Questions tagged [single-cell]

Questions about techniques in which the genetic material of a single cell is analysed individually

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How to convert 10x matrix.mtx.gz files to hdf5 format?

I have a bunch of folders containing barcodes.tsv.gz, features.tsv.gz, and matrix.mtx.gz from the Cellranger Count output for a single-cell dataset that was sent to me from another lab. I need the .h5 ...
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How can I get data of single cell RNA sequence with raw count?

I am using dataset GSE85241, but I can't find the read counts of the dataset. It only provides with RPKM values. How can I find the read counts of a dataset?
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Merging two seurat objects

I want to merge 2 Seurat objects together, the first object is purely aligned to human but the second object comes from PTX samples so aligned to both human and mouse. In order to just get the human ...
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Does the number of RNA reads per cell obtained from the 10X scRNA experiment depend on amount of mRNA in given cell?

As we know, the amount of RNA reads per cell obtained from 10X scRNA experiment vary between cells. I wonder if this is effect of technical issues or does the number of RNA reads per cell obtained ...
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What genes are driving a certain pathway

I've currently run pathway analysis on a set of genes. I'm now trying to figure out how to see which genes are driving each pathway. ...
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Subsetting with harmony

Currently merged two Seurat objects together and then ran Harmony for batch correction. Now I want to subset out a cell type of interest do I re run harmony or just do the standard PCA? ...
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Seurat Integration

I'm following the instructions for integration: https://satijalab.org/seurat/articles/integration_introduction.html And it's taking a while to run: ...
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Is it possible to do cell composition for scATAC-seq data without scRNA-seq data?

Background My understanding was that if I did scATAC-seq and I have some clusters of cell groups, the only way I can label it is by correlating those groups with scRNA-seq data. My lab ordered some ...
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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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Is use_dimred deprecated or is this a runTSNE() bug?

NOTE: I posted the same question on Biostars but so far no replies there so I figured I would ask here, too. So, I was running a pipeline which I developed some time ago and a very weird behaviour was ...
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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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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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2 votes
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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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10X low rate of correct barcodes was observed for the candidate chemistry choices for the input

I am testing a 10x fastq dataset , but cellranger count complains "An extremely low rate of correct barcodes was observed for all the candidate chemistry choices for the input", I have tried ...
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Reading in external single cell data

I'm trying to read in an external single cell dataset from https://www.nature.com/articles/s41467-020-16164-1, but I am having trouble reading in the count matrix. counts found here: https://www.ncbi....
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Error in colSums(cts[[i]]) : 'x' must be an array of at least two dimensions

I'm getting an error when creating the counts from a single cell experiment.I'm identifying shared barcodes as well as lowUMI barcodes with the follow code: ...
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2 votes
3 answers
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In Seurat, how can I export cluster IDs to csv files

I am doing scRNAseq analysis with Seurat. I clustered the cells using the FindClusters() function. What I want to do is to export information about which cells belong to which clusters to a CSV file. ...
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159 views

How to find differential expressed genes within pseudotime trajectory with Seurat cluster?

Did anyone know how to find the differential expressed genes within pseudotime trajectory with Seurat cluster? In monocle tutorial, we can use BEAM to find the differential expressed genes between the ...
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2 answers
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ViolinPlot x-axis

I'm trying to set the x-axis in my Violin Plot to go by each patient rather than it being scattered. ...
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How to calculate species fractions in Seurat

I'm working with a PTX dataset and I'm trying to calculate species fractions based on read count instead of cell count. Would just be a matter of taking the raw counts matrix of cells x gene per ...
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-1 votes
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What is meant by transcriptional changes executed by the cell over a time period?

I read the following line in the research paper - The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells: During differentiation, for example, each ...
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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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380 views

Subsetting a Seurat object based on colnames

I'm trying to subset my seurat object based on colnames. I have gone ahead and labeled each cluster and now I want to subset all the colnames that are in Cancer_human for human_colnames and all the ...
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functions in seurat to calculate the gene count per cluster

I'm running FindAllMarkers ...
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1 vote
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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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1 answer
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converting mouse genes to human genes

I'm trying to convert mouse genes from PTX data to human genes in order to do a comparison with patient data to see what genes are being conserved. I'm using this file for the orthologs. http://www....
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2 answers
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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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1 vote
1 answer
120 views

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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2 answers
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Unifying these files together [closed]

I have two .csv files One for raw read counts of each gene and the other 3114 single cells for four patients which should be the row names of the first file As single cells per patients seems ...
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1 vote
1 answer
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Seurat - subsetting by genes expressed

Originally- I was looking if in at least one of these genes were expressed: ...
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1 answer
317 views

Cellranger gives error

I am trying to run cellranger but I get fastq permission denied error ...
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1 vote
1 answer
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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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1 answer
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Segment cell organelles with pixellib

I've got some images of cell organelles and I really want to avoid labeling them by hand. The images all look something like the image below. Is there already an existing model specifially for cells? ...
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1 answer
244 views

How do I change the identity of a sample from spatial 'Visium' data preprocessing in Seurat v3?

I'm wanting to create a merged object in Seurat using 2 10x Visium 'slices'. However, when I create an object, Seurat assigns an identity "SeuratProject" to the objects (by default I'm ...
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4 answers
96 views

Where can I find Single Cell Data with Location "Coordinates"?

Does single cell data typically have the following meta-data: the "coordinates" (e.g. on a tissue, adjacent tissues) saying where each cell in the sample was located relative to other cells? ...
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1 answer
327 views

Calculating average mito.percentage for each cluster (seurat)

I have a tricky data set with cells that will have a higher percent of mitochondria genes than "typical" data sets. I would like to look at the mito percentage in each cluster without any ...
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1 vote
2 answers
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Single cell RNAseq cell cluster (true cluster or sub cluster)

I am trying to run seurat on ~5000 sinngle cells. I am expecting minimum 15 cell types to be present in the data. I tried to runn it with multiple conditions;I can see there is 27 clusters; I believe ...
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which marker identifications method is recommended when single-nuclei RNA seq datasets are integrated across regions

I'm analyzing Sn-RNA seq datasets which include different brain regions and spinal cord in patients and normal controls. I would have different comparison within and between individuals. I've got a ...
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1 vote
1 answer
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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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1 answer
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Help with setting DimPlot UMAP output into a 2x3 grid in Seurat

On my merged seurat object of 6 samples, when I use the split.by function in tandem with the Dimplot/UMAP plot, all six samples are displayed in series along a commonly labeled 'UMAP_1' x-axis in an ...
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2 votes
2 answers
77 views

How can I take cell number into account to find total RNA expression?

I'm hoping to quantitatively show differences in total RNA expression for a gene in a cluster of interest between different experimental groups. My exported average RNA values for each experimental ...
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0 votes
1 answer
585 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 ...
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2 votes
0 answers
102 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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1 answer
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GSEA enrichr with 10x genomics differential_expression ranks

I am attempting to use GSEA enrichr with 10x genomics differential_expression rankings. Reading what people seem to be doing with GSEA, there seems to be a pre-/post-singlecell gene expression ...
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2 votes
3 answers
405 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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Seurat analysis only with matrix with no barcode and features

I have downloaded a dataset from GEO and only the matrix (mx) files are available. How can I perform the analysis without the barcode and the gene features?
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2 votes
3 answers
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umap and Louvain clustering on normalized data

I know that scaled data must be used for PCA for example as it is based on variance maximization. However I'm wondering if it's the same case for UMAP ? If the data are single-cell RNA seq, after ...
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1 vote
1 answer
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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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