Skip to content

Latest commit

 

History

History
43 lines (34 loc) · 3.36 KB

cellCalling.md

File metadata and controls

43 lines (34 loc) · 3.36 KB

Cell Calling Options

For QuantumScale RNA, single cells are identified by a combination of RT (sample) barcode, bead barcode and library (PCR) index. However not all barcode combinations correspond to real cells, but instead to background, e.g. empty beads. The workflow implements multiple approaches to cells against background barcodes.

CellFinder

CellFinder is an EmptyDrops-like cell calling approach used by default (cellFinder parameter). Cell-barcodes that are above a minimal transcript count (minUTC) but below the provided or calculated UTC threshold can be rescued based on expression differences from the ambient RNA profile. For a detailed description see the ScaleRna workflow handbook. Note that CellFinder is disabled for multi-species (barnyard) samples.

Parameters

parameter description default
cellFinder Enables CellFinder cell calling True
UTC Set a fixed unique transcript count thresholds for barcodes to be called a cell; Optional 0
cellFinderFdr False discovery rate to use for rescuing cells based on deviation from ambient profile. 0.001

TopCell Thresholding

TopCell thresholding sets a hard threshold on the unique transcript count per cell-barcode; all barcodes with counts above this threshold are called as cells, while all other barcodes are background. This threshold is determined dynamically as follows:

  1. Barcodes with counts less than the parameter minUTC are filtered from the data.
  2. Next, the top cell, the cell barcode topCellPercent percentile count (if the expectedCells parameter is set, the percentile is applied to that number).
  3. The UTC top cell is then divided by the parameter minCellRatio to determine the unique transcript count threshold for the dataset.

Parameters

parameter description default
minUTC Minimum number of counts to consider a barcode as a potential cell 100
expectedCells Approximate number of cells expected per sample; Optional; Can be set in samples.csv 0
topCellPercent Percentile to use for the top cell (robust max.) 99
minCellRatio Ratio between transcript counts of top cell and the lower cell threshold 10

Fixed Threshold

A unique transcript count threshold is used when the parameter UTC is greater than 0 and cellFinder is not enabled. In this case every barcode with a total count >= UTC is called as a cell.

Fixed Cell Number Threshold

With this option a fixed number of cell barcodes with the highest UTCs are called. The number is given by expectedCells which can be set per sample in samples.csv

Parameters

parameter description default
fixedCells Flag to enable fixed cell number calling False
expectedCells The number of cells to call 0

Outlier Identification

The workflow can flag potential outlier cells, based on multiple QC metrics. Barcodes are flagged in allcells.csv based on a Median Absolute Deviation (MAD) threshold for number of reads, fraction of usable reads (counted towards a gene), and fraction of mitochondrial reads. These parameters are defined in create_mtx.config. By default cells classified as outliers are not filtered from the UMI count matrix. It is possible to remove cells classified as outliers using the parameter filterOutliers.