High-throughput single-cell technologies provide an unprecedented view into cellular heterogeneity, yet they pose new challenges in data analysis and interpretation. In this protocol, we describe the use of Spanning-tree Progression Analysis of Density-normalized Events (SPADE), a density-based algorithm for visualizing single-cell data and enabling cellular hierarchy inference among subpopulations of similar cells. It was initially developed for flow and mass cytometry single-cell data. We describe SPADE's implementation and application using an open-source R package that runs on Mac OS X, Linux and Windows systems. A typical SPADE analysis on a 2.27-GHz processor laptop takes ∼5 min. We demonstrate the applicability of SPADE to single-cell RNA-seq data. We compare SPADE with recently developed single-cell visualization approaches based on the t-distribution stochastic neighborhood embedding (t-SNE) algorithm. We contrast the implementation and outputs of these methods for normal and malignant hematopoietic cells analyzed by mass cytometry and provide recommendations for appropriate use. Finally, we provide an integrative strategy that combines the strengths of t-SNE and SPADE to infer cellular hierarchy from high-dimensional single-cell data.
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This study was primarily supported by National Institutes of Health (NIH) grant U54CA149145, with S.K.P. as principal investigator. G.P.N. is supported by NIH grants U19 AI057229, 1U19AI100627, U54 CA149145, N01-HV-00242, 1R01CA130826, 5R01AI073724, R01 GM109836, R01CA184968, 1R01NS089533, P01 CA034233, R33 CA183654, R33 CA183692, 41000411217, 201303028, HHSN272201200028C, HHSN272200700038C, and 5U54CA143907; CIRM DR1-01477; Department of Defense grants OC110674 and 11491122; FDA grant HHSF223201210194C; Bill and Melinda Gates Foundation grant OPP1113682; Alliance for Lupus Research grant 218518; and the Rachford and Carlota A. Harris Endowed Professorship. P.Q. is supported by NIH grant R01 CA163481. S.C.B. is supported by the Damon Runyon Cancer Research Foundation Fellowship (DRG-2017-09) and NIH grant R00 GM104148-03.
Integrated supplementary information
Unlabeled subsample bone marrow data set from Bendall et al.2 used to explain the SPADE workflow in Figure 1.
MCM FCS file containing expression data from manually gated normal human bone marrow cells from Bendall et al.2 used for comparison analysis. MCM FCS file of ALL single-cell data from Amir et al.7 used for comparison analysis. Data in FCS file format containing the mouse lung epithelial RNA-seq expression from Treutlein et al.23.
MCM FCS file of ALL single-cell data from Amir et al. (2013)7 used for comparison analysis.
Data in FCS file format containing the Mouse lung epithelial RNA-Seq expression from Treutlein et al. (2014)23
About this article
Journal of Hematology & Oncology (2017)