Jump to content

NifH amplicon sequence: Difference between revisions

From OceanWiki
← Older edit
Hagi BucknWise (talk | contribs)
Bureaucrats, Interface administrators, Administrators
185 edits
Chreaa (talk | contribs)
8 edits
No edit summary
No edit summary
 
(5 intermediate revisions by 2 users not shown)
Line 1: Line 1:
 
{{BreadcrumbsNFixation}}


* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
Line 32: Line 32:


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


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


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


=== Units & currency ===
=== Units & currency ===
• Sequence counts or relative abundance (%)
* Sequence counts or relative abundance (%)
• Reads per sample
* Reads per sample


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


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


e.g. [https://github.com/raw-lab/NFixDB nFixDB]
e.g. [https://github.com/raw-lab/NFixDB nFixDB]


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


== Example Applications & Protocols ==
== Example Applications & Protocols ==


=== Classic examples ===
=== Classic examples ===
• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs
* 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
* 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
* 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 ===
=== Common calculations/conversions ===
• Relative abundance = (reads per nifH ASV) / (total nifH reads per sample)
* Relative abundance = (reads per nifH ASV) / (total nifH reads per sample)
• Rarefaction to standardize sequencing depth
* Rarefaction to standardize sequencing depth
• Conversion of read counts to proportions (%)
* Conversion of read counts to proportions (%)
• Phylogenetic clustering (e.g., nifH clusters I–IV)
* Phylogenetic clustering (e.g., nifH clusters I–IV)


== References ==
== 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.

Latest revision as of 11:45, 11 February 2026

Data types › Nitrogen Fixation › 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

  1. ↑ Zehr, J. P., & McReynolds, L. A. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene. Applied and Environmental Microbiology.
  2. ↑ Zehr, J. P., et al. (2003). Global distribution and diversity of nitrogen-fixing microorganisms. Nature.
  3. ↑ Gaby, J. C., & Buckley, D. H. (2012). A comprehensive evaluation of PCR primers to amplify the nifH gene. PLOS ONE.
Retrieved from "https://biogeoscapes.net//wiki/index.php?title=NifH_amplicon_sequence&oldid=509"