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Questions tagged [scrnaseq]

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

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2answers
33 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 ...
6
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0answers
45 views

scRNA-seq multi-dataset integration for small datasets

There have been a few methods proposed for integration (or batch correction) of scRNA-seq datasets, such as Seurat CCA, MNN Correct, Scanorama, and Harmony. The concern is generally about the maximum ...
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1answer
55 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 ...
2
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1answer
67 views

Understanding PCHeatmap outputs

I am currently trying to understand the purpose of these PCHeatmaps - part of the seurat package in R: All the online documentation I have searched for has only ...
2
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1answer
35 views

scRNASeq expression matrix with decimal values

I am trying to replicate some results of a scRNASeq experiment and, when I looked at the data provided by the author, I noticed that some of the counts in the expression matrix are represented as ...
-1
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1answer
66 views

How do I perform a pathway related grouping of genes?

I have a list of gene names from the analysis of my mouse single seq data. I want to perform a simple grouping of the genes based on the pathways to which they belong or pathways through which they ...
0
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2answers
87 views

How can I obtain the percentage gene expression per identity class in Seurat as further processible numbers (e.g. matrix)?

I am analysing my single cell RNA seq data with the Seurat package. I want to know if there is a possibilty to obtain the percentage expression of a list of genes per identity class, as actual numbers ...
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2answers
55 views

Using preprocessing/alignment functions on the server

I am new to bash and the processes behind cluster computing in general and need some help with understanding some basics. After looking all over the internet and this forum (+ askUbuntu) I found ...
1
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1answer
73 views

Percentage distribution of cells in all clusters based on their treatment condition?

I have 2151 cells, I clustered them by Seurat to 5 clusters. With the code below, I am able to have the number of cells per cluster and per condition: ...
2
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2answers
57 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 ...
3
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1answer
89 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. ...
2
votes
1answer
130 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 ...
2
votes
1answer
112 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 ...
3
votes
2answers
136 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
votes
1answer
99 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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2answers
122 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
50 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
votes
1answer
200 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: ...
3
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0answers
38 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
87 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 ...
2
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2answers
287 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
votes
2answers
69 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
50 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. ...
3
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2answers
152 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
31 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. ...
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3answers
613 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 ...
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1answer
128 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
56 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
votes
1answer
93 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
60 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
58 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
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1answer
38 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
118 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
80 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
187 views

Making a plot cleaner

By this code I produced this tSNE ...
1
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1answer
30 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
46 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
53 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
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1answer
94 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
113 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
96 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
21 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
votes
2answers
157 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
votes
1answer
173 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
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0answers
34 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
74 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
69 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
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2answers
118 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
64 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
351 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: