16S amplicon sequencing of potato tuber
For the preparation of 16S rRNA gene amplicon libraries of seed potato tubers, a first round of PCR was performed using the primer pair 799f (5-AACMGGATTAGATACCCKG-3), which excludes amplification of chloroplast 16S, and 1392r (5-ACGGGCGGTGTGTRC-3), both carrying Illumina adapters, i5 or i7 index, for NexTera barcodes (Bulgarelli et al. 2012; Illumina, 2024; Lundberg et al. 2012).  The reaction conditions were as follows: 25 l reaction volume that contained DNA, 1x KAPA HiFi GC Buffer (5x) with MgCl2, 10 mM dNTPs, 10 M of each primer and 0.5 units of KAPA HiFi DNA polymerase (Kapa Biosystems, Boston, MA, USA). This reaction was carried out with the following settings: 95C for 5 min, 25 cycles at 98C for 20 s, 55C for 15 s, 72C for 1 min and final extension at 72C for 5 min. To visualize amplicons, 25 ul of PCR product were loaded onto a 2% agarose gel (w/v) in 1x TAE, and the bacterial 16S rRNA gene amplicons (approximately 600 bp) were separated from eukaryotic 18S rRNA gene amplicons (1000 bp) by electrophoresis for 50 min at 90 V and excised from the gel using Wizard SV Gel and PCR Clean-up System (Promega, Madison, WI, USA) following manufacturers instructions. For the second round of PCR, 5 l of the eluate was used with the to attach the NexTera i5 forward primer and i7 reverse primer for sample recognition in sequencing. Illumina sequencing (MiSeq 2 x 300 bp) was performed at University of Wisconsin Biotechnology Center, Madison, Wisconsin, USA.