knitr::opts_chunk$set(echo = TRUE, cache = FALSE, eval = TRUE,
                      warning = TRUE, message = TRUE)

Introduction

Although multidimensional single-cell-based flow and mass cytometry have been increasingly applied to microenvironmental composition and stem-cell research, integrated analysis workflows to facilitate the interpretation of experimental cytometry data remain underdeveloped. We present flowSpy, a comprehensive R package designed for the analysis and interpretation of flow and mass cytometry data. We applied flowSpy to mass cytometry and time-course flow cytometry data to demonstrate the usage and practical utility of its computational modules. flowSpy is a reliable tool for multidimensional cytometry data workflows and produces compelling results for trajectory construction and pseudotime estimation.

Overview of flowSpy workflow

The flowSpy package is developed to complete the majority of standard analysis and visualization workflow for FCS data. In flowSpy workflow, an S4 object in R is built to implement the statistical and computational approach, and all computational modules are integrated into one single channel which only requires a specified input data format. Computational modules of flowSpy can be divided into four main parts (Fig. 1): preprocessing, trajectory, analysis and visualization.

## 2020-04-20 15:31:10 [INFO] Number of cells in processing: 600
## 2020-04-20 15:31:10 [INFO] rownames of meta.data and raw.data will be named using column cell
## 2020-04-20 15:31:10 [INFO] Index of markers in processing
## 2020-04-20 15:31:10 [INFO] Creating FSPY object.
## 2020-04-20 15:31:10 [INFO] Determining normalization factors
## 2020-04-20 15:31:10 [INFO] Normalization and log-transformation.
## 2020-04-20 15:31:10 [INFO] Build FSPY object succeed
## FSPY Information:
##  Input cell number: 600  cells 
##  Enroll marker number: 10  markers 
##  Cells after downsampling: 600  markers
## Mapping data to SOM
## 2020-04-20 15:31:16 [INFO] Calculating Pseudotime.
## 2020-04-20 15:31:16 [INFO] Pseudotime exists in meta.data, it will be replaced.
## 2020-04-20 15:31:16 [INFO] The log data will be used to calculate trajectory
## 2020-04-20 15:31:17 [INFO] Calculating Pseudotime completed.
## 2020-04-20 15:31:17 [INFO] 37 cells will be added to leaf.cells .
## 2020-04-20 15:31:17 [INFO] Calculating walk between root.cells and leaf.cells .
## 2020-04-20 15:31:17 [INFO] Generating an adjacency matrix.
## 2020-04-20 15:31:17 [INFO] Walk forward.
## 2020-04-20 15:31:17 [INFO] Calculating walk completed.