The impact of DNA extract homogenization and replication on marine
sediment metabarcoding diversity and heterogeneity
Abstract
Metabarcoding of environmental DNA (eDNA) is an attractive complement to
morphological methods for characterizing marine sediment benthic
communities. However, incomplete sampling is a major concern when
inferring community composition, and metabarcoding results are heavily
dependent on methodology. Using 18S V1-V2 and COI markers, we
investigated the effect on observed alpha- and beta diversity of (A)
homogenization intensity during sediment DNA extraction, (B) extraction
replicates vs larger sediment extraction volume, and (C) pre- and
post-PCR extract pooling. We show that an intermediate Precellys
homogenizer program for DNA extraction can significantly improve
sediment metabarcoding results in terms of captured diversity and
inter-replicate homogeneity compared to vortexing only. This effect was
stronger than that of increased sediment extract volume. Pre-PCR pooling
of DNA extraction replicates increased observed rarefied richness
compared to single extract medians, but not to the extent of amplifying
or sequencing extraction replicates individually before pooling, i.e.
post-PCR, or in silico pooling, respectively. We argue that this
discrepancy was due to both an increased number of PCR artifacts and
reduced PCR drift. Inter-sample heterogeneity was considerably higher
for the COI metazoan dataset, compared to the total eukaryotic 18S
dataset, likely due to a combination of metazoan eDNA distribution,
stochastic effects due to less conserved primer sites, and a high degree
of COI non-target amplification. Based on our results, extraction
replicates of smaller sediment volumes, in combination with firm but
intermediate homogenization and pre-PCR pooling, is a cost-effective way
of maximizing sediment eDNA metabarcoding sample coverage, compared to
increased extraction volume.