Evaluation of accuracy and precision in an amplicon sequencing workflow for marine protist communities

TitleEvaluation of accuracy and precision in an amplicon sequencing workflow for marine protist communities
Publication TypeJournal Article
Year of Publication2019
AuthorsCatlett D, Matson PG, Carlson CA, Wilbanks EG, Siegel DA, M. Iglesias‐Rodriguez D
JournalLimnology and Oceanography: Methods
Volume18
Issue1
Pagination20-40
Date Published12/2019
Type of ArticleEvaluation of Existing Methods
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.

URLhttps://aslopubs-onlinelibrary-wiley-com.proxy.library.ucsb.edu:9443/doi/full/10.1002/lom3.10343
DOI10.1002/lom3.10343