Data from: A broadly applicable COI primer pair and an efficient single‐tube amplicon library preparation protocol for metabarcoding

The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene
makes it ideal for assigning sequences to species. However, this
variability also makes it difficult to design truly universal primers.
Here, we present the forward primer “Sauron-S878”, specifically designed
to facilitate library preparation for metabarcoding. This primer is
modified to improve the coverage of terrestrial species compared to the
primer mCOIintF, optimized for aquatic systems, which raised the in-silico
coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in
3’-region, up to 3 mismatches in remaining primer). When paired with the
reverse primer “jgHCO2198” (fragment length ~313 bp) these primers
amplified 98.4% of 255 tested DNA extracts from various taxa, which is
better than many other common COI barcoding primers. Furthermore, a single
tube protocol was developed, wherein these primers amplify the target
gene, and attach MIDs and Illumina sequencing adapters in one reaction.
This eliminates the need for re-amplification or enzymatic ligation during
library preparation while keeping the flexibility to modularly combine
primers and MIDs. Using the single tube approach, three replicates of
three mock samples were sequenced on a MiSeq platform with no adverse
effects compared to commercial Nextera indexing kits. From this run 75% of
all included taxa could be recovered, with no considerable bias among
taxonomic groups. Despite the fact that 98.4% of the extracts were
confirmed to amplify in-vitro, this number was lower than expected. A
reason for this discrepancy was a clear link between the relative
concentration of a specific DNA type in the template and the number of
returned reads for this DNA. We would argue that such a bias may be
especially problematic in metabarcoding where samples usually contain
trace DNA in unknown amounts. However, how this affects the completeness
of metabarcoding results has yet been poorly investigated.