Limitations and potential improvements
The lower rate of FC sequence recovery compared to BR sequence recovery
implies that factors associated with FC PCRs, rather than sampling or
DNA extraction, were the main cause of failures to obtain complete
barcode sequences. The most likely explanation for this is that one or
both primers used in FC PCRs have suboptimal matches with the specimens
in question [48]. While it is unclear which of the FC primers
(Ill_LCO1490 and Ill_C_R) might cause this problem, deficiencies of
the LCO1490 / HCO2198 primer pair have been noted previously [49,
50], pointing to LCO1490 as problematic. These failures were
concentrated in certain Coleoptera and Hymenoptera families, suggesting
that the primer sequences may need adjustment to improve outcomes for
these groups.
Improvements to our bioinformatic process may be possible. We separately
identified FC and BR amplicons before attempting to align and merge
those with expected taxonomic identities. It may seem intuitively
simpler to merge all detected FC and BR sequences into putative
barcodes, and then to identify the correct barcode among those based on
taxonomy. However, there were unexpectedly high numbers of FC and BR
sequences for many specimens after filtering and denoising, which would
result in exceedingly high numbers of pairwise combinations of sequences
requiring examination for correct taxonomic identity.