Fig. 2: Demultiplexing of HIT-scISOseq concatemers.

a Four types of terminal combinations present after the ligation of cDNA fragments. These structures are distinguished by different combinations of elements (colored rectangles) as listed in the figure and are defined by the combination of 5′ and 3′ primers used. The 5′ (3′) primer forward and reverse strands are represented by 5p + (3p + ) and 5p- (3p-), respectively. Only fragments with 5′ and 3′ primer sequences at two terminals (5p + 3p- and 3p + 5p-) were regarded as FLNC reads. b Barcode assignment statistics for SIRV FLNC reads. The number of all mapped SIRV FLNC reads (Total SIRV) is presented, along with the number of FLNC reads with valid 10× Genomics 16 bp barcodes (Valid barcodes) and that detected with SIRV amplification barcodes (Right barcodes). c Accuracy, specificity and sensitivity of barcode assignment. d Confusion matrix for barcode assignment. e, f Mixed human-mouse test using NGS and HIT-scISOseq. Human and mouse cells were mixed at equal concentrations. Red dots indicate human-specific cells; green dots indicate mouse-specific cells. Only 1.43% (blue dots) are mixed human-mouse cells. g, h The UMI count correlation between NGS and HIT-scISOseq of human-specific (Pearson’s correlation coefficient r = 0.998, n = 761, p = 0) and mouse-specific cells (Pearson’s correlation coefficient r = 0.996, n = 1094, p = 0). Source data are provided as a Source Data file.