16s rRNA sequencing (V3-V4 region) of Maize bacteriobiota
Primers 806R and 515F were elongated with the P1 adapter and with the sequence of linker, barcode and adapter at their 5 end, respectively, to generate amplicon libraries for sequencing on the Ion S5 System (Thermo Fisher Scientific). Order of samples on a PCR plate was randomized to reduce potential batch effects of PCR reactions. Each sample was amplified with a unique barcode in triplicates to reduce PCR bias. Peptide nucleic acid (PNA) PCR clamps, PNAm and PNAp, were added to suppress plant plastid and mitochondrial 16S rRNA gene. Final library was cleaned with Agencourt AMPure XP magnetic beads (Beckman Coulter). Ion 530 chip with DNA library and Ion S5 calibration standard was prepared with Ion OneTouch 2 system using the kit Ion 520 & Ion 530 Kit-OT2 and sequencing was performed on Ion S5 System (Thermo Fisher Scientific).