Unassigned MiFish 12S ASVs
The Anacapa Toolkit failed to assign taxonomy to 49.6% (169/341) of ASVs representing 24.5% (81,002/330,877) of all reads using all three reference databases investigated in this study (Supplemental Table 4). Of the 169 unassigned ASVs, 16 were forward-only reads, and 153 were merged reads. To explore the origins of these unassigned reads, we used BLAST to query all Genbank sequences, revealing that 94.7% (160/169) of these ASVs aligned to marine prokaryotic and eukaryotic 16Ssequences (Max Alignment Scores 87.9-475). Of these aligned ASVs, 85% (136/160) matched to uncultured sequences generated from marine metagenomic studies. 80.0% (128/160) of successfully aligned ASVs matched to bacterial barcodes including those from Psychromonassp., Photococcus caeruleum , Loktanella sp.,Leucothrix sp., and Gimesia sp., and cyanobacteria. A smaller fraction of assigned ASVs (18.8%; 30/160) best aligned to eukaryotic sequences including those from diatoms (e.g. Nitzschia alba and Eucampia antarctica ) and other marine microalgae (e.g. Picobiliphytes, Heterosigma akashiwo , Mesopedinella arctica , and Phacus warszewiczii ). Given that these 169 unassigned sequences were non-vertebrate, we excluded these ASVs from all subsequent comparisons. All remaining 172 ASVs were assigned to a Class of vertebrates by at least one of the three reference databases used. Of these vertebrate ASVs, 58 were merged, 107 were forward-only, and 7 were reverse only reads.