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 [1]
- Zehr et al. (2003) – nifH diversity in marine environments [2]
- Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny [3]
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.