View source for Other diazotroph marker genes: nifD, nifK

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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 ==
nifD and nifK amplicon sequencing targets genes encoding the α- and β-subunits of the molybdenum–iron (MoFe) protein of nitrogenase, respectively. These genes can be complementary to nifH sequencing and provide additional phylogenetic and functional resolution of diazotrophic communities. Environmental DNA is extracted, followed by PCR amplification of nifD and/or nifK using gene-specific primers. Amplicons are sequenced using high-throughput platforms, and resulting sequences are used to assess diazotroph diversity, composition, and relative abundance across environments.

== Output ==
* nifD and/or nifK amplicon sequence variants (ASVs) or operational taxonomic units (OTUs)
* Taxonomic and phylogenetic assignment of diazotrophs
* Relative abundance profiles of nifD/nifK variants


=== Scale of measurement ===
* Molecular / gene-level
* Semi-quantitative (relative abundance; absolute abundance requires complementary quantification)
* Community-level inference of nitrogen fixation potential

=== Data generated ===
* DNA sequence reads (FASTQ files)
* ASV or OTU tables
* Phylogenetic trees (often higher resolution than nifH)
* Metadata-linked abundance matrices

=== Units & currency ===
* Sequence counts
* Relative abundance (%)
* Reads per sample

=== Sample size ===
* Varies by study design
* Biological and technical replication recommended

=== Repositories & databases ===
* nifDdada2, github – focused on nifD
* NCBI GenBank – nifD and nifK reference sequences
* ENA / SRA – raw sequencing reads
* IMG/M – integrated microbial genomes and functional genes

== Limitations ==
* Lower primer coverage and fewer validated primer sets compared to nifH
* Larger gene size can complicate amplification and sequencing
* Lower reference database coverage relative to nifH
* Does not directly measure nitrogen fixation activity
* Horizontal gene transfer complicates evolutionary interpretation

== Example Applications & Protocols ==
* PCR amplification protocols adapted from specific primers
* Custom laboratory SOPs due to limited standardized protocols

=== Classic examples ===

=== Common calculations/conversions ===
* Relative abundance = (reads per nifD or nifK ASV) / (total nifD/nifK reads per sample)
* Rarefaction to standardize sequencing depth
* Phylogenetic clustering based on nifD/nifK sequences
* Comparative analyses with nifH-derived community profiles

== References ==