Diazotroph community characterization via a high-throughput nifH amplicon sequencing and analysis pipeline

The dinitrogenase reductase (nifH) gene is a widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. Bioinformatic analysis of marker gene sequences requires considerable expertise and creation of database tools to facilitate the analysis. A large number of PCR primer sets have been developed for nifH amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. In this study, we advance the state of the art for nifH analysis by evaluating nifH primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters.