...
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0answers
138 views

Normalization of single cell RNASeq data with ERCC spike-ins

I wish to normalize a scRNASeq dataset with respect to ERCC spike-ins, where "for some of the samples, ERCC spike-in RNA was added to the lysis buffer" and was wondering how to do so? I have seen ...
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2answers
101 views

How are UMIs used to dedupulicate in Drop-seq tools?

The module DigitalExpression which is part of the popular Drop-seq tools digitally count gene transcripts. The manual is not very clear on how exactly it resolves ...
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0answers
20 views

Is there a computational method/method/way to predict if a cell population in a tissue immigrated or is enriched locally?

I have single cell data which I have analysed for differential expression. In my experiment, I subjected two groups of mice (a control and a treatment group) to treatment that will lead to the ...
2
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1answer
466 views

How to highlight specific cells in Seurat 2.4

I used Seurat 2.4 on our scRNA dataset to obtain the following tSNE plot.I was able to successfully extract cell IDs from the different clusters, and generate gene expression profiles. The analysis, ...
4
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1answer
563 views

PCA vs tSNE in single cell RNA-seq

What makes tSNE being the preferred dimensional reduction for visualization in single cell RNA-seq over PCA? I am aware that tSNE works better at showing local structures and fails to capture global ...
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1answer
55 views

How I normalize these two sets of data

I have average log fold change for a cluster of cells versus another cluster of cells by Seurat like below ...
2
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1answer
297 views

Why do I have >10,000 cells in the 10X matrix produced by cellranger?

In my scRNA seq experiment, single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3′ Library & Gel Bead Kit v2 and Chip Kit (10x Genomics) according to the ...
2
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1answer
749 views

How do I add a colour annotation bar to the heatmap generated by DoHeatmap function of Seurat v2?

I am using Seurat v2 for professional reasons (I am aware of the availablity of Seurat v3). I am clustering and analysing single cell RNA seq data. How do I add a coloured annotation bar to the ...
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2answers
88 views

residual Squared Coefficient of Variation (rCV²) vs Distance to Median (DM)

I am working with single cell RNA-seq data. I obtained the squared coefficient of variation (CV²) as a measure of gene expression variability: I want a metric to express gene expression noise that ...
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0answers
94 views

scRNA-seq multi-dataset integration for small datasets

There have been a few methods proposed for integration (or batch correction) of scRNA-seq datasets, such as Seurat CCA, MNN Correct, Scanorama, and Harmony. The concern is generally about the maximum ...
1
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1answer
126 views

Discordance in gene signature behavior between bulk and single-cell RNASeq

The objective of the following analysis is to identify an activation signature of a specific phenotype on bulk RNASeq and to apply it to single-cell RNA-Seq, in order to identify the population of ...
3
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1answer
318 views

Understanding PCHeatmap outputs

I am currently trying to understand the purpose of these PCHeatmaps - part of the seurat package in R: All the online documentation I have searched for has only ...
2
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1answer
62 views

scRNASeq expression matrix with decimal values

I am trying to replicate some results of a scRNASeq experiment and, when I looked at the data provided by the author, I noticed that some of the counts in the expression matrix are represented as ...
-1
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1answer
82 views

How do I perform a pathway related grouping of genes?

I have a list of gene names from the analysis of my mouse single seq data. I want to perform a simple grouping of the genes based on the pathways to which they belong or pathways through which they ...
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3answers
797 views

How can I obtain the percentage gene expression per identity class in Seurat as further processible numbers (e.g. matrix)?

I am analysing my single cell RNA seq data with the Seurat package. I want to know if there is a possibilty to obtain the percentage expression of a list of genes per identity class, as actual numbers ...
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2answers
68 views

Using preprocessing/alignment functions on the server

I am new to bash and the processes behind cluster computing in general and need some help with understanding some basics. After looking all over the internet and this forum (+ askUbuntu) I found ...
2
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1answer
300 views

Percentage distribution of cells in all clusters based on their treatment condition?

I have 2151 cells, I clustered them by Seurat to 5 clusters. With the code below, I am able to have the number of cells per cluster and per condition: ...
2
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2answers
86 views

scRNA-seq differential transcript usage

Many of the modern gene-quantification tools (Salmon/Kallisto) output transcript-level (as opposed to gene-level) data. All of the scRNA-seq analysis I have seen just uses the gene-level values. I ...
3
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1answer
172 views

How do I prevent the FeatureHeatmap function from the Seurat package, from sorting my data groups in alphabetical order when plotting data?

How can I prevent a function from sorting my data groups (factors) in alphabetical order without affecting the integrity of the data? I am analysing single cell RNA sequencing data using Seurat 2.3.4. ...
2
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1answer
692 views

How to set the position of groups in a Seurat object on a FeatureHeatmap plot

I am analysing singe cell sequence data and I have followed this tutorial, https://satijalab.org/seurat/pbmc3k_tutorial.html to perform QC and various differential analyses using the Seurat package on ...
2
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1answer
459 views

Visualising gene expression across cell type and conditions in one plot, in Single Cell Sequencing data

I am new to single cell sequencing data analysis but I have basic programming background in R and python. I want to be able to plot differential expression of two genes, Gene1 and Gene2, across three ...
3
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2answers
1k views

Separate boxplots for multiple violin plot

I am using the following function from seurat package to generate multiple violon plots and I am interested in adding box plots to them but it doesn't work when I have plotted different data at once. ...
5
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1answer
370 views

How to normalise scRNASeq data for differential expression analysis

I wish to perform differential expression analysis for cluster-specific gene expression in single-cell data (with a tool such as MAST or SCDE). I have data for 3 biological replicates. I performed ...