16S amplicon sequencing of soil near but outside garlic mustard invasion
We targeted the variable region 4 of the 16S ribosomal gene using the improved 16S rRNA Gene primer recommended by the Earth Microbiome Project. The PCR method we used contained two reactions. The first reaction amplified the 16S region and the second reaction attached the larger barcode, spacer, and Illumina attachment. The delay in attaching the larger segment effectively minimizes genomic interference.  We cleaned the final product with the 1.8x AMPure bead mixture. We measured the molarity of our samples using PicoGreen reagents on the Qubit Fluorometer and pooled all samples, including both ITS and 16S, to a final concentration of 4 pM in EDTA. The library was sequenced on a single lane of the MiSeq platform using the MiSeq Reagent Kit v3, with 600 cycles.