DGCyTOF: deep learning with graphic cluster visualization to predict cell types of single cell mass cytometry data
Authors: Cheng Lijun , Karkhanis Pratik , Gokbag Birkan , Li Lang 
Published: March 2021

AbstractBackgroundSingle-cell mass cytometry, also known as cytometry by time of flight (CyTOF) is a powerful high-throughput technology that allows analysis of up to 50 protein markers per cell for the quantification and classification of single cells. Traditional manual gating utilized to identify new cell populations has been inadequate, inefficient, unreliable, and difficult to use, and no algorithms to identify both calibration and new cell populations has been well established.MethodsA deep learning with graphic cluster (DGCyTOF) visualization is developed as a new integrated embedding visualization approach in identifying canonical and new cell types. The DGCyTOF combines deep-learning classification and hierarchical stable-clustering methods to sequentially build a tri-layer construct for known cell types and the identification of new cell types. First, deep classification learning is constructed to distinguish calibration cell populations from all cells bysoftmaxclassification assignment under a probability threshold, and graph embedding clustering is then used to identify new cell populations sequentially. In the middle of two-layer, cell labels are automatically adjusted between new and unknown cell populations via a feedback loop using an iteration calibration system to reduce the rate of error in the identification of cell types, and a 3-dimensional (3D) visualization platform is finally developed to display the cell clusters with all cell-population types annotated.ResultsUtilizing two benchmark CyTOF databases comprising up to 43 million cells, we compared accuracy and speed in the identification of cell types among DGCyTOF, DeepCyTOF, and other technologies including dimension reduction with clustering, including Principal Component Analysis (PCA), Factor Analysis (FA), Independent Component Analysis (ICA), Isometric Feature Mapping (Isomap), t-distributed Stochastic Neighbor Embedding (t-SNE), and Uniform Manifold Approximation and Projection (UMAP) withk-means clustering and Gaussian mixture clustering. We observed the DGCyTOF represents a robust complete learning system with high accuracy, speed and visualization by eight measurement criteria. The DGCyTOF displayedF-scoresof 0.9921 for CyTOF1 and 0.9992 for CyTOF2 datasets, whereas those scores were only 0.507 and 0.529 for thet-SNE+k-means; 0.565 and 0.59, forUMAP+k-means. Comparison of DGCyTOF witht-SNEandUMAPvisualization in accuracy demonstrated its approximately 35% superiority in predicting cell types. In addition, observation of cell-population distribution was more intuitive in the 3D visualization in DGCyTOF thant-SNEandUMAPvisualization.ConclusionsThe DGCyTOF model can automatically assign known labels to single cells with high accuracy using deep-learning classification assembling with traditional graph-clustering and dimension-reduction strategies. Guided by a calibration system, the model seeks optimal accuracy balance among calibration cell populations and unknown cell types, yielding a complete and robust learning system that is highly accurate in the identification of cell populations compared to results using other methods in the analysis of single-cell CyTOF data. Application of the DGCyTOF method to identify cell populations could be extended to the analysis of single-cell RNASeq data and other omics data.Availability and implementationA Python package (Python 3) and analysis scripts for reproducing the results are available athttps://lijcheng12.github.io/DGCyTOF/.DGCyTOF python package contactLijun.cheng@osu.edu.Supplementary informationSupplementary data are available at journal **** online.Key pointsDGCyTOF has been developed for the automatic identification of known and new cell populations.Optimal dimension reduction that integrates graphic-clustering technology facilitates highly accurate identification of new cell populations.Use of a feedback-calibration system can enhance the homology of cell types for the identification of cell populations.Three-dimensional visualization technology allows vivid depiction of cell types to improve their classification.