16S amplicon sequencing of rhizosphere: Nicotiana tabacum infected with Ralstonia solanacearum
The hypervariable regions V4 and V5 of the 16S rRNA were amplified via polymerase chain reaction (PCR) using a modified version of the primer pair 515F (Parada et. al. 2016) and 926R (Quince et. al. 2011) with the Illumina (Illumina, San Diego, CA) P5 and P7 common adapters appended to the 5 end of the locus-specific primer sequence. The 515F/926R primer pair was ordered from IDTTM (Coralville, IA) with the following specifications: 25nmol DNA oligo and a final dNTP and MgCl2 concentration of 0.3mM and 2.5mM, respectively. PCR reactions, product clean-up, ligation of Nextera XT indices, and final library purification and pooling was carried as described in the Illumina 16S Metagenomic Sequencing Library Preparation protocol. Resulting amplicons were sequenced at the North Carolina State Universitys Genomic Sciences Laboratory using Illumina MiSeq v3 chemistry with 300-bp paired-end reads.