High throughput method of 16S rRNA gene sequencing library preparation for plant root microbial community profiling

Abstract Microbiota are a major component of agroecosystems. Root microbiota, which inhabit the inside and surface of plant roots, play a significant role in plant growth and health. As next-generation sequencing technology allows the capture of microbial profiles without culturing the microbes, profiling of plant microbiota has become a staple tool in plant science and agriculture. Here, we have increased sample handling efficiency in a two-step PCR amplification protocol for 16S rRNA gene sequencing of plant root microbiota, improving DNA extraction using AMPure XP magnetic beads and PCR purification using exonuclease. These modifications reduce sample handling and capture microbial diversity comparable to that obtained by the manual method. We found a buffer with AMPure XP magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. We also demonstrated that purification using exonuclease before the second PCR step enabled the capture of higher degrees of microbial diversity, thus allowing for the detection of minor bacteria compared with the purification using magnetic beads in this step. In addition, our method generated comparable microbiome profile data in plant roots and soils to that of using common commercially available DNA extraction kits, such as DNeasy PowerSoil Pro Kit and FastDNA SPIN Kit for Soil. Our method offers a simple and high-throughput option for maintaining the quality of plant root microbial community profiling.