Fig. 1

Overview of EXB-based molecular barcoding. a Structure of the EXB adapter. The adapter consists of a paired-end Y-adapter structure followed by a 6 bp random nucleotide sequence and three rationally designed 6 bp barcode subunits separated by distinct scaffold sequences. The 6 bp barcode subunits are random combinations of 64 possible sequences as output from the linear generator matrix as shown. The Tn5 transposase recognition sequence at the end of the adapter allows for the generation of sequencing libraries via in vitro Tn5 transposition. b Edit (substitution) distance metrics for all possible 6 bp barcode pairs. Over 93% of pairwise comparisons between barcodes have an edit distance greater than 4. c Schematic of in vitro transposition of EXBs. Tn5 transposase loaded with EXB adapters are incubated with double stranded cDNA. A gap-fill repair reaction then generates paired-end EXB sequencing libraries. After PCR, EXBs are read as inline barcodes, after which the insert sequence is read. d Single-end abundance of EXBs. Single-ended EXB identities were measured by pooling one million reads of each library