Alignment score of barcoded probe sequences with 1 and 2 mismatched bases. A multiplexed experiment containing three barcoded probes ('0MM', GGGTGCACGAGTGCGTGT; '1MM' GGGT A CACGAGTGCGTGT; '2MM' GGGT A CA T GAGTGCGTGT) shows a significant difference between alignment scores. 0MM events had the highest alignment score for the 0MM sequence (ANOVA, F (2, 67,524) = [13,657.25], **** P = 0, n = 4, n total events = 22,509) (i). 1MM events had the highest alignment score for the 1MM sequence (ANOVA, F (2, 33,204) = [2,702.66], **** P = 0, n = 4, n total events = 11,069) (ii). 2MM events had the highest alignment score for the 2MM sequence (ANOVA, F (2, 33,597) = [7,763.95], **** P = 0, n = 4, n total events = 11,200) (iii). Summary statistics for box plots: centre, median; bounds of box, interquartile range (IQR) 25th and 75th percentile; whiskers, minimum and maximum within 1.5 IQR. Barcoded probe concentration was 30 nM in all conditions. All experiments were performed in 2 sequencing buffer (700 mM KCl, 50 mM HEPES, 100 mM MgCl 2 , 100 mM ATP, 4.4 mM EDTA (pH 8.0)).
