11
votes
Accepted
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 ...
- 582
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 ...
- 2,594
9
votes
Accepted
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 ...
- 621
8
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 ...
- 403
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 ...
- 71
7
votes
Accepted
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 ...
- 12.8k
7
votes
Accepted
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.
- 3,241
7
votes
Accepted
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 ...
- 873
6
votes
Accepted
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 ...
- 2,594
6
votes
Accepted
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 ...
- 3,241
5
votes
Accepted
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 ...
- 1,022
5
votes
Accepted
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 ...
- 574
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 ...
- 574
5
votes
Accepted
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 ...
- 66
4
votes
Accepted
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 ...
- 2,594
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 ...
- 4,662
4
votes
Accepted
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 ...
- 1,802
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 ...
- 309
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 ...
- 873
4
votes
Accepted
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
...
- 2,574
4
votes
Accepted
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 ...
- 3,241
4
votes
Accepted
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 ...
- 674
4
votes
scRNA-seq differential transcript usage
I was hoping to find some tool that already exists.
The short answer is, none exist yet. I see a number of hurdles to overcome first.1
For example, no one has yet characterized saturation for ...
- 621
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. ...
- 752
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:
...
- 2,166
3
votes
Accepted
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 ...
- 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 ...
- 2,139
3
votes
Accepted
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 ...
- 19.4k
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:
...
- 2,139
Only top scored, non community-wiki answers of a minimum length are eligible