NifH gene amplicon sequencing and metagenomic approaches are complementary in assessing diazotroph diversity.

Exploring the diversity of diazotrophs is key to understanding their role in supplying fixed nitrogen that supports marine productivity. A nested PCR assay using the universal primer set nifH1-nifH4, which targets the nitrogenase (nifH) gene, is a widely used approach for studying marine diazotrophs by amplicon sequencing. Metagenomics, direct sequencing of DNA without PCR, has provided complementary views of the diversity of marine diazotrophs. A significant fraction of the metagenome-derived nifH sequences (e.g. Planctomycete- and Proteobacteria-affiliated) were reported to have nucleotide mismatches with the nifH1-nifH4 primers, leading to the suggestion that nifH amplicon sequencing does not detect specific diazotrophic taxa and underrepresents diazotroph diversity. Here, we report that these mismatches are mostly located in a single-base at the 5'-end of the nifH4 primer, which does not impact detection of the nifH genes. This is demonstrated by the presence of nifH genes that contain the nucleotide mismatches in a recent compilation of global ocean nifH amplicon datasets, with high relative abundances detected in a variety of samples. While the metagenome- and metatranscriptome-derived nifH genes accounted for 4.4% of the total amplicon sequence variants from the global ocean nifH amplicon database, the corresponding amplicon sequence variants can have high relative abundances (accounting for 47% of the reads in the database). These analyses underscore that nifH amplicon sequencing using the nifH1-nifH4 primers is an important tool for studying diversity of marine diazotrophs, particularly as a complement to metagenomics which can provide taxonomic and metabolic information for some dominant groups.