The "best match" criterion used for genus assignments. a) Selecting a threshold for the "best close match" criterion. We looked at the results of the "best match" criterion and plotted the number of assignments (y axis) against the K2P distance (x axis) between the queries and the "best match" reference library barcodes ("true positives" as positive values above the × axis and "false positives" below the × axis). The distance that maximized the number of TP (which in our case also corresponded to the distance with the lowest proportion of FP) was selected as the threshold based on visual inspection of the histogram (i.e. 0.05) and is indicated by the dashed vertical line. The small inset chart is a cartoon showing how each section of the larger chart corresponds to each of the four "best close match" assignment outcomes (TP, FP, TA and FA) depending on the selection of a threshold i.e. placement of a vertical line. b) DNA barcode genus assignment success based on two sequence direct comparison criteria ("best match", "best close match") for 118 query species, with reference sub-libraries of varying species completeness (10-100% of available species selected randomly).