Fig. 3: Metrological evaluation of uncalibrated RNA-seq quantification.

a Violin plots of the Measurement Error (ME) for the absolute quantification of 100 spike-in transcripts in Mix1 and Mix2 across eight experimental batches. ME is calculated as the percentage difference between the measured (mean normalized FPKM) and certified copy numbers (all determined with log₂ transformed). The green line indicates the ideal value of ME = 0. b Violin plots of the ME for the ratio of measured copy numbers between Mix1 and Mix2, compared against the ratio of their certified values, for each of the 100 spike-in pairs across the eight batches. c Boxplots of the Root Mean Square Error (RMSE) for absolute quantification in Mix1 and Mix2 across the batches. Each box presented the RMSE values for 100 spike-ins between observed and reference values within a batch. d Bar plots of the Spearman Correlation Coefficient (RC) between measured and certified copy numbers for Mix1 and Mix2 across the batches. The dashed line indicates a reference value of RC = 0.89. For violin plots, the white box represents the interquartile range (IQR), the center line is the median, and the shaded area shows the probability density. For all boxplots, the center line indicates the median, the box limits represent the upper and lower quartiles (IQR), and the whiskers extend to 1.5 × IQR. Technical replicates are libraries (n = 3). Source data for this figure is available in the Source Data file.