16S Amplicon Sequencing of Agricultural Soil: Ametoctradin-treated
Amplification of the V1-V2 and V3-V4 regions of the bacterial 16S rRNA gene were performed on total DNA from soil samples. Master mixes for these reactions used the Qiagen HotStar Hi-Fidelity Polymerase Kit with a forward primer composed of the Roche Titanium Fusion Primer A, a 10 bp Multiplex Identifier (MID) sequence unique to each of the samples. The gene-specific part of the primer used the universal bacterial primer 8F to amplify V1-V2 region or 515 F bacterial primer to amplify V3-V4 region. The reverse primer was composed of the Roche Titanium Primer B, the identical 10 bp MID sequence as the forward primer and the reverse bacterial primer 338R, which spans the V1-V2 hyper variable region of the bacterial 16S or 806R bacterial primer for the amplification of V3-V4 region.