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Evaluation of accuracy and precision in an amplicon sequencing workflow for marine protist communities
Author(s) -
Catlett Dylan,
Matson Paul G.,
Carlson Craig A.,
Wilbanks Elizabeth G.,
Siegel David A.,
IglesiasRodriguez M. Debora
Publication year - 2020
Publication title -
limnology and oceanography: methods
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.898
H-Index - 72
ISSN - 1541-5856
DOI - 10.1002/lom3.10343
Subject(s) - amplicon , protist , biology , amplicon sequencing , computational biology , deep sequencing , 18s ribosomal rna , pyrosequencing , dna sequencing , gene , barcode , genetics , ribosomal rna , evolutionary biology , polymerase chain reaction , 16s ribosomal rna , computer science , genome , operating system
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.