16S amplicon Rhizoplane from Plant F
Z. marina rhizosphere, rhizoplane and endosphere, we preformed amplicon sequencing of the full-length region of the 16S rRNA gene. Genomic DNA samples were amplified using Q5 High-Fidelity 2 Master Mix (New England Biolabs) with peptide nucleic acid clamps universal pPNA sequence (5-GGCTCAACCCTGGACAG-3) and universal mPNA clamps (5-GGCA AGTGTTCTTCGGA-3)33 added to a final concentration of 0.5 M to block amplification of host plastid and mitochondrial sequences. The 16S rRNA gene were amplified using GM3F (5-AGAGTTTGATCMTGGC-3) and GM4R (5-TACCTTGTTACGACTT-3)34. PCR amplification was performed under the following cycling conditions: initial denaturation at 98C for 30 s;  35 cycles of 98C for 10 s, 56C for 30 s, and 72C for 1 min; final extension at 72C for 2 min. PCR products were purified using solid-phase reversible immobilization (SPRI) magnetic beads and subsequently prepared for sequencing using the Oxford oore Technology (ONT) sequencing using the SQK-NBD114 Native Barcoding Kit with Short Fragment Buffer to prevent loss of amplicons. Sequencing was performed on the PromethION 2 Solo platform using a FLO-PRO114M flow cell (R10.4.1 chemistry) at SeqCoast Genomics (New Hampshire, USA). Basecalling was performed using Dorado v7.4.1235 in super-accurate basecalling mode and barcode trimming enabled in MinKNOW (v24.06.10).
SUB16055827
Zmarina_16S_Plan_F_5143_006