Abstract
Understanding marine biodiversity is of paramount importance. Recently, we developed a polymerase chain 16 reaction (PCR)-free small-organelle-enriched metagenomics (SoEM) method with enhanced performance in species identification compared to multilocus DNA metabarcoding. However, this method has a problem of applicability for field surveys, for example, the need to filter large volumes of seawater. Here, we propose an improved version of the original SoEM method (SoEM pioneer version [SoEM-pv]) for marine biodiversity assessment. There are three major contributions to this proposed SoEM method (SoEM v1.0): (1) presentation of PCR-free protocols for seawater environmental DNA analysis; (2) provision of widely applicable optimal DNA extraction easy-to-use bioinformatics pipelines for analysis of the SoEM data. To validate the accuracy and sensitivity of SoEM v1.0, we evaluated the performance of the taxonomic capture capacity from seawater samples and found that, given the sample volume of 2 liters, (1) SoEM v1.0 had an approximately 5- to 10-fold higher DNA extraction yield than the SoEM-pv; (2) SoEM v1.0 had the highest taxonomic diversity compared to the other methods, 1.6-fold higher than PCR-based multi-locus DNA metabarcoding and 1.2-fold higher than shotgun metagenomic sequencing; and (3) the number of species identified did not increase proportionally with increasing sample size from 2 to 20 liters. We suggest that SoEM v1.0 is a promising and feasible method for marine eukaryotic diversity studies and expect it to be welcomed by biological oceanographers and coastal resource managers.