Trajectory and Pseudotime Analysis

“Monocle2 (v2.16.0)”, an R package, was applied to conduct single-cell evolutionary trajectory analysis and to estimate the transcriptome evolution in primary and extramedullary MM samples. Monocle is based on the assumption that one-dimensional “time” can depict the multi-dimensional expression values to elucidate the cell state transitions. In the trajectory analysis, we used genes meeting the following standards: mean_expression ≥ 0.1 and dispersion_empirical ≥ 1 * dispersion_fit to sort cells in pseudo-time order. The visualization functions “plot_cell_trajectory” were used to plot the minimum spanning tree on cells. Genes that changed along with the pseudotime were calculated (q-value <0.01) by the “differentialGeneTest” function and visualized with the plot_pseudotime_heatmap and the genes were clustered into subgroups according to the gene expression patterns.