Sequence alignments and validation of PCR primers used to detect phylogenetically diverse nrfA genes associated with dissimilatory nitrate reduction to ammonium (DNRA)

Authors: CANNON, JORDAN - University Of Illinois; SANFORD, ROBERT - University Of Illinois; Connor, Lynn; YANG, WENDY - University Of Illinois; Chee Sanford, Joanne

Submitted to: Data in Brief
Publication Type: Database / Dataset
Publication Acceptance Date: 5/14/2019
Publication Date: 6/7/2019
Citation: Cannon, J., Sanford, R.A., Connor, L.M., Yang, W.H., Chee-Sanford, J.C. 2019. Sequence alignments and validation of PCR primers used to detect phylogenetically diverse nrfA genes associated with dissimilatory nitrate reduction to ammonium (DNRA). Data in Brief. https://doi.org/10.1016/j.dib.2019.104016.
DOI: https://doi.org/10.1016/j.dib.2019.104016

Interpretive Summary: Dissimilatory nitrate reduction to ammonium (DNRA) is a microbial process that has received revived attention due to recent reports showing its occurrence more broadly in upland soils than previously thought. DNRA competes with denitrification as processes that consume N-fertilizer in agricultural systems, with DNRA retaining nitrogen (N) in contrast to gaseous N losses attributed to well-studied denitrifying activity. PCR primer sets were designed to target nrfA, the gene encoding the enzyme NrfA that catalyzes the nitrite ammonification step in the process of DNRA. This study provides further methodological details and validation of the primers recently published (Cannon et al., 2019, J. Microbiol. Meth., https://doi.org/10.1016/j.mimet.2019.03.020) including the use of amplified fragment length polymorphism (AFLP) profiling and high-throughput PCR-based sequencing of genes from environmental samples and selected reference strains. We also provide sequence alignment files available to the public for the first time of the full length nrfA genes, PCR reference amplicon alignment, NrfA amino-acid alignment and NrfA translated PCR amplicon-amino acid alignment for readily available access through the published journal article. The impact of this research is anticipated to be the advancement of studies on DNRA by providing molecular-based tools that have not been previously available for practitioners to examine new nrfA gene sequences corresponding to active regions of the NrfA protein and to aid further primer design modifications for discovery of new nrfA genes.

Technical Abstract: PCR primer sets were designed to target nrfA, the gene encoding the pentaheme nitrite reductase NrfA that catalyzes the nitrite ammonification step in the process of dissimilatory nitrate reduction to ammonium (DNRA). Details of the nucleotide alignments of the primer target regions of 271 nrfA sequences from reference genomes representing 18 distinct clades of NrfA are shown here along with validation of application to PCR-based methodology including the use of amplified fragment length polymorphism (AFLP) profiling and Illumina platform amplicon-based sequencing of environmental samples and selected reference strains. Summary data tables illustrate the specificity of forward primers nrfAF2awMOD and nrfAF2awMODgeo when paired with the new reverse primer nrfAR1MOD in relation to consensus target reference sequences associated with members of 18 NrfA clades. Specificity of the new primers to nrfA sequences in environmental samples is shown in AFLP analysis and amino acid-translated amplicon sequences obtained with the new primer sets. We also provide sequence alignment files of the full length nrfA genes, PCR reference amplicon alignment, NrfA amino-acid alignment and NrfA translated PCR amplicon-amino acid alignment. The full nucleotide and protein alignments contain 271 reference genomes that represent the 18 identified NrfA clades as a tool to further aid practitioners in examining new sequences corresponding to the primer target regions and allow further primer design modifications if deemed pertinent to specific studies. A more comprehensive analysis of this data may be obtained from the article “Optimization of PCR primers to detect phylogenetically diverse nrfA genes associated with nitrite ammonification” (Cannon et al., 2019, Journal of Microbiological Methods, https://doi.org/10.1016/j.mimet.2019.03.020).