View source for NifH amplicon sequence

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* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]:  [[User:Chreaa|Christian Furbo Reeder]]
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! What is being measured in 1 - 3 words
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| '''Approach:''' Sequencing
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| '''Context:''' ''Environmental'', incubation
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| '''Spatial scale:''' stations, across oceans
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| '''Temporal scale:''' days, seasons
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| '''Units:''' 
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| '''Community captured:''' all, can be size-fractionated
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| '''Co-measurements:''' 

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== 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. [https://github.com/raw-lab/NFixDB 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 <ref>Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology.</ref>
* Zehr et al. (2003) – nifH diversity in marine environments <ref>Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature.</ref>
* Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny <ref>Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.</ref>

=== 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 ==