Questions tagged [scrnaseq]

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

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IsoPlexis Data Analysis

Has anyone analyzed the raw data output from IsoPlexis secretome profiling? I want to make use of this data outside of their "end-to-end" software.
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How to manage memory contraints when analyzing a large number of gene count matrices? I keep running out of RAM with my current pipeline

I have several hundred scRNA-seq count matrices, each from a different sample. For my other dataset containg a few dozen samples, I simply merged everything together into one Seurat object, but that ...
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scRNA-Seq pseudobulk for marker detection

A standard approach for scRNA-Seq is to partition the single cells into individual clusters, then use a Wilcoxon test to find markers that characterize each cluster (or other statistical methods that ...
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Subsetting out prefix from genes and merging

I'm working with PTX samples (human and mouse), they're aligned to both transcriptomes and so gene names contain hg38- and mm10- at the beginning of each gene. I have eliminated teh prefixes because I ...
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Normalization methods to combine scRNA-seq experiments with different sequencing depths

I am training a classifier to identify a cell type in a particular state of activity using scRNA-seq. There is a large variation in the sequencing depth (reads average per cell) of the testing data (...
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How will Seurat handle pre-normalized and pre-scaled data?

I want to use data from the following dataset: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE84371 The data has been TPM normalized, which is not ideal for clustering but I have to work with ...
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Why is bulk RNA sequencing reflecting AVERAGE expression but not TOTAL expression of all cells?

When I am reading papers that compares bulk RNA sequencing and single-cell RNA sequencing, we often see papers describe bulk RNA seq measures the average cell expression. For example, in this paper ...
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Pulling normalized gene expression data across cells from Seurat object

I want to pull gene expression out of a seurat object. I created one called combinedObject following their tutorial, I normalized scaled it etc and created some ...
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2 answers
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Plotting a gene in Seurat

I saw in the extensive Seurat documentation for Dimplot (dimensional reduction plot), here, you can plot a gene by specifying it with group.by = "gene" but this does not work in practice. <...
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Featureplot by co-expression of some genes

I want to identify MDSCs cells which don't not express HLA-DRs (HLA-DR negative) but express ...
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Arrange ggplot Figure for scRNA-seq data

I have generated a ggplot for 8 single-cell libraries, with the purpose of visualizing the tSNE facet plot by sample, colored by cell type -- with percentages. The best I could get to is this - ...
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How to solve correlation problems between different samples in scRNA-seq?

I am trying to align and merge different samples from NCBI. I end up having correlation problem with these sample. The picture below shows an heatmap of the R² by doing a linear regression between 2 ...
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Some definitions about RNA-seq

I want to select a 10x single cell kit What does 2x 50 75 100 150 250 mean in paired end sequencing?
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(scRNA-seq) I have a dataset with 4 conditions (experiments) should I be anchoring aka integrating data for each comparison when looking for DEGs?

I have an experiment with 4 conditions in it two of them are controls. When I am correcting for batch effects (either with PCA or CCA) should I restrict my comparisons between particular groups? So ...
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Integrating scRNA-seq data using raw data

I am trying to reporduce the the following article Chen, M. B., Jiang, X., Quake, S. R., & Südhof, T. C. (2020). Persistent transcriptional programmes are associated with remote memory. Nature, ...
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Acquiring & Processing single cell data (not 10x)

I have a question related to processing of Single cell RNA-seq data originating from non 10x platform. I have worked with data that originated from 10x platform and I parse it through cell ranger ...
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Can 5' end 10x be used instead of 3' end

I have PBMCs of some patients who have recovered from cancer, and I want to look at their immune landscapes. I got confused when looking at available kits: can I use 5' end kits from 10x instead of 3' ...
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Assigning subcluster idents to original object

I have a scRNA-seq Seurat object I've analyzed, and I noticed that for some of the clusters, there's more than one cell type. I've created a subset which and run FindClusters again to label the cell ...
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How to split a Seurat cluster in several subclusters?

I've analyzed my scRNA-seq data and have a couple of Seurat clusters that show more than one cell type in each cluster. (for example, cluster 9 shows both NK and CD4 cells) How can I split a cluster ...
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Gene names from components in NMF analysis

I am using Rcppml package in R for my NMF analysis, I have a matrix from single-cell analysis. I have cells scores and I wanted to know how to extract genes instead of the components. i used the ...
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Using different "principal components" in UMAP visualizations

In certain UMAP visualization, it appears that the visualizations show the first UMAP "principal component" plotted against the second. For example, like this: Apparently, this lets us use ...
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How to identify in a RNA seq data in which sample is in which cell using R using qusage

I am using RNA seq data and have been using QuSage in R in hopes to try to identify different samples and figure out which cell it belongs to. I am trying to visualize and utilize the results but I am ...
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FindMarkers from Seurat returns p values as 0 for highly significant genes

I have been working on FindMarkers function for identifying significant genes in the cluster. But some Significant genes have very low p values, so they are returned as 0 in the output.Any value less ...
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CITEseq different sequencing depth normalization -- Seurat

