NifH amplicon sequence
| 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: |
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.