Unveiling errors in soil microbial community sequencing: a case for reference soils and improved diagnostics for nanopore sequencing.

Autor: Manter DK; Soil Management and Sugar Beet Research, United States Department of Agriculture Agricultural Research Service (USDA-ARS), Fort Collins, CO, USA. daniel.manter@usda.gov., Reardon CL; Soil and Water Conservation Research Unit, USDA-ARS, Adams, OR, USA. catherine.reardon@usda.gov., Ashworth AJ; Poultry Production and Product Safety Research Unit, USDA-ARS, Fayetteville, AR, USA., Ibekwe AM; Water Efficiency and Salinity Research Unit, USDA-ARS, Riverside, CA, USA., Lehman RM; North Central Agricultural Research Laboratory, USDA-ARS, Brookings, SD, USA., Maul JE; Sustainable Agricultural Systems Laboratory, USDA-ARS, Beltsville, MD, USA., Miller DN; Agroecosystem Management Research Unit, USDA-ARS, Lincoln, NE, USA., Creed T; Soil Management and Sugar Beet Research, United States Department of Agriculture Agricultural Research Service (USDA-ARS), Fort Collins, CO, USA., Ewing PM; Food Systems Research Unit, USDA-ARS, Burlington, VT, USA., Park S; Water Efficiency and Salinity Research Unit, USDA-ARS, Riverside, CA, USA., Ducey TF; Coastal Plains Soil, Water and Plant Research Center, USDA-ARS, Florence, SC, USA., Tyler HL; Crop Production Systems Research Unit, USDA-ARS, Stoneville, MS, USA., Veum KS; Cropping Systems and Water Quality Research Unit, USDA-ARS, Columbia, MO, USA., Weyers SL; Soil Management Research Unit, USDA-ARS, Morris, MN, USA., Knaebel DB; Federal government retiree, Fayetteville, NY, USA.
Jazyk: angličtina
Zdroj: Communications biology [Commun Biol] 2024 Jul 28; Vol. 7 (1), pp. 913. Date of Electronic Publication: 2024 Jul 28.
DOI: 10.1038/s42003-024-06594-8
Abstrakt: The sequencing platform and workflow strongly influence microbial community analyses through potential errors at each step. Effective diagnostics and experimental controls are needed to validate data and improve reproducibility. This cross-laboratory study evaluates sources of variability and error at three main steps of a standardized amplicon sequencing workflow (DNA extraction, polymerase chain reaction [PCR], and sequencing) using Oxford Nanopore MinION to analyze agricultural soils and a simple mock community. Variability in sequence results occurs at each step in the workflow with PCR errors and differences in library size greatly influencing diversity estimates. Common bioinformatic diagnostics and the mock community are ineffective at detecting PCR abnormalities. This work outlines several diagnostic checks and techniques to account for sequencing depth and ensure accuracy and reproducibility in soil community analyses. These diagnostics and the inclusion of a reference soil can help ensure data validity and facilitate the comparison of multiple sequencing runs within and between laboratories.
(© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
Databáze: MEDLINE
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