README.md

In jmiguelj/FRAMEtags: FRAME-tags Analysis Pipeline

FRAMEtags

This package automatically gates and quantifies flow cytometry or microscopy data captured from live cells barcoded with FRAME-tags (FTs) using openCyto pipeline.

Installation

1. Download and install R v3.3.3 or greater at https://cloud.r-project.org/

2. Open R

3. Paste and execute the following commands to install required packages from bioconductor (update packages if necessary)

source("https://bioconductor.org/biocLite.R")
biocLite(c("openCyto"
          , "ggcyto"
          , "devtools"
          , "flowWorkspace"
          , "flowCore"
          , "flowClust"
          , "ncdfFlow"))

1. Then the following commands to install FRAMEtags

library("devtools")
install_github("jmiguelj/FRAMEtags")
library("FRAMEtags")

Required input files

(You can download sample data zip file with required input folder/files here: https://github.com/jmiguelj/FT-data/raw/master/FT%20data.zip)

In general, you need to prepare a folder on your computer containing the following files:

1. .fcs files to be analyzed

   • with at least 2 fluorescence, 1 forward scatter and 1 side scatter parameter

2. "Tag-Strain.csv"

   • containing a list of the FTs present in the sample and their corresponding user-specified identities or strain names

3. "Tube-Treatment.csv"

   • containing a list of the "Tube Name" or "Well IDs" and their corresponding user-specified sample treatment or experimental condition, each row corresponds to one input .fcs file and the "treatment" column is used mainly for labeling output statistics

How to run

Into the R command line paste and execute the following command:

getFRAMEtagData()

then follow onscreen directions.

Output files

In main input folder you should see:

1. "gating_template.csv""

   • can be modified and getFRAMEtagData() rerun to test modifications

2. "files-analyzed_log.csv"

   • includes list of .fcs files succesfully analyzed, getFRAMEtagData() will ignore these files
   • to reanalyze specific files, remove them from the log, or delete log entirely

3. "output" folder containing:

   i. "counts.csv"; final event counts for each FT and total parent population "nonMaxF"

   ii. "FT-gate.png"; visual overview of final FT gates for each .fcs + use this to determine any potential anomalous gating

   iii. "full-stats.csv"; event counts for every gate leading to and including the final FT gates

   iv. "percent.csv"; relative population fractions of each FT

Leading number indicates processing round as listed in the log file. Output folder will also include detailed gating plots if user chooses to output them.

jmiguelj/FRAMEtags documentation built on May 28, 2019, 8:45 a.m.