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Targeted metagenomics using probe capture detect a larger diversity of nitrogen and methane cycling genes in complex microbial communities than traditional metagenomics

Siljanen, Henri M P ; Manoharan, Lokeshwaran LU orcid ; Hilts, Angus S ; Bagnoud, Alexandre ; Alves, Ricardo J E ; Jones, Christopher M ; Kerou, Melina ; Sousa, Felipa L ; Hallin, Sara and Biasi, Christina , et al. (2025) In ISME Communications 5(1).
Abstract

Microorganisms are key players in the global cycling of nitrogen and carbon, controlling their availability and fluxes, including the emissions of the powerful greenhouse gases nitrous oxide and methane. Standard sequencing methods often reveal only a limited fraction of their diversity, because of their low relative abundance, the insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage of DNA amplification-based assays. Here, we developed and tested a targeted metagenomics approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic nitrogen and methane cycling. We designed comprehensive probe... (More)

Microorganisms are key players in the global cycling of nitrogen and carbon, controlling their availability and fluxes, including the emissions of the powerful greenhouse gases nitrous oxide and methane. Standard sequencing methods often reveal only a limited fraction of their diversity, because of their low relative abundance, the insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage of DNA amplification-based assays. Here, we developed and tested a targeted metagenomics approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic nitrogen and methane cycling. We designed comprehensive probe libraries for each of the 14 target marker genes comprising 264 111 unique probes. In validation experiments with mock communities, targeted metagenomics yielded gene profiles similar to the original communities. Only GC content had a small effect on probe efficiency, as low GC targets were less efficiently detected than those with high GC, within the mock communities. Furthermore, the relative abundances of the marker genes obtained using targeted or traditional shotgun metagenomics were significantly correlated. In addition, using archaeal
amoA genes as a case-study, targeted metagenomics identified a substantially higher taxonomic diversity and a larger number of sequence reads per sample, yielding diversity estimates 28 or 1.24 times higher than shotgun metagenomics or amplicon sequencing, respectively. Our results show that targeted metagenomics complements current approaches to characterize key microbial populations and functional guilds in biogeochemical cycles in different ecosystems, enabling more detailed, simultaneous characterization of multiple functional genes.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
in
ISME Communications
volume
5
issue
1
article number
ycaf183
publisher
Nature Publishing Group
external identifiers
  • pmid:41221508
ISSN
2730-6151
DOI
10.1093/ismeco/ycaf183
language
English
LU publication?
yes
additional info
© The Author(s) 2025. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.
id
58c9b63e-ee27-44bb-9a46-ba2927c3ceb1
date added to LUP
2025-11-18 08:31:08
date last changed
2025-11-18 09:34:13
@article{58c9b63e-ee27-44bb-9a46-ba2927c3ceb1,
  abstract     = {{<p>Microorganisms are key players in the global cycling of nitrogen and carbon, controlling their availability and fluxes, including the emissions of the powerful greenhouse gases nitrous oxide and methane. Standard sequencing methods often reveal only a limited fraction of their diversity, because of their low relative abundance, the insufficient sequencing depth of traditional metagenomes of complex communities, and limitations in coverage of DNA amplification-based assays. Here, we developed and tested a targeted metagenomics approach based on probe capture and hybridization to simultaneously characterize the diversity of multiple key metabolic genes involved in inorganic nitrogen and methane cycling. We designed comprehensive probe libraries for each of the 14 target marker genes comprising 264 111 unique probes. In validation experiments with mock communities, targeted metagenomics yielded gene profiles similar to the original communities. Only GC content had a small effect on probe efficiency, as low GC targets were less efficiently detected than those with high GC, within the mock communities. Furthermore, the relative abundances of the marker genes obtained using targeted or traditional shotgun metagenomics were significantly correlated. In addition, using archaeal<br>
 amoA genes as a case-study, targeted metagenomics identified a substantially higher taxonomic diversity and a larger number of sequence reads per sample, yielding diversity estimates 28 or 1.24 times higher than shotgun metagenomics or amplicon sequencing, respectively. Our results show that targeted metagenomics complements current approaches to characterize key microbial populations and functional guilds in biogeochemical cycles in different ecosystems, enabling more detailed, simultaneous characterization of multiple functional genes.<br>
 </p>}},
  author       = {{Siljanen, Henri M P and Manoharan, Lokeshwaran and Hilts, Angus S and Bagnoud, Alexandre and Alves, Ricardo J E and Jones, Christopher M and Kerou, Melina and Sousa, Felipa L and Hallin, Sara and Biasi, Christina and Schleper, Christa}},
  issn         = {{2730-6151}},
  language     = {{eng}},
  number       = {{1}},
  publisher    = {{Nature Publishing Group}},
  series       = {{ISME Communications}},
  title        = {{Targeted metagenomics using probe capture detect a larger diversity of nitrogen and methane cycling genes in complex microbial communities than traditional metagenomics}},
  url          = {{http://dx.doi.org/10.1093/ismeco/ycaf183}},
  doi          = {{10.1093/ismeco/ycaf183}},
  volume       = {{5}},
  year         = {{2025}},
}