Simple amplicon sequencing library preparation for plant root microbial community profiling

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 developed a novel high-throughput method based on a two-step PCR amplification protocol, involving DNA extraction using magnetic beads and PCR purification using exonuclease, for 16S rRNA gene amplicon sequencing of plant root microbiota. This method reduces sample handling and captures microbial diversity comparable to that obtained by the standard method. We found that using a buffer with magnetic beads enabled efficient extraction of microbial DNA directly from plant roots. In addition, we 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. Our method offers a simple and high-throughput solution for maintaining the quality of plant root microbial community profiling. ### Competing Interest Statement The authors have declared no competing interest. * Amplicon-seq : amplicon sequencing TE : Tris-EDTA LBB : Lysate Binding Buffer ISO_AMP : Isopropanol method for DNA extraction and AMPure XP beads method for PCR purification ISO_EXO : Isopropanol method for DNA extraction and exonuclease method for PCR purification AMP_AMP : AMPure XP beads method for DNA extraction and AMPure XP beads method for PCR purification AMP_EXO : AMPure XP beads method for DNA extraction and exonuclease method for PCR purification ASV : Amplicon Sequencing Variant PCoA : Principal Coordinate Analysis LEfSe : Linear Discriminant Analysis Effect Size