soil microbiome of organic vineyard plot aggregate size of 0.5 mm
A nested PCR amplification protocol was carried out to target the V1-V2 hypervariable region of bacterial 16s rDNA. Initially, the bacterial 16s rDNA was amplified using the standard primers, 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and 1100R (5`-GGGTTNCGNTCGTTG-3`). This was followed by nested amplification using the same forward primer but barcoded at the 5` end and the 338R reverse primer (5'-TGCTGCCTCCCGTAGGAGT-3).  PCR was carried out on an Applied Biosystems Veriti 96-Well Thermal Cycler. The 25 l reaction mix used for both PCR runs consisted of 16.25 l ultrapure water, 5 l 5X Phusion HF reaction buffer, 0.5 l dNTPs mix, 1 l forward primer, 1 l reverse primer, 0.25 l Phusion HF DNA polymerase, and 1 l DNA for each sample. A 1 l aliquot from the first PCR cycle was used as a template for the nested reaction. The thermal PCR profile was as follows: initial denaturation at 98 C for 3 min, followed by 20 cycles of denaturation at 98 C for 30 sec, primer annealing at 57 C for 30 sec, and elongation at 72 C for 1 min. The final elongation step was executed for 5 min at 72 C.  All final PCR products were run on an agarose gel to verify amplification specificity and quality, in parallel with negative control. The final PCR products were conducted by Hylabs Inc., using the Fluidigm Access Array primers for Illumina to generate libraries compatible for sequencing on the Miseq. Samples were measured for concentration by Qubit and size by Tapestation and then sequenced on the Illumina Miseq using a Miseq V2 sequencing kit (500 cycles) to generate 2x250 paired-end reads.