title: soil microbiome from Organic Farming
design: Microbial community analysis of soil  was determined by DNA extraction from 0.5 g soil from each soil sample, using and Exgene soil SV kit from GeneAll (Songpa-gu, Korea), 550 L SL buffer (extraction buffer), 50 L RH buffer, 300 L PD buffer, 900 L TB buffer (tissue binding), 500 L NW buffer (wash buffer N), and 50 L elution buffer. Centrifugation was carried out between each extraction step on a 5810R Eppendorf centrifuge. After extraction the samples were stored at -20oC until PCR amplification.  The amplification was undertaken by using SimpliAmpTM thermal cycler (Thermo Fisher Scientific, Walham, MA, USA), by mixing 12.5 L HS Taq Mix Red (PCR Biosystems, London, UK), 9.5 L ultrapure water, 1.0 L extracted DNA, 1 l CS1-515F (ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGT) and 1 l CS2-806R (TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCT). The thermal cycling program was set to 95C for 3 min, 24 cycles of 98C for 10 sec, 55C for 10 sec, 72C for 20 sec, and after the cycles, 72C for 1 min. The amplified DNA was stored at 20C until sequencing.  All final PCR products were run on an agarose gel to verify amplification specificity and quality, in parallel with a 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. The data were de-multiplexed using the Illumina base space cloud to generate two FASTQ files for each sample.