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ABSTRACT DNA metabarcoding of zooplankton biodiversity is used increasingly for monitoring global ocean ecosystems, requiring comparable data from different research laboratories and ocean regions. The MetaZooGene Intercalibration Experiment (MZG‐ICE) was designed to examine1 and analyse patterns of variation of DNA sequence data resulting from multi‐gene metabarcoding of 10 zooplankton samples carried out by 10 research groups affiliated with the Scientific Committee for Ocean Research (SCOR). Aliquots of DNA extracted from the 10 zooplankton samples were distributed to MZG‐ICE groups for metabarcoding of four gene regions: V1‐V2, V4 and V9 of nuclear 18S rRNA and mitochondrial COI. Molecular protocols and procedures were recommended; substitutions were allowed as necessary. Resulting data were uploaded to a common repository for centralised statistics and bioinformatics. Based on proportional sequence numbers for abundant phyla, overall patterns of variation were consistent across many—but not all—MZG‐ICE groups. V9 showed highest similarity, followed (in order) by V4, V1‐V2, and COI. Outlier data were hypothesised to result from the use of different PCR protocols and sequencing platforms, and possible contamination. MZG‐ICE results indicated that DNA metabarcoding data from different laboratories and research groups can provide reliable, accurate and valid descriptions of biodiversity of zooplankton throughout the ocean. Recommendations included: pre‐screening QA/QC of raw data, detailed records for laboratory protocols, reagents, and instrumentation, and centralised bioinformatics and multivariate statistics. In the absence of universal agreement on standardised protocols or best practices, intercalibration is the best way forward toward validation of DNA metabarcoding of zooplankton diversity for global ocean monitoring.
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Comparison of advanced methodologies for diatom identification within dynamic coastal communities
Abstract Diatom community composition has a critical influence on global ocean health and ecological processes. Developing accurate and efficient methods for diatom identification under dynamic environmental conditions is essential to understanding the implications of diatom community changes. Two developing methods for identifying and enumerating phytoplankton, cell imaging and molecular sequencing, are experiencing rapid advancements. This study aims to compare diatom taxonomic composition results within natural assemblages derived from rapidly advancing methods, FlowCam imaging and metabarcoding of the V4 region of the 18S rRNA gene, with traditional light microscopy cell counting techniques. All three methods were implemented in tandem to analyze changes in dynamic diatom assemblages within simulated upwelling experiments conducted in the California upwelling zone. The results of this study indicate that, summed across all samples, DNA sequencing detected four times as many genera as morphology‐based methods, thus supporting previous findings that DNA sequencing is the most powerful method for analyzing species richness. Results indicate that all three methods returned comparable relative abundance for the most abundant genera. However, the three methods did not return comparable absolute abundance, primarily due to barriers in deriving quantities in equal units. Overall, this study indicates that at the semi‐quantitative level of relative abundance measurements, FlowCam imaging and metabarcoding of the V4 region of the 18S rRNA gene yield comparable results with light microscopy but at the qualitative and quantitative levels, enumeration metrics diverge, and thus method selection and cross‐method comparison should be performed with caution.
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- Award ID(s):
- 1751805
- PAR ID:
- 10474443
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Limnology and Oceanography: Methods
- Volume:
- 21
- Issue:
- 11
- ISSN:
- 1541-5856
- Format(s):
- Medium: X Size: p. 687-702
- Size(s):
- p. 687-702
- Sponsoring Org:
- National Science Foundation
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