New technician ruined his first 10X CITEseq experiment on cells counting level and the ratio between replicates is 14k:2.2k:0.8k ncells. How should I now normalize protein data (ADT) from experiment ...
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2 votes
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Calculating percent mouse cells per sample

I want to see the correlation between % mouse reads per sample vs % mouse cells per sample. I've already calculated the % mouse reads per sample but I'm stuck on calculating the % of mouse cells per ...
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A very large number of clones in BCR reportorie

I am using mixcr to convert fq.gz raw data file (single cell BCR sequencing) to txt files with the names such as JX01_d3-B.clonotypes.IGH.txt , Then I use the immunarch to load the file to explore the ...
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Problem with Seurat reference mapping

I have 10X scRNA-seq multiome's 3' poly-A capture (scRNA-seq+ATAC-seq) from PBMC Using Seuratreference mapping, I mapped my scRNA-seq part on reference PBMC (for ...
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Understanding the dot plot from seurat

I am working with single cell data and using seurat to analyze the results. Often in manuscript, we see the dotplots showing the expression of the marker genes or genes of interest across the ...
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sctransform - mitochondrial expression filtering / transformation

I have a Seurat object which has a high expression of mitochondrial genes ...
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Creating new seurat object with new matrix

I'm trying to do a cross-species comparison between the patient TME vs. the PTX TME to understand gene expression conservation. I have already converted between mouse and human genes giving me a ...
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Avoiding the warning 'The following features were omitted as they were not found in the scale.data slot for the SCT assay' in Seurat

I have a Seurat object I want to plot a list of genes but I got a warning saying most of genes removed I have tried with both my genes of interest and all variable genes but the results is very ...
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Find corresponding symbol for gene used in Seurat

I have a Seurat object ...
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TCR-seq or scRNA-seq

I need idea, intuition, suggestion please I have 10X scRNA-seq of PBMC (multiome's 3' poly-A capture), can I capture ...
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Sample versus subject in scRNA-seq

I am reading this paper, where the authors mention We apply SAUCIE to the batch correcting, denoising and clustering of an 11-million cell mass cytometry dataset with 180 samples from 40 subjects in ...
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Making a box plot of the proportion of cells in each cell types in two groups

I have number of cells in three cell types T, B, M in a Seurat object For two groups of patients, cancers and controls like ...
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Seurat heatmap for two conditions

I have a Seurat object of four cancers and four controls ...
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Help with Seurat QC ambiguity

I have four PBMC samples from 10X scRNA-seq ...
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2 votes
1 answer
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SCTransform Warning: in theta.ml(y = y, mu = fit$fitted) : iteration limit reached

Running SCTransform on my seurat object produces the warning: Warning in theta.ml(y = y, mu = fit$fitted) : iteration limit reached What does this warning mean? ...
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Merging two dataframes in R

I have a file with cluster number of a scRNA-seq and corresponding annotated cell type like ...
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Filtering cells from values in metadata

I metadata slot of Seurat object I have mapping score of each cell to a reference PBMC data like ...
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What does this Seurat argument mean

I have extensively read about percent mito in Seurat but I got more and more confused Let's say we want to keep cells with ...
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Seurat Dimplot with different clustering IDs

In Seurat metadata I have assigned cells to some cell types with different resolutions I have added the cluster identities to the object via Idents: ...
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The confusion of using TPM (transcripts per million)

It is shown that TPM values are not suitable for DEG analysis but good for within-sample comparison since TPM normalized the gene length. My question is first: if TPM is not suitable for across ...
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10X scRNAseq: Sample mix-up

The student who was working on scRNA seq of KO and WT lines has made a mistake and he mixed both lines and generate the final sequencing data. Now, we are having gene expression data but don't know ...
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color by clusters and sampled in Seurat

I have a Seurat object I want to have both cluster numbers and coloured cells by sample names like this figure (from a Nature paper) I have tried group.by argument ...
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Adding metadata to Seurat object

I have a Seurat object of 8 patients. I want to add metadata to that so that I have origin of each cell. At the moment UMAP just shows a bunch of cells while I want to color clusters by sample This is ...
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1 vote
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How to run GSEA analysis on R studio using DEG file list generated from scRNAseq analysis

I want to draw the standard plot of GSEA on Rstudio. I have a data frame that consists of a list of DEGs as below. Actually, I put the DEG list directly to PANTHER that resulted in the GO or pathway ...
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Is there an easy way to identify nuclear genes using scanpy?

This question has also been asked on Biostars Scanpy has documentation where mitochondrial genes could be easily filtered out using ["MT"]. I was wondering if anybody has information on ...
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1 vote
1 answer
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Seurat clustering Methods-resolution parameter explanation

I am learning the Seurat algorithms to cluster the scRNA-seq datasets. I found this explanation, but am confused. Can someone explain it to me, "The FindClusters function implements the procedure,...
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Removing cells zero for a gene from a scRNA-seq data

I have a big single-cell RNA seq data ...
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