Questions tagged [scrnaseq]

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

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2answers
185 views

How are UMIs used to dedupulicate in Drop-seq tools?

The module DigitalExpression which is part of the popular Drop-seq tools digitally count gene transcripts. The manual is not very clear on how exactly it resolves ...
1
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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 ...
2
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1answer
1k views

How to highlight specific cells in Seurat 2.4

I used Seurat 2.4 on our scRNA dataset to obtain the following tSNE plot.I was able to successfully extract cell IDs from the different clusters, and generate gene expression profiles. The analysis, ...
5
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1answer
817 views

PCA vs tSNE in single cell RNA-seq

What makes tSNE being the preferred dimensional reduction for visualization in single cell RNA-seq over PCA? I am aware that tSNE works better at showing local structures and fails to capture global ...
-1
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1answer
62 views

How I normalize these two sets of data

I have average log fold change for a cluster of cells versus another cluster of cells by Seurat like below ...
2
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1answer
761 views

Why do I have >10,000 cells in the 10X matrix produced by cellranger?

In my scRNA seq experiment, single-cell libraries were generated using the GemCode Single-Cell Instrument and Single Cell 3′ Library & Gel Bead Kit v2 and Chip Kit (10x Genomics) according to the ...
2
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2answers
2k views

How do I add a colour annotation bar to the heatmap generated by DoHeatmap function of Seurat v2?

I am using Seurat v2 for professional reasons (I am aware of the availablity of Seurat v3). I am clustering and analysing single cell RNA seq data. How do I add a coloured annotation bar to the ...
0
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2answers
195 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 ...
13
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1answer
200 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 ...
1
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1answer
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 ...
3
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1answer
1k 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
131 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 ...
0
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1answer
120 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 ...
2
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3answers
3k 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 ...
1
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2answers
86 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 ...
3
votes
1answer
1k 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
votes
2answers
122 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
votes
1answer
202 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. ...
3
votes
1answer
2k 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
1k 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
3k 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
772 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 ...
2
votes
4answers
2k views

Low custom tdtomato gene content

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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2answers
445 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
542 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: ...
6
votes
2answers
183 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
votes
1answer
648 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 ...
3
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2answers
2k 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
244 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
1answer
208 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
846 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 ...
4
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0answers
51 views

Simulating 3' end tag-based scRNA-seq reads

Are there any tools that will simulate 3' end tag-based single-cell RNA-seq reads? For example, Drop-seq, 10X Chromium, ...
5
votes
3answers
7k 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
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 ...
0
votes
1answer
112 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
271 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
163 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
77 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
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 ...
2
votes
2answers
306 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
806 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 ...
1
vote
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 ...
-1
votes
1answer
219 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 ...
2
votes
2answers
755 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
352 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
108 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 ...
6
votes
2answers
220 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
890 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 ...
1
vote
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....
3
votes
1answer
261 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 ...