Genomic recovery from rare terrestrial microbes enabled by DNA-based GC-fractionation

Paul O Sheridan; Yiyu Meng; David Coutts; Tom Williams; Cecile Gubry-Rangin

doi:10.1093/ismeco/ycaf152

Genomic recovery from rare terrestrial microbes enabled by DNA-based GC-fractionation

Paul O Sheridan, Yiyu Meng, David Coutts, Tom Williams, Cecile Gubry-Rangin

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

Abstract

Genome reconstruction from metagenomic samples has dramatically increased our understanding of uncultivated lineages of life. However, untargeted metagenomic sequencing is biased towards the more abundant microbes, neglecting less abundant lineages playing important ecological roles, such as the ammonia-oxidising archaea. Here, we demonstrate that separating soil molecular DNA using a bisbenzimide-CsCl guanine-cytosine (GC)-content-based DNA fractionation approach separates microbial DNA along a GC-content gradient. The fractions from both extremes of the GC-content gradient possess different 16S rRNA gene composition than the original unfractionated DNA. The high diversity in the lower GC-content fractions (< 45%) contrasts with the higher DNA abundance in the higher GC-content fractions (50%–70%), highlighting the low GC fractions as an enriched source of rare microbe DNA. Metagenomic sequencing of specific low- and high-GC fractions enabled the reconstruction of 204 taxonomically diverse metagenome-assembled genomes from 31 microbial phyla, with at least 63 of these originating from rare (< 0.1% relative abundance) or very rare (< 0.01% relative abundance) microbial families. Therefore, this approach facilitates genomic assembly of rare taxa in resulting pseudo-communities. Ultimately, this technique enables a semi-targeted metagenomic approach to recover genomes from low-abundance microbes with GC-contents that differ significantly from the environmental microbial community of interest. As mounting evidence suggests that rare microbes drive critical ecosystem functions, this approach will facilitate a deeper understanding of their metabolic potential in the environment.

Original language	English
Article number	ycaf152
Journal	ISME Communications
Volume	5
Issue number	1
Early online date	3 Sept 2025
DOIs	https://doi.org/10.1093/ismeco/ycaf152
Publication status	E-pub ahead of print - 3 Sept 2025

Data Availability Statement

Scripts for general manipulation of the 16S rRNA gene diversity have been deposited at https://github.com/bodington/gc_16S/, and scripts specific to the genome novelty have been deposited at https://github.com/SheridanPO-Lab/Genome-novelty. The 16S rRNA gene sequencing data and the 204 new genome sequences presented in this work have been deposited under the NCBI BioProject PRJNA1160233.

Acknowledgements

The authors would also like to acknowledge the support of the Maxwell computer cluster funded by the University of Aberdeen. In addition, we acknowledge Scotland’s Rural College (SRUC) as the custodian of the Aberdeen Cropping Experimental (ACE) Platform which hosts both the pHoenix and Tulloch Organic Rotation long-term experiments (LTE’s) and was the source of some of the soil samples used as part of this study. The maintenance of these LTEs is funded, in part, from the Scottish Government’s RESAS programme (Healthy soil for a Green Recovery). For the purpose of open access, the authors have applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising from this submission.

Funding

UKRI financially supported P.O.S. and Y.M. through the NERC grant (NE/R001529/1). In addition, C.G.-R. was supported by a Royal Society University Research Fellowship (URF150571) and D.B. received a PhD studentship from the Royal Society (RG160625).

Funders	Funder number
Natural Environment Research Council	NE/R001529/1
Royal Society	URF150571 , RG160625

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10.1093/ismeco/ycaf152Licence: CC BY

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title = "Genomic recovery from rare terrestrial microbes enabled by DNA-based GC-fractionation",

abstract = "Genome reconstruction from metagenomic samples has dramatically increased our understanding of uncultivated lineages of life. However, untargeted metagenomic sequencing is biased towards the more abundant microbes, neglecting less abundant lineages playing important ecological roles, such as the ammonia-oxidising archaea. Here, we demonstrate that separating soil molecular DNA using a bisbenzimide-CsCl guanine-cytosine (GC)-content-based DNA fractionation approach separates microbial DNA along a GC-content gradient. The fractions from both extremes of the GC-content gradient possess different 16S rRNA gene composition than the original unfractionated DNA. The high diversity in the lower GC-content fractions (< 45\%) contrasts with the higher DNA abundance in the higher GC-content fractions (50\%–70\%), highlighting the low GC fractions as an enriched source of rare microbe DNA. Metagenomic sequencing of specific low- and high-GC fractions enabled the reconstruction of 204 taxonomically diverse metagenome-assembled genomes from 31 microbial phyla, with at least 63 of these originating from rare (< 0.1\% relative abundance) or very rare (< 0.01\% relative abundance) microbial families. Therefore, this approach facilitates genomic assembly of rare taxa in resulting pseudo-communities. Ultimately, this technique enables a semi-targeted metagenomic approach to recover genomes from low-abundance microbes with GC-contents that differ significantly from the environmental microbial community of interest. As mounting evidence suggests that rare microbes drive critical ecosystem functions, this approach will facilitate a deeper understanding of their metabolic potential in the environment.",

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Genomic recovery from rare terrestrial microbes enabled by DNA-based GC-fractionation

Abstract

Data Availability Statement

Acknowledgements

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