polygate-debarcoder

Takes cyTOF data and polygon gates drawn on flowjo and outputs gated data with positive three tag barcodes Originally intended for use in debarcoding data obtained from cyTOF using protein barcodes introduced in Wroblewska et al. 2018.

Required Python 3.7 +

To install from source:

git clone https://github.com/Qiucwu/polygate-debarcoder 
cd polygate-debarcoder
pip install -r requirements.txt 

Install dependencies using pip install

pip install FlowCytometryTools
pip install fcswrite 

Quick Start

Open terminal and navigate to path of the program. Run init.py file

 python init.py 

You will be prompted to provide the following input files:

1. Gate coordinate files placed into folder ('coordinates')(refer to list section below for coordinate file formatting)
2. CyTOF FCS file placed into folder ('fcs_file')
3. Folder name

Outputs will be saved into folder ('Debarcoded Files'):

1. Individual debarcoded files
2. Distribution of barcodes within cell population
3. List of Missing Barcodes

Gate Coordinate Requirement

Gates are currently obtained from flowjo by gating on (x) metal of interest and (y) NGFR(metal 149). You may change the metal on the y-axis that you are gating for, but be sure to change it in the gate functions. Standard preprocessing should be done to remove doublets and dead cells prior to gating for epitotes of interest.

Drawing a polygon gate example

Gate Coordinate File Format

The formating of the gate coordinates files is essential for running the program. Make sure that you have your gate coordinates file in an .xls file in the format dictacted below. An example gate coordinates file will be provided in the folder "coordinates"

Metal	Name	Statistic	#Cells	Gate Coordinates
mass of metal	name of tag	% of cells positive for that metal	absolute number of cells positive for that metal	**quatrilateral gate coordinates exported from flowjo/FCSexpress

** Program can only take gates with 4 vertices! Modify the ray_tracing_method function with your own function