NifH amplicon sequence - Revision history

Hagi BucknWise at 19:45, 11 February 2026

2026-02-11T19:45:57Z

← Older revision		Revision as of 12:45, 11 February 2026
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	* Varies by study design		* Varies by study design
	* Commonly:		* Commonly:
	o 10–100 environmental samples~~<br>~~		o 10–100 environmental samples
	o Thousands to millions of reads per sequencing run~~<br>~~		o Thousands to millions of reads per sequencing run
	* Replication often includes biological and technical replicates		* Replication often includes biological and technical replicates

Hagi BucknWise at 19:45, 11 February 2026

2026-02-11T19:45:00Z

← Older revision		Revision as of 12:45, 11 February 2026
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	=== 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<br>
	o Thousands to millions of reads per sequencing run		o Thousands to millions of reads per sequencing run<br>
	• 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 ==
Line 81:		Line 81:

	=== 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 ==

Hagi BucknWise at 19:42, 11 February 2026

2026-02-11T19:42:23Z

← Older revision		Revision as of 12:42, 11 February 2026
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	== 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 ===
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	=== Classic examples ===		=== 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 & 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>		* 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>		* 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 ===

Hagi BucknWise at 19:41, 11 February 2026

2026-02-11T19:41:09Z

← Older revision		Revision as of 12:41, 11 February 2026
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	== 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.~~

Hagi BucknWise at 19:40, 11 February 2026

2026-02-11T19:40:31Z

← Older revision		Revision as of 12:40, 11 February 2026
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	=== 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 ===

Kate Evans at 19:34, 11 February 2026

2026-02-11T19:34:40Z

← Older revision		Revision as of 12:34, 11 February 2026
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			{{BreadcrumbsNFixation}}

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

Chreaa at 09:30, 10 February 2026

2026-02-10T09:30:35Z

← Older revision		Revision as of 02:30, 10 February 2026
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	\| '''Community captured:''' all, can be size-fractionated		\| '''Community captured:''' all, can be size-fractionated
	\|-		\|-
	\| '''Co-measurements:''' ~~Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history~~		\| '''Co-measurements:'''

	\|}		\|}
	</div>		</div>

Chreaa at 09:30, 10 February 2026

2026-02-10T09:30:01Z

← Older revision		Revision as of 02:30, 10 February 2026
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	~~{{NifH amplicon sequence}}~~

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

Chreaa at 09:28, 10 February 2026

2026-02-10T09:28:51Z

← Older revision		Revision as of 02:28, 10 February 2026
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	{{~~BreadcrumbsPhotoautotrophy~~}}		{{NifH amplicon sequence}}

	* [[Page authors\|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]		* [[Page authors\|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
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	== Method Overview ==		== 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 ==		== 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 ===		=== 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 ===		=== Data generated ===
			• DNA sequence reads (FASTQ files)
			• ASV or OTU tables
			• Phylogenetic trees (commonly used for nifH)
			• Metadata-linked abundance matrices

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

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

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

	=== Classic examples ===		=== Classic examples ===
	* Classic/original papers		• Zehr & McReynolds (1989) – First use of nifH as a molecular marker for diazotrophs
			• Zehr et al. (2003) – nifH diversity in marine environments
	~~=== Recent applications ===~~		• Gaby & Buckley (2012) – Evaluation of nifH primers and phylogeny
	* New papers + protocol links (~~e.g. word doc used in lab~~) ~~if applicable~~

	=== Common calculations/conversions ===		=== Common calculations/conversions ===
	* E.g. ~~conversion to standard units~~		• 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 ==		== References ==

	~~[[Category:Main Pages\|Model types]]~~		• 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.

Chreaa: Created page with "{{BreadcrumbsPhotoautotrophy}} * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- TOC
{| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' Sequencing |- | '''Context:''' ''Environmental'', incubation |- | '''Spatial scale:''' stations, across oceans |- | '''Temporal scale:''' days, seasons |- | '''Units:..."

2026-02-10T09:22:50Z

Created page with "{{BreadcrumbsPhotoautotrophy}} * Page authors: Christian Furbo Reeder, PRIMO * Responsible curator: Christian Furbo Reeder ---- __TOC__ <div class="model-box"> {| class="model-ib" ! What is being measured in 1 - 3 words |- | '''Approach:''' Sequencing |- | '''Context:''' ''Environmental'', incubation |- | '''Spatial scale:''' stations, across oceans |- | '''Temporal scale:''' days, seasons |- | '''Units:..."

New page

{{BreadcrumbsPhotoautotrophy}}

* [[Page authors|Page authors]]: [[Christian Furbo Reeder]], [[PRIMO]]
* [[Responsible curator|Responsible curator]]: [[User:Chreaa|Christian Furbo Reeder]]
----

__TOC__
<div class="model-box">
{| class="model-ib"
! 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:''' Other measurements required for interpretation of results e.g., temperature, salinity, wind speed history
|}
</div>
<div style="clear:both"></div>

== Method Overview ==

== Output ==

=== Scale of measurement ===

=== Data generated ===

=== Units & currency ===

=== Sample size ===

=== Repositories & databases ===

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

== Limitations ==

== Example Applications & Protocols ==

=== Classic examples ===
* Classic/original papers

=== Recent applications ===
* New papers + protocol links (e.g. word doc used in lab) if applicable

=== Common calculations/conversions ===
* E.g. conversion to standard units

== References ==

[[Category:Main Pages|Model types]]