Question: In Seurat, How Do nCount_RNA Differ from nFeature_RNA?
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gravatar for bioinformatics2020
5 days ago by
bioinformatics20200 wrote:

I'm reading up on the Seurat user guide: https://satijalab.org/seurat/v3.1/pbmc3k_tutorial.html And they mention for QC utilizing

The number of unique genes detected in each cell and The total number of molecules detected within a cell

They then refer to them as nCount_RNA and nFeature_RNA, but I'm not sure which is which. So my question is:

1.) What are the nCount_RNA and what are the nFeature_FNA 2.) Later in the pipeline, when you're normalizing the data, it says they "normalizes the feature expression measurements for each cell by the total expression." Can anybody explain that?

seurat single-cell • 70 views
ADD COMMENTlink modified 5 days ago by jared.andrews073.7k • written 5 days ago by bioinformatics20200
2
gravatar for jared.andrews07
5 days ago by
jared.andrews073.7k
St. Louis, MO
jared.andrews073.7k wrote:

nFeature_RNA is the number of unique genes detected in each cell. nCount_RNA is the total number of molecules detected within a cell. Low nFeature_RNA for a cell indicates that it may be dead/dying or an empty droplet. High nCount_RNA and/or nFeature_RNA indicates that the "cell" may in fact be a doublet (or multiplet). In combination with %mitochondrial reads, removing outliers from these groups removes most doublets/dead cells/empty droplets, hence why filtering is a common pre-processing step.

The NormalizeData step is basically just ensuring expression values across cells are on a comparable scale. By default, it will divide counts for each gene by the total counts in the cell, multiply that value for each gene by the scale.factor (10,000 by default), and then natural log-transform them.

ADD COMMENTlink modified 5 days ago • written 5 days ago by jared.andrews073.7k

Hey Jared, appreciate the easy-to-understand response! Quick question, do you know how exactly Seurat determines the number of molecules within a cell?

ADD REPLYlink written 5 days ago by bioinformatics20200

You know, I hunted around a bit and couldn't find exactly where nCount_RNA was defined. Presumably, it pulls that info during Read10x (from the .mtx file) or ReadAlevin and summarized for each cell to nCount_RNA during CreateSeuratObject.

ADD REPLYlink written 5 days ago by jared.andrews073.7k
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