The impact of DNA extract homogenization and replication on marine sediment metabarcoding diversity and heterogeneity

Metabarcoding of environmental DNA (eDNA) is an attractive complement to morphological methods for surveys and routine monitoring of marine sediment benthic communities. However, metabarcoding and other genetic techniques are heavily affected by choices made during sampling, processing, and analysis. Here, we investigated the effect of different eDNA extraction protocols on observed alpha- and beta diversity of replicates from the same grab. Specifically, we compared (A) homogenization intensity during sediment DNA extraction, (B) extraction replicates vs larger sediment extraction volume, and (C) pre- and post-PCR extract pooling. Using the 18S V1-V2 region marker, we show that a Precellys homogenizer protocol during DNA extraction can significantly improve sediment metabarcoding results in terms of captured diversity and inter-replicate homogeneity compared to vortexing only. This effect superseded that of increased sediment extract volume. Pre-PCR pooling of DNA extraction replicates increased observed rarefied richness compared to data from single extracts only, but not to the extent of sample extract replicates amplified individually before pooling. We argue that this discrepancy was due to a reduction both in recovered sample diversity, but also the number of PCR artifacts and PCR drift. Our results demonstrate that extraction replicates of smaller sediment volumes, in combination with moderate Precellys homogenization and pre-PCR pooling, are a cost-effective way to increase the amount of organism diversity that is recovered from sediment eDNA metabarcoding samples.