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Questions tagged [scrnaseq]

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

2
votes
1answer
29 views

Using Seurat to compare mutant vs.wt

I am interested in using Seurat to compare WT VS. Mutant. I don't know how to use any code or package to test whether when mutant mice, that have deleted gene, got the cluster to combine?
2
votes
2answers
34 views

Can I get the graph generated by cellranger

I’ve run the cellranger analysis pipeline on single cell RNASeq datasets. I can import the matrix and graph-based clusters into R. Doing this I can optimise the dimension reduction and plot cells with ...
3
votes
0answers
21 views

Problem: “Pair-end” reads scRNA seq data (Drop-seq)

In case of Drop-seq, we have paired end data. Read 1: Cell code + UMI (unique molecule identifier) Read 2: The transcript information But I have a problem/doubt with the sample I am working on. ...
1
vote
2answers
81 views

Changing a wide range of colours to a limited gradient

I returned a FeaturePlot from Seurat to ggplot. My plot has a weird range of colours as below I produced this plot by this code ...
3
votes
0answers
26 views

Simulating 3' end tag-based scRNA-seq reads

Are there any tools that simulate 3' end tag-based single-cell RNA-seq reads? For example, Drop-seq, 10X Chromium, CEL-seq, etc.. I see there are tools that simulate scRNA-seq gene count data (e.g. ...
1
vote
1answer
62 views

Script to allow gene set enrichment analysis of 10x genomics data in R

I have 10x single cell RNA seq data. Which R package is best suited for analysis of the 10x data matrix. What is the script to prepare the data for downstream GSEA analysis. I have already processed ...
1
vote
1answer
54 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 ...
0
votes
1answer
29 views

Low percentage of reads with consistent barcodes in Split-Seq run

I am following this answer to select reads with allowed barcodes from a Split-Seq run. In particular, I have a whitelist for each round of barcodes (3 rounds) and I filter out reads which have more ...
1
vote
1answer
42 views

The visualisation of a list of genes on URD object

Seurat R package has some functions like FeaturePlot, DimPlot and DoHeatmap by which we can plot the expression of a list of genes on cell clusters. I am working with URD that likely does not have ...
2
votes
3answers
40 views

How to detect mismatch before mapping in RNA-Seq data

In the methods of this paper, the authors say: Reads were then filtered based on quality score in the UMI region. Any read with >1 low-quality base (phred <=10) were discarded. Reads with more ...
5
votes
1answer
47 views

What is and how to detect a dephased read

In the methods of this paper, the authors say: To simplify analysis, we first removed any dephased reads in our library (last 6 bases of read did not match the expected sequence). I have read this ...
1
vote
1answer
20 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 ...
2
votes
2answers
34 views

RNA velocity: competing explanations for variable ratio of spliced to unspliced transcript

In "RNA velocity of single cells", La Manno et al. look at ratios between spliced and unspliced mRNA as a way of estimating the velocity of a cell through transcriptome space. In checking for a ...
1
vote
2answers
59 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 ...
0
votes
0answers
158 views

Making a plot cleaner

By this code I produced this tSNE ...
1
vote
1answer
26 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 ...
0
votes
0answers
27 views

ValidateCluster function in Seurat

I have used Seurat for clustering my cells in different time points at the same resolution (0.7) that gave me 2 or 3 clusters in each time point. I fount that by ValidateCluster function in Seurat ...
-1
votes
1answer
37 views

Convert TPM-normalized matrices back to UMI in python

I want to process a plenty of scRNAseq datasets in python, and I want to run TMM ...
1
vote
1answer
45 views

VlnPlot problem

I was confused about the VlnPlot that it shows like the screenshot. It only shows the dot but no distribution of the data, is that normal?
0
votes
1answer
61 views

Fitting a principal curve into a diffusion map

I returned a Seurat object with a diffusion map by seurat_object <- RunDiffusion(seurat_object,genes.use = seurat_object@var.genes) as below Now, how I ...
-1
votes
1answer
85 views

A strange error while clustering of single cell RNA-seq using URD package

I am using URD R package on my single cell RNA-seq data hoping to be able to trace gene expression changes during the development. After dimension reduction step by PCA and diffusion map whatever I am ...
0
votes
0answers
19 views

Identifying distinct principal components related to time and cell type

I have 2000 cells and 9 time points. I have clustered my cells in each time point to 2 or 3 clusters by Seurat R separately. within each time point likely we must have clusters of cells just because ...
5
votes
2answers
134 views

How I can test my hypothesises computationally

I have single cell RNA-seq data on about 2000 cells in 9 time point. I have clustered my cells in each time point by Seurat. I am seeing in some time points I have 3 clusters while in another time ...
2
votes
1answer
53 views

Why DoHeatmap Does not show all genes in genes.use?

