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Questions tagged [scrnaseq]

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

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0answers
11 views

scRNA-seq differential transcript usage

Many of the modern gene-quantification tools (Salmon/Kallisto) output transcript-level (as opposed to gene-level) data. All of the scRNA-seq analysis I have seen just uses the gene-level values. I ...
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0answers
33 views

How do I prevent the FeatureHeatmap function from the Seurat package, from sorting my data groups in alphabetical order when plotting data?

How can I prevent a function from sorting my data groups (factors) in alphabetical order without affecting the integrity of the data? I am analysing single cell RNA sequencing data using Seurat 2.3.4. ...
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1answer
66 views

How to set the position of groups in a Seurat object on a FeatureHeatmap plot

I am analysing singe cell sequence data and I have followed this tutorial, https://satijalab.org/seurat/pbmc3k_tutorial.html to perform QC and various differential analyses using the Seurat package on ...
1
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1answer
56 views

Visualising gene expression across cell type and conditions in one plot, in Single Cell Sequencing data

I am new to single cell sequencing data analysis but I have basic programming background in R and python. I want to be able to plot differential expression of two genes, Gene1 and Gene2, across three ...
2
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2answers
56 views

Separate boxplots for multiple violin plot

I am using the following function from seurat package to generate multiple violon plots and I am interested in adding box plots to them but it doesn't work when I have plotted different data at once. ...
5
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1answer
58 views

How to normalise scRNASeq data for differential expression analysis

I wish to perform differential expression analysis for cluster-specific gene expression in single-cell data (with a tool such as MAST or SCDE). I have data for 3 biological replicates. I performed ...
1
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1answer
51 views

scRNA-seq,10x cellranger pipelines,low custom tdtomato gene content! looking for help!

I have a set of scRNA-seq samples expressing TdTomato, which has high content in microscope. I followed the 10x cellranger pipelines to finsh the work, my procedures are as follows: added TdTomato on ...
3
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1answer
42 views

PCA plot shows big difference but not many differentially expressed genes are found

I got a PCA plot of bulk RNA-seq experiment that looks the following way: It was generated by the following code: ...
3
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2answers
155 views

scRNA-seq, 10x cellranger pipelines

I'm starting to do sc-rnaseq using 10x cellranger pipelines, and i add TdTomato sequence to mouse reference genome and add an entry in the gtf. My code is But when I makref, it remindered that: ...
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0answers
35 views

Why are TPMs per 10k or 100k in many scRNA-seq studies?

I noticed that many scRNA-seq papers normalize TPMs to 10k or 100k as opposed to 1M (as the abbreviation defines them). It doesn't really matter since you are just moving the decimal point, so why ...
5
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1answer
64 views

Derive a GTF containing protein coding genes from a GTF file with Exons and CDS

Why I need a compatible file I’m trying to run velocyto with the R package to analyse RNA velocity (cell trajectories) with single cell RNASeq data. I have performed single cell analysis from 10x ...
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2answers
117 views

Using Seurat to compare mutant vs.wt

I am interested in using Seurat to compare wild type vs Mutant. I don't know how to use the package. How can I test whether mutant mice, that have deleted gene, cluster together?
2
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2answers
58 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
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0answers
34 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. ...
2
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2answers
103 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
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0answers
27 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. ...
4
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3answers
222 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
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1answer
80 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
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1answer
41 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 ...
2
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1answer
55 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
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3answers
46 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
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1answer
57 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 ...
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1answer
24 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
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2answers
70 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 ...
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2answers
68 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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0answers
174 views

Making a plot cleaner

By this code I produced this tSNE ...
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1answer
27 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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0answers
37 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 ...
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1answer
42 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 ...
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1answer
60 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
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1answer
81 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
90 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
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0answers
20 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 ...
6
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2answers
145 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 ...
3
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1answer
93 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 ...
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0answers
30 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....
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0answers
70 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
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1answer
49 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 ...
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2answers
69 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 ...
2
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1answer
58 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 ...
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3answers
180 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:
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1answer
269 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 ...
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0answers
95 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
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0answers
29 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 ...
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1answer
85 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? ...
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0answers
58 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 ...
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0answers
93 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-...
4
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1answer
166 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 ...
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0answers
67 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 ...
2
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2answers
178 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 ...