Questions tagged [scrnaseq]

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

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3
votes
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?
1
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1answer
51 views

Is it possible to use spike-ins with droplet technologies?

By reading Bacher & Kendziorski (2016), I found the following statement: recent droplet technologies are not yet able to accommodate spike-ins What's the reason why current droplet ...
3
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2answers
841 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 ...
1
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2answers
795 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
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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
351 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 ...
6
votes
1answer
988 views

Order of batch effects removal, data imputation and library size normalization in scRNA-seq data

I am preprocessing scRNA-seq data. What is the best practice in use to run both ComBat for batch effects removal, data imputation (to mitigate dropout) and library size normalization? I thought that ...
0
votes
1answer
111 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 ...
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 ...
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 ...
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
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1answer
59 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 ...
-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 ...
-1
votes
1answer
1k 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 ...
3
votes
1answer
889 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
2answers
383 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
votes
2answers
3k 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:
-3
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1answer
592 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? ...
8
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2answers
7k views

Are mitochondrial genes to exclude in scRNA-seq such as ribosomal genes?

In this answer, it is stated that ribosomal genes should be excluded prior to normalization in scRNA-seq as contaminants. Do mitochondrial genes have to be excluded as well? I plotted the top 50 ...
1
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0answers
99 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-...
6
votes
1answer
212 views

Scaling by linear regression against the number of reads

I am trying to build the preprocessing pipeline presented in The Tabula Muris Consortium et al. (pp). It is a pipeline to preprocess single-cell sequencing data. There is one step that is not clear: ...
2
votes
2answers
2k 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 ...
1
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0answers
80 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 ...
3
votes
2answers
3k views

What are RIKEN genes?

I am using data from the Tabula Muris Consortium. These are gene counts from scRNA-seq of mouse cells. There are some specific genes with name ending with suffix 'Rik' (e.g. 0610005C13Rik or ...
4
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1answer
2k 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 ...
3
votes
2answers
2k views

Handling sample identity in aggregated 10x libraries?

cellranger aggr can combine multiple libraries (samples), and appends each barcode with an integer (e.g. AGACCATTGAGACTTA-1). The sample identity is not recorded in ...
12
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2answers
3k views

determining doublets in single-cell RNA-seq

Doublets are a known problem with scRNA-seq experiments, where 2 or more cells are sometimes captured instead. To determine their presence, there are studies that mix multiple species (such as human ...
1
vote
1answer
154 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 ...
3
votes
1answer
3k views

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

I am trying to run UMAP in the following way: ...
7
votes
1answer
104 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 ...
4
votes
1answer
2k 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 ...
0
votes
1answer
205 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 ...
5
votes
1answer
387 views

Which tools for differential expression analysis in scRNA-Seq?

I am starting to run analysis for differential expression in scRNA-Seq. Which tools are available for this kind of analysis? Can tools for bulk RNA-Seq like DESeq be used for scRNA-Seq?
5
votes
3answers
1k 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, ...
6
votes
1answer
638 views

Drawbacks of upper quartile normalization for scRNA-seq data

I would like to use Upper Quartile normalization for scRNA-seq data defined as: The upperquartile (UQ) was proposed by (Bullard et al. 2010). Here each column is divided by the 75% quantile of the ...
2
votes
2answers
2k views

ComBat for batch effects removal on scRNA-seq data

Is it possible to use sva's ComBat for batch effects removal on scRNA-seq data? Is there any difference between RNA-seq and scRNA-seq data which doesn't allow to use ComBat on single-cell data? (I am ...
6
votes
1answer
2k views

What's a template switching site?

Reading Islam et al. (2011): From each transcript, a single read is obtained, corresponding to a template-switching site located preferentially at the 59 end of the mRNA. By reading this page I ...
2
votes
1answer
2k views

What is a poly(T) primer anchor sequence?

I am reading Tang et al. (2009). In the method description it is stated that: A single cell is manually picked under a microscope and lysed. Then mRNAs are reverse-transcribed into cDNAs using a ...
4
votes
1answer
133 views

Why is ribosomal RNA difficult to remove even with Poly(A) selection?

In this answer (actually in a comment), it is stated that: As you've noticed from your own analysis, the ribosomal genes have quite variable expression across cells. They're expressed everywhere, ...
3
votes
2answers
116 views

Detect transcript isoform abundance for a specific gene in scRNA-seq

I want to detect the count of isoform transcripts for a specific gene in scRNA-seq data. Data is coming from cells of Mus Musculus. For transcript isoforms I mean the different alternatives provided ...
0
votes
1answer
44 views

Looper: can't specify the individual path for one pipeline run

I have multiple samples that I need to run one pipeline on. I am using Looper: https://github.com/pepkit/looper I have a real trouble specifying a separate location for each pipeline output files. ...
1
vote
1answer
37 views

Looper: missing argument error

While trying to run looper I am getting the following error: Error (missing attribute): 'sandro_rna_seq' requires sample attribute '/scratch/nv4e/sandro_looper/pipeline/jyvo_experiment-metadata....
1
vote
1answer
20 views

Looper: Duplicate sample name error

I am using Looper. While running looper run project_config.yaml I am getting duplicate sample name error. I have two ...
0
votes
1answer
87 views

What is the detection limit of protocol in scRNA-seq

In Shapiro et al., when discussing about loss of molecules as source of error in single-cell sequencing, it is written that: Another source of error is losses, which can be severe. The detection ...
6
votes
3answers
496 views

Improve scRNA-seq dataset for further analysis

I got a dataset from C.Elegans scRNA-seq paper: GSM2599701_Gene.count.matrix.celegans.cell.Rdata in ...
1
vote
1answer
59 views

Error in .checkedCall : subset indices out of range [closed]

After normalization the way it is described here: https://bioconductor.org/packages/3.7/bioc/vignettes/scran/inst/doc/scran.html Using quickCluster() function ...
1
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2answers
4k views

How to filter ribosomal RNA from scRNA-seq data

I want to filter out ribosomal RNA from scRNA-seq data (downloaded from here). Is there a list of known ribosomal RNA? The only solution I found is SortMeRNA, however it works with raw sequencing ...