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Questions tagged [scrnaseq]

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

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35 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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0answers
22 views

Human Cell Atlas - cell annotations

Human Cell Atlas Preview Datasets have been available for a while now (there was some discussion about that earlier). However, although the raw data is available, cell labels are not (for example, ...
2
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1answer
38 views

How to load and split my two individual datasets when integrating datasets in Seurat?

I want to do comparative analyses of my two individual single cell RNA-Seq datasets in Seurat. And I load and list my data as follows: ...
3
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1answer
25 views

public multi-modal single-cell data

There is a scRNAseq Bioconductor package with a few different example scRNA-seq datasets. Are there any R packages that offer multiple modalities of single-cell data? For example, hashtags or ADTs or ...
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0answers
39 views

Want to make a tsne plot show subset

I am new to R. I make a tsne plot for my data "yfp". And there are two subsets in my data (yfppt and yfpng). I want to show their distribution or location in yfp tsne plot but failed. And I even did ...
2
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2answers
57 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
1
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3answers
78 views

Understanding Single Cell RNAseq Plots

I have just started some single cell RNAseq analysis with 500 cells. I have used Seurat for analysis. Following the guideline I have generated few plots. Now I am trying to understand how to read ...
0
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2answers
107 views

how to generate an scRNA counts file

I have barcodes.tsv, genes.tsv and matrix.mtx file from my RNA seq data and I want to generate a scRNA counts files using R. When I tried using the readMM function, ...
2
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0answers
45 views

High percentage of poly A sequences in 10X chromium R2 read

I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality. According to the Cell Ranger summary 56% of the reads can be mapped to ...
1
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0answers
87 views

PCA on large sparse matrix of single cell RNA-seq

I received a large sc-RNA-seq data matrix, as of the nature of sc-RNA-seq compared to bulk-RNA-seq the data matrix is very sparse. Moreover, due to the fact that this is single cell data the cell ...
2
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1answer
40 views

Single Cell RNA seq Analysis for low input Cells?

I am having a single cell RNA seq data from ~500 Cells. I do understand this number is very low for present day analysis tools like Seurat. So, I want to know what if one have a such a small number of ...
1
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1answer
79 views

Hierarchical clustering for outlier detection - single cell RNASeq & WGCNA

I wish to conduct WGCNA on a single cell RNASeq dataset and, when choosing the optimal beta parameter after running pickSoftThreshold am presented with a rather ...
1
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0answers
75 views

Normalization of single cell RNASeq data with ERCC spike-ins

I wish to normalize a scRNASeq dataset with respect to ERCC spike-ins, where "for some of the samples, ERCC spike-in RNA was added to the lysis buffer" and was wondering how to do so? I have seen ...
1
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2answers
70 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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0answers
16 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
128 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, ...
4
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1answer
351 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
53 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 ...
1
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1answer
149 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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1answer
369 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
43 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 ...
7
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0answers
79 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
107 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
185 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
50 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
73 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
381 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
63 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
153 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
72 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
152 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
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1answer
449 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
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1answer
306 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
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2answers
598 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
226 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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3answers
395 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
61 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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1answer
302 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: ...
5
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0answers
46 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
132 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
732 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
119 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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1answer
90 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
310 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
33 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
2k 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
199 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
80 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
160 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
81 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 ...