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Questions tagged [single-cell]

Questions about techniques in which the genetic material of a single cell is analysed individually

-1
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0answers
38 views

why the gene expression pattern wrong in the feature plot

I have no idea that the gene was wrong in the featureplot, which according to publishing data that it should not be expressed so many around all clusters. According to publishing data it should ...
0
votes
1answer
26 views

how to write the script to know each sample's cluster number?

I have followed the Seurat Tutorial:Integrating stimulated vs. control PBMC datasets to learn cell-type specific responses by using my data. And I am confused about how to write the script to ...
2
votes
2answers
87 views

Why are the clusters in the left side of a tSNE plot and not in the centre?

I have no idea why I used the subsetdata in Seurat after using CCA the TSNEPlot shows the cluster all in the left side and merge, did I miss something? Here are the code and figure I got. ...
2
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1answer
45 views

Why does my SplitDotPlotGG plot show differential expression of a gene but Seurat::FindMarkers() function does not return it?

I have analysed and clustered my single cell rna seq data with methods in the Seurat package. After clustering, I obtained two main clusters: 0 and 1. I used the Seurat SplitDotPlotGG function to ...
0
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1answer
22 views

GEOquery errors due to list input

I want to extract the expression matrix from the GSE object in GEOquery. ...
0
votes
1answer
67 views

how to obtain only one cluster's data (.csv file) in Seurat

I was using Seurat to analysis single-cell RNA Seq. And I was interested in only one cluster by using the Seurat. Does anyone know how to achieve the cluster's data(.csv file) by using Seurat or any ...
2
votes
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 ...
0
votes
1answer
61 views

how to write italic script in Rstudio [closed]

I have got the problem on how to write the italic script in RStudio. I used the script in the screenshot and got the error "Error in grobs[[i]]: subscript out of bounds."
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
vote
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 ...
5
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0answers
30 views

Database of Cell Volumes by Cell Ontology?

Are there any databases that have aggregated information on cell volumes? Most useful would be a database keyed by cell ontology (e.g., CL:0000236 for B cell). ...
5
votes
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
votes
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 ...
2
votes
1answer
37 views

What does that mean if there is no bifurcations in my time series single cell data?

Sorry, I have analysed my time series single cell data with Scuba algorithm but that says that there is no bifurcations in my data and outputted a single chain of 4 states with no branching like ...
3
votes
2answers
160 views

Collapse cell barcodes distribution within 1 Hamming distance

I have a barcode distribution from single-cell data, e.g: ...
3
votes
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
votes
1answer
72 views

about dotplot legend meaning

Here is the code I used: ...
3
votes
0answers
35 views

What is the correct way of dealing with the analysis of data from flow cytometry?

I would like to detect a change in expression of a molecule present on a cell type by flow cytometry. Assuming I am able to detect, using an antibody, a signal that represents the amount of the ...
3
votes
1answer
49 views

Why is cell count stated relative to animal size?

Why do some single-cell RNA-seq papers give the number of cells sampled as a fraction of the size of the organ, animal, or embryo, as opposed to just saying how many cells they sampled? For instance,...
1
vote
1answer
48 views

How is PCR duplication rate computed in scATAC-seq?

Reading Cusanovich et al. (2015) I encountered the sentence: We mixed pairs of cell lines (HEK293T or HL-60 versus GM12878), performed combinatorial cellular indexing, and sequenced the resulting ...
1
vote
1answer
584 views

Violinplot of gene expression

I have plotted the log normalized expression of two genes by violonplot for 4 clusters. I have links to my pictures and Seurat object too. My problem is this; in violin plot I can not see the mean or ...
3
votes
2answers
1k views

Mapping a list of cells in seurat featureplot

I have 209 cells, I clustered them by Seurat to 4 clusters. By Featureplot I am able to track a gene in clusters: Higher color shows higher expression. Now, for some genes I want to highlight some ...
7
votes
1answer
63 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
2answers
1k views

Resolution parameter in Seurat's FindClusters function for larger cell numbers

In Seurats' documentation for FindClusters() function it is written that for around 3000 ...
5
votes
1answer
91 views

Which are the use cases for the methods for DE in Seurat

In Seurat we can specify multiple methods for finding DE genes. I am wondering when should we use which one, the use cases. As ...
3
votes
2answers
522 views

What is cellranger doing in comparison to other methods?

I've recently started working with the 10X-Genomics platform with Illumina (MiSeq and HiSeq) for single-cell RNA-Seq. I've been recommended the "cellranger" (version 2.1.0) which I understand handles ...
4
votes
2answers
1k views

Manually define clusters in Seurat and determine marker genes

I want to define two clusters of cells in my dataset and find marker genes that are specific to one and the other. Is there a way to do this in Seurat? Say, if I ...
3
votes
1answer
585 views

Subset on multiple genes in Seurat

I know that I can do subsetting on just one gene in Seurat: ...
5
votes
3answers
383 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, ...
11
votes
2answers
1k 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 ...
4
votes
2answers
922 views

What are doublets in single cell RNA-seq data?

I am reading The Tabula Muris Consortium et al. (pp). In some organs, cells with more than 2 million reads were also excluded as a conservative measure to avoid doublets. How exactly is a “doublet”...
6
votes
1answer
149 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: ...
5
votes
1answer
253 views

Single-cell RNA sequencing (scRNA-seq): filtering cells by transcript counts, how to choose cutoffs?

I am running a notebook with example for the MAGIC algorithm. In the data preprocessing step, a filtering operation is required to filter out cells with a small count of transcripts. My question is ...
5
votes
1answer
67 views

How to compute the chance of failing to detect a gene given the detection limit of a protocol

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 ...
2
votes
1answer
99 views

Mini-bulk RNA-seq and scRNA-seq analysis differences?

We have RNA-seq data from libraries prepared using the Smartseq2 single-cell protocol on 500 cells (mini-bulk) / library. The complexity is much better than with single-cells (~14k genes for 1.5M ...
6
votes
1answer
371 views

How to trim adapter sequences from GSE65360 in order to map the reads?

I am trying to map single-cell chromatin accessibility data from doi:10.1038/nature14590, obtained using scATAC-seq, to the reference genome. Example paired-end reads are here. The reads contain ...
16
votes
3answers
354 views

What is the actual cause of excessive zeroes in single cell RNA-seq data? Is it PCR?

First, sorry if I am missing something basic - I am a programmer recently turned bioinformatician so I still don't know a lot of stuff. This is a cross post with a Biostars question hope that's not ...
2
votes
2answers
75 views

Getting hierarchy of cell populations with Drop-seq data

I plan on collecting some drop-seq data from brain tissue (from mice and humans). Using only the 75-85% of genes that have 1:1 orthology between mice and humans, I'd like to cluster the cells by ...
2
votes
1answer
737 views

Cellranger aggr and Seurat merge difference?

I have multiple libraries of 10x Chromium single-cell RNA-seq data, which I'd like to combine. One option is using cellranger aggr which by default does a depth ...