Questions tagged [scrnaseq]

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

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43 views

Alternatives to imputation for single-cell RNA-Seq analysis to increase statistical power of DE analysis

I have a small scRNA-Seq dataset (n = 357, inhibitory neurons). This set of cells is split almost evenly between two conditions (Case and Control). I would like to test for differential expression ...
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2answers
3k views

Subsetting from seurat object based on orig.ident?

I am pretty new to Seurat. I want to subset from my original seurat object (BC3) meta.data based on orig.ident. however, when i use subset(), it returns with Error. ...
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1answer
1k views

How to add cluster name to metadata in Seurat?

I'm working on a Seurat object and want to name the clusters according to 2 values alone (yes/no). So I want to add a new column to metadata and annotate the clusters (UMAP) with it. ...
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1answer
480 views

Adding treatment groups via metadata to Seurat object?

I'd like to add metadata to 6 individual Seurat objects so that after I merge the objects into one, I can later label or split by using these identifiers. For example, I'd like to append an age group ...
2
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0answers
91 views

scRNA-Seq: Account for sequencing depth and gene length?

Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length. For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
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0answers
54 views

gene set enrichment in single-cell data

Gene set or pathway analysis in scRNA-seq data has its own challenges. Since the data is sparse and many genes will be missing for any given cell, some people simply add up all the counts across the ...
2
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3answers
372 views

How to reduce the occupied RAM when you are dealing with a very sparse matrix in a single-cell Experiment in R?

I'm dealing with a very large and sparse dataset and the first issues I met occurred when I tried to use quickCluster that reported me this error: ...
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0answers
19 views

minimum number of neighbors for scRNA-seq analysis

Many scRNA-seq clustering/visualization methods are based on nearest neighbors. The maximum number of neighbors is limited by the neighborhood size. Is there a method to determine the minimum number ...
2
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0answers
73 views

Clustering issue about overlapping CD4, CD8, and Foxp3 genes in mouse tumor models

bioinformatics scientists. I hope your work goes smoothly well and be safe. I'm stuck in trouble at the step of clustering. Briefly, I downloaded fastq files from a published study (https://www.ncbi....
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1answer
1k views

Error: Insufficient values in manual scale. 24 needed but only 1 provided?

folks. Can you give me idea about an issue that I encountered? I ran single-cell RNA sequencing data using Suerat in R, and when I tried to draw violin plot, there is an error as below. VlnPlot(...
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1answer
395 views

PCA plot did not work in single cell RNA-seq

I run plotPCA for single cell RNA-seq data, while I get error message (I use R 4.0). I attached the code and error message here. Did anyone know the reason and how to deal with it. Thanks! ...
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2answers
1k views

calculateQCMetrics defunct, how to calculate the quality metrics by perCellQCMetrics

I am trying to follow a tutorial from Sanger institute (from May 2019) on analysis of single cell RNA Seq data. They use calculateQCMetric function to calculate the quality metrics, but I am getting ...
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1answer
453 views

How to remove zero value of gene on FeatureScatter plot using Seurat?

Nowadays, I am trying to calculate Pearson correlation values between two genes of my interest from single-cell RNA-data (features.tsv, barcodes.tsv, and matrix.mtx files) which are obtained from the ...
3
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1answer
604 views

Receptor-Ligand R-package for visualization

I was wondering if there was any R-package for receptor-ligand interaction similar to SingleCellSignal (https://github.com/SCA-IRCM/SingleCellSignalR_v1/blob/master/SingleCellSignalR/vignettes/...
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3answers
997 views

isSpike function in SingleCellExperiment package is deprecated?

I am trying to follow a tutorial from Sanger institute (from May 2019) on analysis of single cell RNA Seq data. They use isSpike function to filter out ERCC (control) and MT (mitochondrial RNA) reads, ...
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1answer
256 views

Is it important to filter out poor quality cells before performing an integration analysis on single cell RNA sequencing data?

In order to perform an integration analysis of single cell RNA seq data, is it important to check the percentage expression of mitochondrial genes of cells as well as the feature counts to exclude ...
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29 views

How do I generate a list of post-synaptic gene markers that will guide me in my search for these markers in intestinal stem cells?

I would like to generate a list of gene markers that represent post-synaptic related genes. I will then use this list to search through single cell sequencing data, in order to assess whether certain ...
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1answer
50 views

Is there a way to get the code from "github.mit.edu"?

everyone. I am a postdoc who just begins to analyze single-cell RNA-seq data. Nowadays, I found a really interesting paper (https://doi.org/10.1016/j.celrep.2018.10.047, PMID: 30404002). So, I ...
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1answer
37 views

Is there a single cell RNAseq equivalent of GTEx or TCGA? [closed]

Or do I need to find individual studies and obtain data the long way!
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1answer
158 views

What column and row naming requirements exist with Seurat (context: when loading SPLiT-Seq data)

I'm trying to use Seurat for the first time and am learning about single-cell analysis for the first time, and I'm doing so with split-seq data. (Full disclosure: I'm also a lightweight when it comes ...
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1answer
701 views

Cell Ranger) Error during performing a test run

I am interested in the analysis of single-cell RNA-sequencing data. I installed Cell Ranger according to the instruction (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/...
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3answers
2k views

How do I pull singe cell RNA sequencing data from GEO database?

I am new to R and computational biology. I am trying to look through a published data set to check for gene expression for my own project. I am having trouble finding materials to teach me how to ...
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3answers
771 views

How to import data from cell ranger to R (Seurat)?

I will have some scRNA-seq data. The goal of the experiment will be to see if there is any difference in gene expression between treatment groups using the package Seurat from R. I have read a ...
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1answer
384 views

Set new Idents based on gene expression in Seurat and mix n match identities to compare using FindAllMarkers

I am relatively new to Bioinformatics and scRNA-seq data analysis. I am using Seurat V3 to analyze a scRNA-seq dataset in R. Currently, I have merged three scRNA-seq samples from the same donor into ...
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1answer
106 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
75 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 ...
1
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1answer
1k 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 ...
2
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1answer
153 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
104 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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2answers
241 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 ...
2
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2answers
86 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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2answers
2k 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
57 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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0answers
523 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
34 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
3k 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
2k 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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1answer
2k 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
1k 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
32 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
920 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
55 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 ...
2
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2answers
92 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
862 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
538 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, ...
4
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2answers
188 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
147 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
129 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
397 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
256 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 ...