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

Authors: JOHNSON, F - Purdue University; LERCH, ROBERT - Retired ARS Employee; MOTAVALLI, P - National Institute Of Food And Agriculture (NIFA); Veum, Kristen; SCHARF, P - University Of Missouri

Submitted to: Environmental Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2024
Publication Date: 2/7/2024
Citation: Johnson, F.E., Lerch, R.N., Motavalli, P.P., Veum, K.S., Scharf, P.C. 2024. Comparative analysis of three next-generation sequencing techniques to measure nosZ gene abundance in Missouri claypan soils. Environmental Research. 249. Article 118346. https://doi.org/10.1016/j.envres.2024.118346.
DOI: https://doi.org/10.1016/j.envres.2024.118346

Interpretive Summary: Denitrification is the process by which nitrate is converted to gases under conditions of low soil oxygen. This can be a major loss path for nitrogen fertilizer. For decades, researchers have developed and applied various methods for measuring denitrification in field soils to understand the potential for it to occur in a given field. However, these methods were laborious, so researchers over the last 25 years focused on the potential for DNA gene sequencing techniques to measure the quantity of denitrification genes in soil and relate the gene abundance to the extent of denitrification in soils. Gene abundance data generated by the DNA-based methods has been shown to be poorly correlated to actual or potential denitrification in soils. Part of the disconnect is related to the fact that DNA based methods may be measuring gene abundance from inactive genetic material rather than actively expressed genes. This prompted the use of RNA-based methods to measure denitrification genes in soil. Since RNA is less stable than DNA in soil, any genes measured based on RNA sequencing methods are more likely to be actively expressed genes that may better correlate to actual denitrification in field soils. In this study, we used three RNA-based gene sequencing methods to measure a key gene, nosZ, in the denitrification pathway in soils from two fields in the claypan region of northeastern Missouri. To do this, we first quantified the amount of RNA in the soil at 0-15 and 15-30 cm depths. We also measured actual denitrification in the same soil samples to compare the abundance of nosZ to the occurrence of denitrification across the landscape. The three methods were real-time quantitative polymerase chain reaction (RT-qPCR), droplet digital polymerase chain reaction (dd-PCR), and nanostring sequencing (NS). Results showed that RNA concentrations were 3-5 times greater in the surface soil (0-15 cm) compared to the high clay subsoil (15-30 cm), and only the surface soil had enough RNA for performing the gene sequencing methods. The RNA data also indicated low biological activity in the claypan subsoil. Comparison among the gene sequencing methods showed the NS method resulted in a very narrow range of nosZ abundances that were 10-20 times lower than RT-qPCR and dd-PCR at each landscape position (i.e., summit, backslope, and toeslope) in both fields studied. The abundance of nosZ measured with RT-qPCR and dd-PCR better correlated to actual denitrification and these methods successfully captured the spatial variability of actual denitrification within the fields, indicating that these two methods were useful techniques for measuring nosZ abundance and potentially predicting denitrification. These results could lead to more rapid and accurate assessments of denitrification potential in soils which benefits growers by helping them to improve nitogen use effeciency and benefits the climate by leading to reductions in greenhouse gas emissions.

Technical Abstract: Quantitative next-generation sequencing techniques have been critical in gaining a better understanding of microbial ecosystems. The objectives of this research were to: 1) understand how depth influences RNA concentration and nosZ abundance; and 2) compare and evaluate multiple RNA-based sequencing methods for quantifying nosZ abundance in Missouri claypan soils. Research sites consisted of two claypan soil fields in Central Missouri. 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 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. Average RNA concentrations were (at 0-15 cm and 15-30 cm, respectively) 71.3 and 21.3 mg/kg in Field 1 and 40.1 and 8.14 mg/kg in Field 3. These data indicated low overall soil microbial activity below 15 cm. Due to low quantities of extractable soil RNA in the subsoil, nosZ abundance was only determined in the 0-15 cm depths. Average NS nosZ abundance measurements were constrained to a narrow range and were 10-20 fold lower than ddPCR and RT-qPCR at each landscape position within each field. The abundance of nosZ measured with RT-qPCR and ddPCR successfully captured the spatial variability of denitrification within these two fields, suggesting they are more useful techniques than NS for measuring nosZ abundance in soils.