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 %).