Massively parallel single-cell genomics of microbiomes in rice paddies

Abstract

Plant growth-promoting microbes (PGPMs) have attracted increasing attention because they may be useful in increasing crop yield in a low-input and sustainable manner to ensure food security. Previous studies have attempted to understand the principles underlying the rhizosphere ecology and interactions between plants and PGPMs using ribosomal RNA sequencing, metagenomic sequencing, and genome-resolved metagenomics; however, these approaches do not provide comprehensive genomic information for individual species and do not facilitate detailed analyses of plant–microbe interactions. In the present study, we developed a pipeline to analyze the genomic diversity of the rice rhizosphere microbiome at single-cell resolution. We isolated microbial cells from paddy soil and determined their genomic sequences by using massively parallel whole-genome amplification in microfluidic-generated gel capsules. We successfully obtained 3,237 single-amplified genomes in a single experiment, and these genomic sequences provided insights into microbial functions in the paddy ecosystem. Our approach offers a promising platform for gaining novel insights into the roles of microbes in the rice rhizomicrobiome and to develop microbial technologies for improved and sustainable rice production.

Uncultured prokaryotic genomes in the spotlight: An examination of publicly available data from metagenomics and single-cell genomics

“…3 e), SAG had fewer CDSs per genome (1469 CDSs) than MAG (2108 CDSs), which is consistent with the lower genome completeness of SAG compared to MAGs. SAGs obtained using SAG-gel had a similar number of CDSs (2470, 2329, and 1875 CDSs) to MAGs in humans [17] , [100] , soil [16] , and mammal-associated samples [13] , respectively. In mammals, SAGs obtained using WGA-X also had 2025 CDSs [15] .…”

“…Although SAG datasets are one order of magnitude smaller than MAG datasets, the two datasets derived from the ocean [12] and soil environments [16] are larger than other SAG collections, with some projects acquiring thousands to tens of thousands of SAGs. To date, no cross-habitat microbial or large cohort-based genome collection projects, such as the GEM catalog or UHGG, have been undertaken in studies collecting SAGs.…”

“…Single-cell genomics is a method of obtaining uncultured microbial genomes by physically isolating single cells from individual microbial species, amplifying DNA, and sequencing. [10] , [11] , [12] , [13] , [14] , [15] , [16] . Although it requires more complex techniques than metagenomics because it treats single cells and their tiny DNA, recent advances in technology have resulted in a large amount of single-cell genome sequencing data.…”

Architecture of soil microaggregates: Advanced methodologies to explore properties and functions

“…Very Obtaining taxonomic or functional information with a spatial resolution at the single-cell level is even more challenging but conceivable in the future based on recent advances in single-cell approaches, which could also be suitable for microaggregates. Genomic sequencing of individual cells can successfully be performed, even for soil samples (Aoki et al, 2022;Jing et al, 2021;Kaster & Sobol, 2020). One major challenge will be the sampling of individual cells being associated with aggregates because currently available approaches to select single cells for sequencing rely on cell suspensions.…”

Tools for microbial single-cell genomics for obtaining uncultured microbial genomes

“…The gel capsule can also be aliquoted into a The advantage of SAG-gel over conventional single-cell genome sequencing techniques is the ability to obtain a large number of SAGs with superior completeness. Using this technology, a wide variety of SAGs have been obtained, including those from human commensals (intestinal (Chijiiwa et al 2020;Hosokawa et al 2022;Kogawa et al 2023), oral, and skin-associated (Arikawa et al 2021;Ide et al 2022b)), coral/marine sponge commensals (Kogawa et al 2022;Ide et al 2022a), insect commensals (Arai et al 2023), soil/rhizosphere bacteria (Yoda et al 2020;Aoki et al 2022), and river/marine bacteria. SAG-gel yields individual unknown microbial genomes even from samples containing a wide variety of microbes (including bacteria, archaea, and viruses) that are difficult to segregate by metagenomic analysis.…”