Evaluation of Filtration and DNA Extraction Methods for Environmental DNA Biodiversity Assessments across Multiple Trophic Levels

Frontiers in Marine Science

Ofelia Romero-Maraccini¹  Jesse Port¹  Mya Breitbart²  Dawn B. Goldsmith²  Anni Djurhuus²  Collin J. Closek³  Alexandria B. Boehm³  Reiko Michisaki⁴  Kristine R. Walz⁴  Francisco P. Chavez⁴  Kevan M. Yamahara⁴ 

[1] Center for Ocean Solutions, Stanford University, Stanford, CA, United States;College of Marine Science, University of South Florida, St. Petersburg, FL, United States;Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, United States;Monterey Bay Aquarium Research Institute, Moss Landing, CA, United States;

关键词: environmental DNA;    microorganisms;    vertebrates;    phytoplankton;    trophic levels;    marine ecosystems;   

DOI  :  10.3389/fmars.2017.00314

来源: DOAJ

【 摘 要 】

Metabarcoding of marine environmental DNA (eDNA), originating from tissue, cells, or extracellular DNA, offers the opportunity to survey the biological composition of communities across multiple trophic levels from a non-invasive seawater sample. Here we compare results of eDNA metabarcoding of multiple trophic levels from individual seawater samples collected from a kelp forest in Monterey Bay, California in order to establish methods for future cross-trophic level eDNA analysis. Triplicate 1 L water samples were filtered using five different 47 mm diameter membrane filters (PVDF, PES, GFF, PCTE, and NC) and DNA was extracted from triplicates of each filter-type using three widely-used extraction methods (the DNeasy Blood and Tissue kit, the MoBio PowerWater DNA Isolation kit, and standard phenol/chloroform methods) resulting in 45 individual eDNA samples prepared with 15 workflow combinations. Each DNA extract was amplified using PCR primers for the 16S rRNA gene (microorganisms; Bacteria and Archaea), 18S rRNA gene (phytoplankton), and the 12S rRNA gene (vertebrates), and PCR products were sequenced on an Illumina MiSeq platform. The richness and community composition of microbial, phytoplankton, and vertebrate OTUs were not significantly different between any of the 0.2 μm pore-size filter types extracted with the DNeasy or MoBio kits. However, phenol/chloroform extraction resulted in significantly different community structures. This study provides insight into multiple choices for extraction and filtration methods to use eDNA metabarcoding for biodiversity assessment of multiple trophic levels from a single sample. We recommend any combination of either DNeasy or MoBio with PES, PCTE, PVDF, or NC filters for a cross trophic level comparison.

【 授权许可】

Unknown