Uncovering plant microbiomes using long-read metagenomic sequencing

The microbiome of plants plays a pivotal role in their growth and health. Despite its importance, many fundamental questions about the microbiome remain largely unanswered, such as the identification of colonizing bacterial species, the genes they carry, and the location of these genes on chromosomes or plasmids. To gain insights into the genetic makeup of the rice leaf microbiome, we performed a metagenomic analysis using long-read sequences, and developed a genomic DNA extraction method that provides relatively intact DNA for long-read sequencing. 1.8 Gb reads were assembled into 26,067 contigs, including 136 circular sequences of less than 1 Mbp, as well as 172 large ([&ge;] 1 Mbp) sequences, six of which were circularized. Within these contigs, 669 complete 16S rRNA genes were clustered into 166 bacterial species, 130 of which showed low identity to previously defined sequences, suggesting that they represent novel species. The large circular contigs contain novel chromosomes and a megaplasmid, and most of the smaller circular contigs (<1 Mbp) were defined as novel plasmids or bacteriophages. One circular contig represents the complete chromosome of an uncultivated bacterium in the candidate phylum Candidatus Saccharibacteria. Our findings demonstrate the efficacy of long-read-based metagenomics for profiling microbial communities and discovering novel sequences in plant-microbiome studies.