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

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.