An R-based reproducible and user-friendly preprocessing pipeline for CyTOF data

Abstract

Mass cytometry (CyTOF) has become a method of choice for in-depth characterization of tissue heterogeneity in health and disease, and is currently implemented in multiple clinical trials, where higher quality standards must be met. Currently, preprocessing of raw files is commonly performed in independent standalone tools, which makes it difficult to reproduce. Here, we present an R pipeline based on an updated version of CATALYST that covers all preprocessing steps required for downstream mass cytometry analysis in a fully reproducible way. This new version of CATALYST is based on Bioconductor’s SingleCellExperiment class and fully unit tested. The R-based pipeline includes file concatenation, bead-based normalization, single-cell deconvolution, spillover compensation and live cell gating after debris and doublet removal. Importantly, this pipeline also includes different quality checks to assess machine sensitivity and staining performance while allowing also for batch correction. This pipeline is based on open source R packages and can be easily be adapted to different study designs. It therefore has the potential to significantly facilitate the work of CyTOF users while increasing the quality and reproducibility of data generated with this technology.

A distinct innate immune signature marks progression from mild to severe COVID-19

“…Mass cytometry data preprocessing and automated cell classification Individual FCS files collected from each set of samples were pre-processed using a semi-automated R pipeline based on CATALYST to perform individual file concatenation, bead based normalization, compensation, debarcoding, and batch correction as previously Cell Reports Medicine 2, 100166, January 19, 2021 e4 Article ll OPEN ACCESS described. 76 During this process, inter-sample doublets were excluded based on the debarcoding scheme and intra-sample doublets were excluded based on DNA content. Spillover matrix for CyTOF compensation was assessed on all antibodies used in this study as previously suggested.…”

Profound dysregulation of T cell homeostasis and function in patients with severe COVID‐19

“…Samples were pre-processed as described in the Appendix S1 and mass cytometry analysis was performed as previously described. 17,[60][61][62][63][64][65][66][67] A detailed description of the procedure is provided in the Appendix S1.…”

From bench to bedside: Single-cell analysis for cancer immunotherapy

“…Briefly, these analysis methods compare the distribution of expression values for each protein or gene between treatment groups for the subset of cells annotated as a given cell type. The optimal statistical test for this differential expression analysis remains an open question, although approaches based upon negative binomial tests are emerging as providing the best model of the distribution of the molecular abundances of both scRNA-seq (Hafemeister and Satija, 2019;Risso et al, 2018) and CyTOF data (Crowell et al, 2020;Nowicka et al, 2017). Standard pathway analysis tools can then be applied to determine the molecular pathways that were altered based upon the results of these differential expression analyses (Irizarry et al, 2009;Subramanian et al, 2005).…”