Importance of full-length amplicons
Although FishCARD typically increased taxonomic resolution of ASVs from
our eDNA samples, in one specific case the inclusion of additional
California Current-specific 12S barcodes led to substantially
reduced taxonomic resolution, with 10 ASVs initially assigned toOxyjulis californica subsequently assigned only to family,
Labridae. This result appears to suggest that inclusion of barcodes from
local fauna decreases assignment accuracy. However, all 10 of these ASVs
were forward sequences only. The Señorita (Oxyjulis californica )
and Rock wrasse (Halichoeres semicinctus ), both native to the
California Current share a high degree of similarity in the forward12S sequence fragment, and the diagnostic SNPs occur in the
reverse sequence fragment. This issue is resolved when using the
full-length merged reads which capture diagnostic SNPs between these two
species. Given genetic similarities between these two species, a recent
phylogenetic study of new world Halichoeres wrasses has even
argued for synonymizing Oxyjulis into Halichoeres(Wainwright et al., 2018).
Despite the above, single direction reads can provide accurate taxonomic
assignment for many California Current fishes. For example, within
Labridae, the same family as Señorita and Rock wrasse, forward-reads
provided species-level resolution for the California sheephead,Semicossyphus pulcher . Such variation in taxonomic assignment
based on single-direction sequences highlights the importance of
full-length merged reads. To ensure the most accurate taxonomic
classifications, researchers should maximize the generation of merged
reads by choosing the appropriate sequencing platform (e.g. paired end
300 bp vs. paired end 75 bp).
Although we conducted 300 bp paired-end high throughput sequencing for a
176 bp amplicon, we still obtained a substantial number of orphaned
single end reads. This result is largely due to the generation of low
quality reverse sequence reads, a persistent issue found across Illumina
sequencing platforms (Callahan et al., 2016; Kwon, Park, Lee, & Yoon,
2013). As such, it is important to think carefully about whether or not
to include single end reads on a case-by-case and potentially
species-by-species basis. In the present study, we were only able to
make this determination because of the relative completeness of the
FishCARD database, which included all labrids native to the California
Current.