title: 16S: soil metagenomes: historic agriculture
design: Soil genomic DNA was extracted from 0.3g of soil using a DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) according to the manufacturer instructions. Blank samples were introduced in the DNA extraction process to avoid the inclusion of contaminated samples in downstream analyses. To study the taxonomic profile of both bacterial and archaeal communities, the V4 hypervariable region of the 16S rRNA gene was amplified by PCR using the 515-F (GTGCCAGCMGCCGCGGTAA) and 806-R (GGACTACHVGGGTWTCTAAT) primer pair (Martin 2011). These primers included the Illumina adapters, with the reverse primers also having an error-correcting 12-bp barcode unique to each sample, which permits later demultiplexing. Negative controls (tubes that didnt contain the DNA template) were included in each batch of PCR reactions to avoid undetected contamination. All PCR products were cleaned using an UltraClean PCR Clean-Up Kit (MoBio Laboratories, Carlsbad, CA, USA). Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) was used to quantify DNA. Purified PCR products from all samples were pooled together in equimolar concentrations and sequenced on a 2 x 150 bp Illumina MiSeq platform (Illumina, San Diego, CA, USA). Sequencing was conducted at the Microbiome Core, Steele Children Research Center, University of Arizona.