Comparing diversity levels in environmental samples: DNA sequence capture and metabarcoding approaches using 18S and COI genes.

Environmental DNA studies targeting multiple taxa using metabarcoding provide remarkable
insights into levels of species diversity in any habitat. The main drawbacks
are the presence of primer bias and difficulty in identifying rare species. We tested
a DNA sequence-capture method in parallel with the metabarcoding approach to
reveal possible advantages of one method over the other. Both approaches were performed
using the same eDNA samples and the same 18S and COI regions, followed
by high throughput sequencing. Metabarcoded eDNA libraries were PCR amplified
with one primer pair from 18S and COI genes. DNA sequence-capture libraries were
enriched with 3,639 baits targeting the same gene regions. We tested amplicon sequence
variants (ASVs) and operational taxonomic units (OTUs) in silico approaches
for both markers and methods, using for this purpose the metabarcoding data set.
ASVs methods uncovered more species for the COI gene, whereas the opposite
occurred for the 18S gene, suggesting that clustering reads into OTUs could bias
diversity richness especially using 18S with relaxed thresholds. Additionally, metabarcoding
and DNA sequence-capture recovered 80%–90% of the control sample
species. DNA sequence-capture was 8x more expensive, nonetheless it identified
1.5x more species for COI and 13x more genera for 18S than metabarcoding. Both
approaches offer reliable results, sharing ca. 40% species and 72% families and retrieve
more taxa when nuclear and mitochondrial markers are combined. eDNA metabarcoding
is quite well established and low-cost, whereas DNA-sequence capture
for biodiversity assessment is still in its infancy, is more time-consuming but provides
more taxonomic assignments.