I am heatmaping a list of genes by DoHeatmap function in Seurat R package. I am sure I have 212 genes but heat map shows only a few of my genes ...
0
votes
0answers
27 views

how to legend the feature plot and do.hover in 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....
1
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0answers
64 views

Create an S4 object

In this R documentation: xu-lab/SINCERA I want to replace my own expression data but I don't know how do that ...
2
votes
1answer
33 views

how to filter out cell after doing QC

I was wondering that the threshold that we set after doing QC in Seurat. there is an example ...
1
vote
2answers
54 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 ...
1
vote
1answer
52 views

RCurl issue when installing SCnorm

I am working on the Rivanna cluster that is of SLURM-type. Because of that I do not have write permissions and need to install ...
1
vote
3answers
110 views

Interpretation of the violin plots from sc-RNA-seq

I'm confused about the meaning of the black dots and the red shape in the violin plots from the seurat tutorial:
-2
votes
1answer
177 views

Changing the colour of each cell in tSNE plot

I have plotted a tSNE plot of my 1643 cells from 9 time points by seurat like below as 9 clusters. But, you know I should not expected each cluster of cells contains only cells from one distinct time ...
0
votes
0answers
85 views

How I can reproduce this heat map

I have single cell RNA-seq on 8 time points. I have clustered each time point to 2-3 clusters by seurat. I also have a list of differentially expressed genes between all my 1562 cells in 8 time points....
2
votes
0answers
24 views

Hemoglobin subunits genes in scRNA-seq

In one scRNA-seq sample I encountered the genes: Hbb-bs, Hba-a1 and Hba-a2. These genes appear on top of the list of the highest expressed genes but having up to the 75% percentile of cells having ...
-4
votes
1answer
69 views

Installing a R package from git

I am trying to install STEMNET R package but a permanent error stops me, what can I do please? ...
0
votes
0answers
55 views

Making two heat maps more similar

I have two matrices of gene expression data from RNA-seq. I have made a heat map with the same order of genes by these code like below ...
0
votes
0answers
90 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-...
1
vote
0answers
94 views

No counts for added gene in cellranger (scRNA-seq)

I have a set of scRNA-seq samples enriched with FACS for cells expressing a specific gene reporter (TdTomato). In particular the gene I want to report has positive counts in the resulting matrix for ...
0
votes
0answers
61 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 ...
1
vote
2answers
104 views

Which data is being used for violin plot?

Sorry I just got totally confused conceptually. If this is my raw count data Likely, Seurat divides each value by sum of the column afterward times by 10000. which gives so ...
3
votes
1answer
109 views

Is it possible to identify cells that are expressing two or more genes in Seurat?

I'm interested in looking into cells that are positive for two (or in some cases more) genes. I know I have some double positive just by looking at the FeaturePlot of those genes, but now I'm trying ...
1
vote
1answer
44 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 ...
6
votes
1answer
49 views

Network comparison of single cells (from sequencing data)

What methods can we use to compare networks (PPI, gene regulatory etc) created from single-cell gene expression (or proteomics) data? There are methods that construct networks by comparing two ...
5
votes
1answer
67 views

Specific cell type identification in Single Cell Sequencing

In order to define which cell is of which type we need to identify a set of rules, for instance neurons should express one of the following: Thy1, ...
1
vote
1answer
359 views

RunUMAP in Seurat not working: module 'umap' has no attribute 'UMAP'

I am trying to run UMAP in the following way: ...
3
votes
1answer
173 views

Raw vs Filtered in the output of cellranger count

After running cellranger count I got two relevant for further analysis folders: filtered_gene_bc_matrices and ...
6
votes
1answer
171 views

Pathway level analysis of single-cell gene expression

I'm looking for single-cell specific methods to construct (using gene expression data) new features that express pathway "level" or "activity", and then use these for clustering cells. One example ...
1
vote
1answer
83 views

What process and input data is required for a cellranger reference transcriptome?

I'm analysing single-cell RNA-Seq data using the 10X Genomics cellranger platform. While they provide reference data for Mouse and Humans, other species require a ...
0
votes
1answer
56 views

Demultiplexing and preprocessing for custom single-nucleus Drop-seq data

I am trying to reproduce the preprocessing of paired-end sequencing reads in Lake et al. (ref. 1). First, paired-end reads were filtered out if read 1 had more than four non-T bases in the last ten ...
9
votes
1answer
138 views

How to decide number of neighbors and resolution for Louvain clustering

I am using Louvain clustering (1,2) to cluster cells in scRNAseq data, as implemented by scanpy. One of the parameter required for this kind of clustering is the number of neighbors used to construct ...
4
votes
3answers
211 views

Regressing out unwanted sources of variation in single cell RNA-seq data

I have a dataset of single cell count data and I want to regress out the variation caused by the number of UMI's and the percentage of mitochondrial genes. I know that count data is discrete data, ...