PCR and sequencing outcomes
Initial PCR success rates differed between the FC and BR amplicons and among different taxa (Table 1). Visible PCR products were amplified from 233 of 450 specimens (51.7 %) in FC PCRs, compared to 384 specimens (85.3 %) in BR PCRs. Both FC and BR PCRs visibly succeeded for 205 specimens (45.6 %). Visible FC PCR success rates were lowest for Coleoptera (25.6 %) followed by Diptera (38.1 %), and highest for Annelida (90.1 %). In contrast, visible BR PCR success rates exceeded 70 % for all Orders except for Psocoptera (with only two specimens), including 90 % for Coleoptera, 72.6 % for Diptera, and 86.2 % for Annelida.
The numbers of sequences per amplicon and specimen after filtering and denoising varied widely, from zero (for 67 FC PCRs and five BR PCRs) to several thousand, with means of 72 in FC PCRs and 161 in BR PCRs. High numbers of denoised FC sequences per specimen were strongly correlated with high numbers of denoised BR sequences per specimen (Pearson correlation coefficient = 0.85, p < 0.001). The numbers of pairwise alignments between FC and BR amplicons with expected characteristics (overlap of 85 bp and 100 % identity) per specimen ranged between zero (for 92 specimens) to 180 (for a Hymenoptera specimen), with a mean of 14. Numbers of pairwise alignments were not obviously correlated with numbers of denoised FC and BR sequences. Low to moderate numbers of alignments were detected for 140 specimens from which FC PCRs did not produce visible products, and 39 specimens from which BR PCRs did not produce visible products.
After taxonomic filtering of sequences and pairwise alignments to identify optimal barcodes, full-length COI barcodes were recovered for 334 of 450 specimens (74 %). This included full-length barcodes for 146 specimens from which FC PCRs (109), BR PCRs (22), or both (15) did not result in a visible PCR product. Partial barcodes in the form of BR sequences were only recovered for a further 88 specimens, and FC sequences only for another 17 specimens.
No evidence of DNA extraction methodology effects on barcoding outcomes was observed. Rather, the most obvious factor affecting successful barcode detection was a combination of PCR amplicon and taxonomy (Table 2). FC sequences (either full-length or FC-only barcodes) were successfully detected for 75 % of specimens on average across 19 different orders and families considered, compared to > 94 % for BR sequences. Rates of FC sequence detection were lower than rates of BR sequence detection in 13 groups, the same in five, and higher in only one (Annelida, by 7 %). Among insect taxa, FC sequence detection rates were the same as BR sequence detection rates for two orders (Hemiptera and Lepidoptera) and one family (Ichneumonidae), and lower for all other insect groups. The largest difference between successful FC and BR sequence detection rates was observed for Staphylinidae (10 specimens, -100 %), with disparities ≥ -20 % observed for a further five insect groups including Chrysomelidae (107 specimens, -30 %), and Braconidae (87 specimens, -23 %).