Fig. 1: 4NBoost significantly reduces ligation bias and achieves accurate quantification.

A The CV of the distribution of 30 (sequences with 2′-OH or 2′-Ome at the 3′ ends) or 7 (sequences with 2′-Ome at the 3′ ends) equimolar spike-ins, using total HEK293 cellular RNA as background, is shown in the box plots. In these plots, the center line represents the median, while the box edges correspond to the 25th and 75th percentiles. The whiskers extend to the minimum and maximum values. The biological replicates for these experiments were n = 2 for TruSeq and n = 4 for 4NBoost. B Pie charts illustrating the proportion of 30 equimolar spike-ins detected by TruSeq and 4NBoost in representative samples. C The correlations between the ground truth and predicted abundances of ratiometric spike-ins in various samples are illustrated, with blue points representing unmodified sequences and red points indicating sequences with 2′-Ome at the 3′ ends. D Scatter plots illustrating the correlation between the ground truth and predicted abundances of additional external spike-ins. E Scatter plot illustrating the correlation between the predicted abundances of the selected miRNA as measured by 4NBoost and RT-qPCR, with different colors representing distinct cell lines. The number of biological replicates per cell line was three for Caco-2 and four for all other cell lines. All p-values were calculated using two-tailed Student’s t-tests. Source data are provided as a Source Data file.