Abstract
Advances in high-throughput DNA sequencing methods reveal the vast diversity of marine protists. Amplicon sequencing of “barcode” genes, such as the 18S small subunit ribosomal RNA gene (henceforth, 18S gene), is a cost-effective and widely used genetic method for assessing the composition of marine protist communities. This method is now being applied from local to global scales to interrogate the causes and consequences of protist community variations. Significant efforts have been made to validate amplicon methods targeting prokaryotes, but the precision, accuracy, and quantitative potential of 18S gene amplicon sequencing methods for marine protists remain unclear. Here, we use artificial (mock) communities and environmental samples collected from the Santa Barbara Channel, CA to evaluate the precision and accuracy in an amplicon workflow targeting the V9 hypervariable region of the 18S gene for marine protists. Overall, we find that this amplicon workflow has high precision and reasonable accuracy, but the magnitude of analytical uncertainty can increase significantly unless certain procedural issues are avoided. Finally, we demonstrate the value of positive and negative controls in, and the quantitative potential of, amplicon sequencing assessments of marine protist communities.
| Original language | English |
|---|---|
| Pages (from-to) | 20-40 |
| Number of pages | 21 |
| Journal | Limnology and Oceanography: Methods |
| Volume | 18 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2020 |
| Externally published | Yes |
Funding
We thank Emma Wear and Anni Djurhuus for advice on sample preparation and analyses, Thomas Lankiewicz for assistance with bench work, Alexis Pasulka for helpful comments regarding mock community composition, Alyson Santoro for providing laboratory space, and the Plumes and Blooms team (especially Nathalie Guillocheau and Sarah Amiri) for assistance with field sampling and supplying ancillary environmental data. We thank Alexandra Worden at MBARI, Avery Tatters at USC, and Holly Bowers at Moss Landing Marine Lab for providing algal isolates for culturing. This research was supported by the National Aeronautics and Space Administration Biodiversity and Ecological Forecasting program (Grant NNX14AR62A), the Bureau of Ocean and Energy Management Ecosystem Studies program (BOEM award MC15AC00006), and NOAA in support of the Santa Barbara Channel Biodiversity Observation Network. D.C. is supported on a NASA Earth and Space Science Fellowship (Grant NNX16AO44HS02). Most DNA sequencing was carried out by the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center, supported by NIH Shared Instrumentation (Grant 1S10OD010786‐01). We acknowledge our general use of, and the assistance of the staff members from, the DNA Technologies Core at the UC Davis Genome Center, and the Biological NanoStructures Lab within the California NanoSystems Institute supported by UC Santa Barbara and the University of California, Office of the President, for all sequencing analysis. Raw DNA sequencing data are publicly available in the National Center for Biotechnology Information's Sequence Read Archive under accession number PRJNA532583. We thank Emma Wear and Anni Djurhuus for advice on sample preparation and analyses, Thomas Lankiewicz for assistance with bench work, Alexis Pasulka for helpful comments regarding mock community composition, Alyson Santoro for providing laboratory space, and the Plumes and Blooms team (especially Nathalie Guillocheau and Sarah Amiri) for assistance with field sampling and supplying ancillary environmental data. We thank Alexandra Worden at MBARI, Avery Tatters at USC, and Holly Bowers at Moss Landing Marine Lab for providing algal isolates for culturing. This research was supported by the National Aeronautics and Space Administration Biodiversity and Ecological Forecasting program (Grant NNX14AR62A), the Bureau of Ocean and Energy Management Ecosystem Studies program (BOEM award MC15AC00006), and NOAA in support of the Santa Barbara Channel Biodiversity Observation Network. D.C. is supported on a NASA Earth and Space Science Fellowship (Grant NNX16AO44HS02). Most DNA sequencing was carried out by the DNA Technologies and Expression Analysis Cores at the UC Davis Genome Center, supported by NIH Shared Instrumentation (Grant 1S10OD010786-01). We acknowledge our general use of, and the assistance of the staff members from, the DNA Technologies Core at the UC Davis Genome Center, and the Biological NanoStructures Lab within the California NanoSystems Institute supported by UC Santa Barbara and the University of California, Office of the President, for all sequencing analysis. Raw DNA sequencing data are publicly available in the National Center for Biotechnology Information's Sequence Read Archive under accession number PRJNA532583.
| Funders | Funder number |
|---|---|
| Alexandra Worden at MBARI | |
| Alyson Santoro | |
| Biological NanoStructures Lab | |
| Cores | |
| Energy Management Ecosystem Studies program | |
| NASA Earth and Space Science | |
| NIH Shared Instrumentation | 1S10OD010786‐01 |
| National Center for Biotechnology Information's Sequence Read Archive | PRJNA532583 |
| Plumes and Blooms team | |
| Santa Barbara Channel Biodiversity Observation Network | |
| University of California, Office of the President | |
| National Science Foundation | 1831937 |
| National Institutes of Health | 1S10OD010786-01 |
| National Aeronautics and Space Administration | NNX14AR62A, NNX16AO44HS02 |
| National Oceanic and Atmospheric Administration | |
| University of California, Santa Barbara | |
| University of South Carolina | |
| Bureau of Ocean Energy Management | MC15AC00006 |
| Genome Center, University of California, Davis |