16S Ribosomal Metagenome sequencing of Beta vulgaris - Leaf
Genomic DNA (gDNA) from sugar beet leaf, root, and soil (rhizosphere) samples collected at early and late infection stages were extracted using the Plant/Fungi DNA Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada) and Soil DNA Isolation Plus Kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturers protocol. Samples were stored at 20C until further use. Primer pairs, 338F_5-ACTCCTACGGGAGGCAGCAG-3 and 806R_5-GGACTACHVGGGTWTCTAAT-3 were used for PCR amplification of the V3V4 hypervariable region of 16S rRNA [Drewes et al. 2017]. The 5 ends of the primers were tagged with sample-specific indexing barcodes and universal adapters. Polymerase chain reaction (PCR) was performed in a total reaction volume of 25 L that contained 25 ng of template DNA per reaction. The PCR conditions were initial denaturation at 98C for 30 s; 32 cycles of denaturation at 98C for 10 s, annealing at 54 C for 30 s, and extension at 72C for 45 s; followed by a final extension step at 72C for 10 min. The PCR products were confirmed through 2% agarose gel electrophoresis, purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA), and quantified using a Qubit (Invitrogen, Waltham, MA, USA) system. The size and quantity of amplicon libraries were measured with an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and a Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA) respectively. The libraries were sequenced on a NovaSeq PE250 platform with paired-end reads (2  250 bp) according to the manufacturers protocols (LC Sciences, Houston, TX, USA).