Simple amplicon 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 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.

Stochastic variational variable selection for high-dimensional microbiome data

“…DNA extraction DNA extraction was performed according to our custom protocol using magnetic beads [37]. Briefly, the collected root tissues were ground to a fine powder using a multi-beads shocker (MB2200(S); Yasui Kikai Co. Osaka, Japan).…”

“…16S rRNA gene amplicon sequencing Library preparation using a two-step PCR amplification protocol has been reported in our previous publication [37,38]. Briefly, the V4 region of the bacterial 16S rRNA gene was amplified using 515f and 806rB primers (forward primer: 5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-[3-6-mer Ns] -GTG YCA GCM GCC GCG GTA A -3'; reverse primer: 5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G [3-6-mer Ns] -GGA CTA CNV GGG TWT CTA AT -3') [39,40].…”

Stochastic Variational Variable Selection for High-Dimensional Microbiome Data

“…DNA extraction DNA extraction was performed using our custom protocol with magnetic beads [35]. Briefly, the collected root tissues were ground to a fine powder using a Multi-Beads Shocker (MB2200(S), Yasui Kikai Co. Osaka, Japan).…”

“…16S rRNA gene amplicon sequencing Library preparation using a two-step PCR amplification protocol was reported in our previous publication [35,36]. Briefly, the V4 region of bacterial 16S rRNA gene was amplified with 515f and 806rB primers (forward primer: 5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG-[3-6-mer Ns] -GTG YCA GCM GCC GCG GTA A -3'; reverse primer: 5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G [3-6-mer Ns] -GGA CTA CNV GGG TWT CTA AT -3') [37,38].…”