Questions tagged [scrnaseq]

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

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186 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 ...
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1answer
252 views

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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1answer
66 views

how to get normcounts for singlecellexperiment object?

I need to perform differential expression analysis using the scDD package from R, but I am not able to since I miss the normcounts assay in my SCE object (of course in the example they show, the assay ...
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1answer
52 views

Challenging benchmarks for supervised learning on sparse scRNA-seq data

One challenging aspect of modeling scRNA-seq data is data sparsity, that is, scRNA-seq measurements typically suffer from large fractions of observed zeros (i.e. dropouts), where a given gene in a ...
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1answer
97 views

What is the best way to address the question of doublets and multiplets in a single cell RNA seq data set?

I have attached a histogram plot of the number of genes per cell in a single cell RNA seq data set of lung endothelial cells. I do not find a bimodal or multimodal distribution of the number of genes ...
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1answer
3k views

Changing active.ident in Seurat

Im trying to change the active.ident to another column in metadata but this error keeps popping up! I recently upgraded to R version 4.0.2 from 3.6.1 The older version was working but the new one isn'...
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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 ...
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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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1answer
60 views

Article GEO gives counts that are not integers. Should UMI counts be integers?

I thought that UMI counts are always integers, but when I opened several datasets provided by GEO I got confused because in some ...
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2answers
384 views

SC-RNA seq percent.mito

Is that possible that the percent.mito level is zero? I have done vlnplot and it shows that its percent.mito level is zero. Does that mean my mitochondrial gene didn't detect? It is violin plot. And ...
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1answer
154 views

How to search a specific sequence in BAM files for 10X experiment

I have to search a specific sequence in a set of cells (> 1k cells) from a single-cell experiment done with 10X genomics. As input file I have a single bam file, and 24 fastqs, therefore each file ...
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1answer
37 views

Looper: missing argument error

While trying to run looper I am getting the following error: Error (missing attribute): 'sandro_rna_seq' requires sample attribute '/scratch/nv4e/sandro_looper/pipeline/jyvo_experiment-metadata....
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1answer
20 views

Looper: Duplicate sample name error

I am using Looper. While running looper run project_config.yaml I am getting duplicate sample name error. I have two ...
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1answer
59 views

Error in .checkedCall : subset indices out of range [closed]

After normalization the way it is described here: https://bioconductor.org/packages/3.7/bioc/vignettes/scran/inst/doc/scran.html Using quickCluster() function ...
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1answer
58 views

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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1answer
32 views

Is there a difference between any scRNA-seq visualization and pseudotime?

Is there a good definition of pseudotime? Some tools are clearly labeled as pseudotime and produce values along a trajectory, but there are more complex approaches that involve branching and ...
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1answer
626 views

Seurat FindMarkers() output interpretation

I am using FindMarkers() between 2 groups of cells, my results are listed but i'm having hard time in choosing the right markers. Do I choose according to both the p-values or just one of them? If one ...
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1answer
381 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 ...
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1answer
33 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 ...
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1answer
388 views

Compare clusters from different datasets

I have two scRNAseq datasets, and I select a cluster from one of them, and another cluster from the other. I want to find differentially expressed genes between the ...
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1answer
186 views

TagReadWithGene missing when using latest version of Drop-seq_tools

I am using Drop-seq_tools to analyze scRNA-seq in a similar way of this paper. I am using the same data (GSE97930) to get used to Drop-seq_tools. On the GEO repo the authors provide the relevant code ...
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2answers
808 views

Find a cutoff value for genes that are expressed in single cell RNA-seq?

I want to find a cutoff value for each gene, above which we can consider a gene expressed. The problem is that not all effectively non-expressed genes will have 0 counts due to sequencing errors for ...
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1answer
51 views

Is it possible to use spike-ins with droplet technologies?

By reading Bacher & Kendziorski (2016), I found the following statement: recent droplet technologies are not yet able to accommodate spike-ins What's the reason why current droplet ...
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1answer
39 views

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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0answers
119 views

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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0answers
83 views

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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0answers
16 views

Clustering by individual tissue population post integration of two datasets

Sorry if this question has been asked. Post batch correction and integration of two datasets, where cells of multiple different tissue types are present, I want to run clustering on cells of ...
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0answers
41 views

10x Single-cell RNA-Seq - Updating gene symbol with latest ensembl release - Ensembl IDs missing

I am trying to update the gene symbols provided by the 10x human precompiled reference (2020-A), using the mapping Ensembl ID to gene symbol provided in release 102. However, I found out that 56 of ...
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0answers
42 views

Clusters in scRNA of Glioblastoma

There is a scRNA dataset on Glioblastoma from 10xgenomics. Unfortunately, there seems to be no research paper related to that data. Question 0 : May be there is paper ? How to find it ? I am not ...
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0answers
60 views

Genes associated with digestion in single cell RNASeq

I am reading Saunders et al. (2018). In the methods' subsection "ICA based analysis and clustering" (independent component analysis), a set of independent components from ICA is labelled as &...
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1answer
54 views

Bootstrapping cell type identifications (scRNA-seq)

I have scRNA-seq data held in a SEURAT object, and cell-types were identified with SingleR (essentially a vector of strings - ...
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1answer
120 views

Differentially expressed genes analysis in Seurat

For the differentially expressed genes analysis, is it possible to check for DEGs based on the levels already identified in the object? For example, my dataset contains cells from 11 subjects, which ...
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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 ...
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1answer
156 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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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, ...
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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 ...
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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 ...
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0answers
283 views

How to create a legend in FeaturePlot with do.hover at the same time?

you can see in my feature plot I can only see the cells from which sample but I cannot see the legend. And if I change the command I can see the legend but cannot see the sample identities....
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0answers
99 views

Variability in genes across platforms

I have a matrix of gene expression for single cell (cells in column and genes in row) in 9 time points with about to 200 cells for each time point. I have also a matrix of gene expression in bulk RNA-...
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0answers
80 views

Ordering the cells by the expression of specific genes

I have 209 cells. I want to show the expression of two genes by ordering my cells. Imagine something like a plot on which y axis shows the read counts range from 0 to 10000 and x axis shows the number ...
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3answers
766 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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3answers
119 views

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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2answers
61 views

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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1answer
62 views

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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3answers
993 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
254 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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2answers
196 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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1answer
25 views

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 ...