Integrating DNA Metabarcoding with Morphological Identification for Comparative Analysis of Extraction and PCR Protocols

Phytoplankton are critical in shaping global biogeochemical cycles through the uptake of carbon and inorganic compounds, as well as the production of oxygen. Traditional assessments, relying on microscopy, require considerable taxonomic expertise and often fail to document the genetic diversity within phytoplankton communities. DNA metabarcoding offers a promising alternative for assessing community composition, though inconsistent sampling protocols hinder cross-study comparisons. Here we examine the effects of DNA extraction replication on assessing community composition and the detection of rare species in phytoplankton communities using DNA metabarcoding in samples collected from the Ocean Springs Harbor Pier in Ocean Springs, MS. Prior to DNA extractions, replicate plankton samples were either divided into thirds before extraction or maintained as a single volume for DNA extraction.  Each sample was then metabarcoded using two loci (i.e., rbcL, and 23S). Results from the metabarcoding analysis are compared between those amplified from 1/3^rd sample DNA extractions and full volume DNA extractions and estimates of community composition determined for each.  These results are then compared with community composition estimates from traditional morphological analyses conducted at the Hollings Marine Lab in Charleston, SC, to evaluate the strengths and limitations of each approach. Results suggests that metabarcoding of multiple smaller extractions yields a more diverse community estimate, but it is unclear if the substantially higher costs are justified. Keywords: Phytoplankton, DNA metabarcoding, DNA extraction, PCR replication, sequencing depth, phytoplankton community composition, morphological identification, ecological assessment

Presentation Preference: Oral

Primary Presenter: Kambrial Love, Jackson State University (J00828901@students.jsums.edu)

Authors:

Kambrial Love, Jackson State University  (kambrialjonet024@gmail.com)

Andrew Shuler, NOAA Affiliate, HAB Monitoring and Reference Branch  (andrew.shuler@noaa.gov)

Steve Morton, NOAA Federal, HAB Monitoring and Reference Branch  (steve.morton@noaa.gov)

Brent Thoma, Jackson State University, Associate Professor of Biology  (brent.p.thoma@jsums.edu)