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Evaluation and redesign of the primers for detecting nitrogen cycling genes in environments

A better understanding of how nitrogen (N) cycling genes are involved in ecological processes is one of the crucial areas of microbial ecology. Currently, most molecular biological techniques investigating N cycling genes in the environment heavily rely on the accuracy of the polymerase chain reaction (PCR) primers; however, their specificity and coverage have not been comprehensively evaluated. Here, we collected a sequence database, NcycFunGen, aimed for primer evaluation and redesign. NcycFunGen was based on hidden Markov model profiles for 22 marker genes involved in N cycling, which included 607,359 paired nucleotide and protein sequences with their taxonomic information. Then, a total of 608 published primers were fully evaluated through NcycFunGen, as well as against full‐length sequences collected from KEGG. The new primers were designed by DegePrime. In the experiment, the updated ureC gene primer pair ureC607F/ureC898R and nifH gene primer pair nifH107F64/nifH379R64 was applied to a urea amendment site using droplet digital PCR and high‐throughput amplicon sequencing. The results showed that the majority of primer pairs cover less than 30% sequences of target genes and that 22.55% were inappropriate for quantitative PCR and amplicon sequencing (<100 bp or >550 bp). In general, this in‐silico evaluation demonstrated that although many primers have been adopted in published studies, some of them should be validated and updated as needed according to the updated gene database. Therefore, new degenerate primer pairs for ureC targeting urease, bacterial and archaeal amoA targeting ammonium monooxygenase, and nifH targeting nitrogenase were designed through NcycFunGen. These new primer pairs showed higher coverage and amplification efficiency, as well as amplicon lengths that were applicable for high‐throughput amplicon sequencing. Furthermore, the experimental results displayed better characteristics than commonly used published ureC and nifH gene primer pairs. In conclusion, primer evaluation and redesign are highly recommended to improve the accuracy of primers targeting N cycling genes, which could facilitate amplicon‐based N cycling studies in various environments. The bioinformatics framework developed in this study can also be applied to build functional gene databases for other biogeochemical pathways.