1185EMPLEM24-168 16S_V4 amplicon; culture=C; compartment=RHIZO; treatment_soil=SUB; treatment_ferti=ENS; block=3
Bacterial amplicon libraries targeted the V4 region of the 16S rRNA gene using primers 515FB and 806RB from soil DNA extracts. Libraries were prepared using a two-step dual-indexed PCR workflow developed for Illumina sequencing by the Plateforme d'analyses genomiques (IBIS), Universite Laval, Quebec, QC, Canada. The first PCR used locus-specific primers with Illumina-compatible overhang adapters (95C 3 min; 25 cycles of 95C 30 s, 55C 30 s, 72C 30 s; final extension 72C 5 min). Amplicons were purified with AMPure XP beads. Dual indices and Illumina adapters were added in a second PCR using the Nextera XT Index Kit (95C 3 min; 8 cycles of 95C 30 s, 55C 30 s, 72C 30 s; final extension 72C 5 min). Indexed products were purified again with AMPure XP beads, quantified with Qubit, pooled in equimolar amounts, checked on an Agilent Bioanalyzer, and sequenced on an Illumina MiSeq using MiSeq Reagent Kit v3 (600 cycles) to generate 2 x 300 bp paired-end reads.