Assessment of Skimmed Milk Flocculation for Bacterial Enrichment from Water Samples, and Benchmarking of DNA Extraction and 16S rRNA Databases for Metagenomics

Water samples for bacterial microbiome studies undergo biomass concentration, DNA extraction, and taxonomic identification steps. Through benchmarking, we studied the applicability of skimmed milk flocculation (SMF) for bacterial enrichment, an adapted in-house DNA extraction protocol, and six 16S rRNA databases (16S-DBs). Surface water samples from two rivers were treated with SMF and vacuum filtration (VF) and subjected to amplicon or shotgun metagenomics. A microbial community standard underwent five DNA extraction protocols, taxonomical identification with six different 16S-DBs, and evaluation by the Measurement Integrity Quotient (MIQ) score. In SMF samples, the skimmed milk was metabolized by members of lactic acid bacteria or genera such as <i>Polaromonas</i>, <i>Macrococcus</i>, and <i>Agitococcus</i>, resulting in increased relative abundance (<i>p</i> < 0.5) up to 5.0 log fold change compared to VF, rendering SMF inapplicable for bacterial microbiome studies. The best-performing DNA extraction protocols were FastSpin Soil, the in-house method, and EurX. All 16S-DBs yielded comparable MIQ scores within each DNA extraction kit, ranging from 61-66 (ZymoBIOMICs) up to 80-82 (FastSpin). DNA extraction kits exert more bias toward the composition than 16S-DBs. This benchmarking study provided valuable information to inform future water metagenomic study designs.