Fig. 4

Effect of sampling depth on SCRS-based measurements. Quantitative relationship between sampling depth and the observed diversity of SCRS for an in silico population that includes all the cells sampled for SCRS over the 16 time points (a) or for the actual populations sampled for SCRS at each time point (b). At each sampling depth, 1000 permutations of sampling trials were performed, and the mean and standard deviations of the cumulative diversity index (DI) were calculated to estimate the extent to which the diversity of SCRS was observed and variation of the observation. In addition, quantitative relationship between sampling depth and the population average measurements of single-cell quantitative traits (c) and of their heterogeneity (d) is shown. Similarly, at each sampling depth, 1000 permutations of sampling trials were performed, and the cumMean and cumHI were calculated to estimate the observed values of quantitative traits and the variation of such observations. Gray areas represent the standard deviation of such measurements from the 1000 trials. The sampling depths of 3, 20 and 60 cells, each highlighted by a vertical line, were used as examples to quantitatively assess how the choice of sampling depth affects the accuracy and reliability of measuring the traits. For a and b, the minimal sampling depth, defined as the depth when no more than 1% of gain in cumulative DI will be gained by sampling one more cell (“Methods”; Additional file 9: Figure S5a, S5b), is highlighted for the 960-cell virtual population and for each time point. For c and d, the minimal sampling depth, defined based on the boundary condition of deviation of no more than 5% from the true means or true HI of metabolite contents (“Methods”; Additional file 9: Figure S5c, S5d), is highlighted for each time point. Minimal sampling depth was highlighted with red point and the corresponding text