Comparative analysis of three next-generation sequencing techniques to measure nosZ gene abundance in Missouri...

Quantitative next-generation sequencing techniques have been critical in gaining a better understanding of microbial ecosystems. In soils, denitrifying microorganisms are responsible for dinitrogen (N₂) production. The nosZ gene codes for nitrous oxide reductase, the enzyme facilitating the reduction of nitrous oxide (N₂O) to N₂. The objectives of this research were to: 1) understand how soil depth influences RNA concentration and nosZ gene abundance; 2) assess the spatial dependence of nosZ gene abundance in two claypan soil fields; and 3) compare and evaluate multiple RNA-based sequencing methods for quantifying nosZ gene abundance in soils in relation to dinitrogen (N₂) production. Research sites consisted of two intensively studied claypan soil fields in Central Missouri, USA. Soil cores were collected from two landscape transects across both fields and analyzed for extractable soil RNA at two depths (0-15 cm and 15-30 cm). Measurements of nosZ gene abundance were obtained using real-time quantitative polymerase chain reaction (RT-qPCR), droplet digital polymerase chain reaction (ddPCR), and nanostring sequencing (NS). In both fields, soil RNA concentrations were significantly greater at 0-15 cm depth compared to 15-30 cm. These data indicated low overall soil microbial activity below 15 cm. Due to low quantities of extractable soil RNA in the subsoil, nosZ gene abundance was only determined in the 0-15 cm depth. Sequencing method comparisons of average nosZ gene abundance showed that NS results were constrained to a narrow range and were 10-20-fold lower than ddPCR and RT-qPCR at each landscape position within each field. Droplet digital PCR appears to be the most promising method, as it reflected changes in N₂ production across landscape position.