NifH amplicon sequence

• Page authors: Christian Furbo Reeder, PRIMO
• Responsible curator: Christian Furbo Reeder

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What is being measured in 1 - 3 words
Approach: Sequencing
Context: Environmental, incubation
Spatial scale: stations, across oceans
Temporal scale: days, seasons
Units:
Community captured: all, can be size-fractionated
Co-measurements: Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history

Method Overview

nifH amplicon sequencing targets the nifH gene, which encodes the iron protein subunit of nitrogenase and is widely used as a functional marker for biological nitrogen fixation. Environmental DNA is extracted, the nifH gene is amplified a nested PCR approach with degenerate primers (outer primers: NifH3 and NifH4 and inner primers: NifH1 and NifH2), and amplicons are sequenced (typically via high-throughput sequencing). Resulting sequences are used to assess the diversity, composition, and relative abundance of diazotrophic microorganisms across environments.

Output

• nifH amplicon sequence variants (ASVs) or operational taxonomic units (OTUs) • Taxonomic or phylogenetic assignment of diazotrophs • Relative abundance profiles of nifH variants

Scale of measurement

• Molecular / gene-level • Semi-quantitative (relative abundance, not absolute gene copy number unless combined with qPCR) • Community-level inference of functional potential

Data generated

• DNA sequence reads (FASTQ files) • ASV or OTU tables • Phylogenetic trees (commonly used for nifH) • Metadata-linked abundance matrices

Units & currency

• Sequence counts or relative abundance (%) • Reads per sample

Sample size

• Varies by study design • Commonly: o 10–100 environmental samples o Thousands to millions of reads per sequencing run • Replication often includes biological and technical replicates

Repositories & databases

• nFixDB – curated nifH sequences and annotations • NCBI GenBank – raw nifH sequences • ENA / SRA – raw sequencing reads • IMG/M – integrated microbial genomes and marker genes

e.g. nFixDB

Limitations

• Primer bias due to high nifH sequence diversity • Degenerate primers can amplify non-target genes • Does not directly measure nitrogen fixation activity • Limited taxonomic resolution for some diazotroph groups • Horizontal gene transfer complicates phylogenetic interpretation

Example Applications & Protocols

Classic examples

• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs • Zehr et al. (2003) – nifH diversity in marine environments • Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny

Common calculations/conversions

• Relative abundance = (reads per nifH ASV) / (total nifH reads per sample) • Rarefaction to standardize sequencing depth • Conversion of read counts to proportions (%) • Phylogenetic clustering (e.g., nifH clusters I–IV)

References

• Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology. • Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature. • Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.