11 votes
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PCA vs tSNE in single cell RNA-seq

tSNE often offers better visual representation (separation) on such complicated data than PCA. As Micheal pointed out, computing a tSNE embedding over 20.000 gene dimensions is computationally ...
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  • 504
10 votes

determining doublets in single-cell RNA-seq

Expected rates of doublets / duplets / multiplets Fluidigm C1 doublet rate: around 1-5% depending on chip type used. More information: Fluidigm white paper: Redesign of C1 Medium-Cell 96 IFCs ...
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  • 2,574
8 votes
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What is the actual cause of excessive zeroes in single cell RNA-seq data? Is it PCR?

It may be necessary to distinguish between methods that use unique molecular identifiers (UMIs), such as 10X's Chromium, Drop-seq, etc, and non-UMI methods, such as SMRT-seq. At least for UMI-based ...
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  • 561
7 votes

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

The Biostars thread turned out helpful. The most interesting possible cause, not mentioned in the Ian Subery's answer, is that due to bursty nature of transcription, the true distribution of ...
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7 votes

What are doublets in single cell RNA-seq data?

"Doublet" is commonly used to describe a droplet in droplet-based sequencing that has captured atleast 2 cells. 10x states their doublet rate to be 0.8% per 1000 cells: There is a tradeoff between ...
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  • 71
7 votes
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What are doublets in single cell RNA-seq data?

Is doublet a set of cells sequenced as a single cell? Yes. Depending on the method of single cell sequencing it may be more or less likely for groups of cells to be captured and barcoded with the ...
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7 votes
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What does that mean if there is no bifurcations in my time series single cell data?

The most obvious interpretation is that the four cell lines are related in a linear pathway rather than a branching one.
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  • 3,211
7 votes
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Using Seurat to compare mutant vs.wt

Single-cell analysis to compare samples is a long a difficult process. There is very good documentation for 10x Genomics cellranger, the DropSeq Pipeline and the Seurat R package. These tools all have ...
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  • 873
6 votes
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Resolution parameter in Seurat's FindClusters function for larger cell numbers

Assuming you have an informative selection of variable genes from which you have constructed a number of useful PCs, I'd run a number of iterations with ...
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  • 2,574
6 votes
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Subset on multiple genes in Seurat

I was able to achieve this in the following way: ...
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5 votes

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

I know of no references for this, but in general, I would say that your reasoning is sound. I would just add that in contrast to what I suspect you have simulated, not all transcripts are equally ...
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  • 3,211
5 votes
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Which are the use cases for the methods for DE in Seurat

You can take a look at the recently published article: Bias, robustness and scalability in single-cell differential expression analysis. We evaluated 36 approaches using experimental and synthetic ...
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  • 1,012
5 votes
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Mapping a list of cells in seurat featureplot

To color the TSNEPlot, you can generate a new column in metadata with the expression levels (High, low, etc). Then use pt.shape to set a shape for each identity. To show binary expression based on ...
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5 votes

public multi-modal single-cell data

These tutorials on Seurat multimodal data and the wrapper Seurat data are easy ways to start. The wrapper has some cite-seq data preinstalled making it easy to work with benchmarked data sets If you ...
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5 votes
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How to reduce the occupied RAM when you are dealing with a very sparse matrix in a single-cell Experiment in R?

Ah, looks like I can't even procrastinate on StackExchange anymore without seeing work-related stuff. Oh well. Anyway, the other answers and comments are way off. scran has supported sparse matrices ...
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4 votes
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Violinplot of gene expression

You want to (1) see the mean for each gene, and also to (2) calculate a ratio of expression levels of two genes, then compare it between clusters. (1) First, notice that ...
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  • 2,574
4 votes

Network comparison of single cells (from sequencing data)

You can't build a network of a single cell only with the expression of a single cell. You either need previous known interactions or pathways or you need to use several cells/samples. If you use ...
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  • 4,602
4 votes
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What is cellranger doing in comparison to other methods?

cellranger mkfastq is not necessary anymore. It used to be that the cellranger software wanted the reads to be interleaved, and you could use cellranger to do that for you if you couldn't do it ...
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  • 1,662
4 votes

Getting hierarchy of cell populations with Drop-seq data

This article uses the freely available R package dropbead for filtering and then Seurat to perform a principal component analysis that groups together affine transcriptomes. It could be what you are ...
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4 votes

determining doublets in single-cell RNA-seq

We cannot assume that doublets will produce more UMIs I would caution the assumption that all doublets will have twice the UMI levels of isolated single-cells. Many "doublets" could contain ...
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  • 873
4 votes
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How to trim adapter sequences from GSE65360 in order to map the reads?

First the Nextera adapters and the custom barcode adapters overlap each other ...
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  • 2,556
4 votes
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Derive a GTF containing protein coding genes from a GTF file with Exons and CDS

In your case, I would definitely suggest following @Emily_Ensembl's advice and using the Arabidopsis GTF from Ensembl. But for future reference, if an Ensembl GTF wasn't available, you could build ...
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  • 3,211
4 votes
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how to write italic script in Rstudio

The title is misleading as this error doesn't have anything to do with making the font italic. In Seurat v2, FeaturePlot does not return a ggplot2 object by default, so ...
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  • 674
4 votes

Single-cell sequencing dataset has too many barcodes

The BAM file you downloaded has all of the detected 10X barcodes instead of those that represent cells. In a given 10X experiment the number of input barcodes vastly outnumbers the input cell count. ...
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  • 727
4 votes

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

I wrote a python script based on pysam that is free for anyone to use: ...
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  • 2,051
3 votes
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Scaling by linear regression against the number of reads

I don't know if this question has been solved already, but what they try to do is equalize the depth of sequencing for each cell. Therefore, they scale for the total number of reads. If you regress ...
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  • 200
3 votes

Cellranger aggr and Seurat merge difference?

Cell Ranger aggregate subsamples reads (unless you select none), so you will end up with less total reads in samples that have more initially. The output is still ...
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  • 2,099
3 votes
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How to compute the chance of failing to detect a gene given the detection limit of a protocol

A 90% loss can be rephrased as a 10% chance of detecting anything. So what we want to find is the probability of detecting 0 molecules, when we start with 7 and have 10% probability of success. Once ...
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  • 19.2k
3 votes

What is cellranger doing in comparison to other methods?

The best approach would be to contact 10x Genomics support: support@10xgenomics.com . They usually respond within a few hours. Otherwise, you can check the methods from the first 10x Genomics paper: ...
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  • 2,099
3 votes

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

That is a very general recommendation. Depending on your experiment, you can get a very different number of clusters with the same number of cells at the same resolution. You can actually use a ...
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  • 2,099

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