Questions tagged [single-cell]

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

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19
votes
3answers
2k 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 ...
11
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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 ...
7
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1answer
95 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 ...
7
votes
1answer
817 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 ...
6
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2answers
4k 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
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1answer
209 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
783 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 ...
5
votes
3answers
9k 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
168 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 ...
5
votes
2answers
9k 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 ...
5
votes
1answer
531 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 ...
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, ...
5
votes
1answer
690 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 ...
5
votes
1answer
76 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 ...
5
votes
1answer
471 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 ...
4
votes
2answers
2k 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
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2answers
499 views

Collapse cell barcodes distribution within 1 Hamming distance

I have a barcode distribution from single-cell data, e.g: ...
4
votes
1answer
2k views

Subset on multiple genes in Seurat

I know that I can do subsetting on just one gene in Seurat: ...
4
votes
2answers
881 views

Is there a command line tool to split a SAM/BAM file by CB (cell barcode) tag?

I have a BAM file from a single cell sequencing experiment. Each read has had the cell barcode annotated in the CB tag. Some reads do not have a ...
4
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0answers
63 views

Database of Cell Volumes by Cell Ontology? [closed]

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). ...
3
votes
2answers
1k 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?
3
votes
3answers
4k 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 ...
3
votes
2answers
769 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
48 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 ...
3
votes
1answer
721 views

about dotplot legend meaning

Here is the code I used: ...
3
votes
1answer
52 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,...
3
votes
0answers
51 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 ...
2
votes
3answers
361 views

How to reduce the occupied RAM when you are dealing with a very sparse matrix in a single-cell Experiment in R?

I'm dealing with a very large and sparse dataset and the first issues I met occurred when I tried to use quickCluster that reported me this error: ...
2
votes
1answer
44 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 ...
2
votes
3answers
319 views

umap and Louvain clustering on normalized data

I know that scaled data must be used for PCA for example as it is based on variance maximization. However I'm wondering if it's the same case for UMAP ? If the data are single-cell RNA seq, after ...
2
votes
2answers
110 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 ...
2
votes
3answers
1k 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 ...
2
votes
3answers
93 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
95 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 ...
2
votes
1answer
2k 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 ...
2
votes
2answers
223 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
votes
1answer
217 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 ...
2
votes
2answers
214 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
224 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 ...
2
votes
1answer
77 views

Using cellranger for non-10x data

I'm trying to use cellranger to mkfastq and then count and aggregate single-cell data. This ...
2
votes
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, ...
2
votes
2answers
67 views

How can I take cell number into account to find total RNA expression?

I'm hoping to quantitatively show differences in total RNA expression for a gene in a cluster of interest between different experimental groups. My exported average RNA values for each experimental ...
2
votes
0answers
66 views

scRNA-Seq: Account for sequencing depth and gene length?

Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length. For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
2
votes
0answers
353 views

Understanding BuildClusterTree of Seurat

I am trying to understand how to use BuildClusterTree of Seurat to understand the relationship between clusters. Being from neither a bioinformatics or statistical ...
1
vote
1answer
2k 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 ...
1
vote
2answers
114 views

Single cell RNAseq cell cluster (true cluster or sub cluster)

I am trying to run seurat on ~5000 sinngle cells. I am expecting minimum 15 cell types to be present in the data. I tried to runn it with multiple conditions;I can see there is 27 clusters; I believe ...
1
vote
1answer
93 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
37 views

Challenging benchmarks for supervised learning on sparse scRNA-seq data

One challenging aspect of modeling scRNA-seq data is data sparsity, that is, scRNA-seq measurements typically suffer from large fractions of observed zeros (i.e. dropouts), where a given gene in a ...
1
vote
1answer
34 views

Segment cell organelles with pixellib

I've got some images of cell organelles and I really want to avoid labeling them by hand. The images all look something like the image below. Is there already an existing model specifially for cells? ...
1
vote
1answer
63 views

What is the best way to address the question of doublets and multiplets in a single cell RNA seq data set?

I have attached a histogram plot of the number of genes per cell in a single cell RNA seq data set of lung endothelial cells. I do not find a bimodal or multimodal distribution of the number of genes ...