title: 16SrRNA barcoding soil microbiome
design: All 16S rRNA illumina-tag PCR reactions were performed on the DNA extracts per the Earth Microbiome Projects protocol using 515f GTGCCAGCMGCCGCGGTAA, 806r primers (Walters et al. 2016; Caparoso et al., 2011 ). PCR products were pooled, and gel purified on a 2% agarose gel using the Qiagen Gel Extraction Kit (Qiagen, Germantown, Maryland, USA). Two PCR negatives were also pooled due to yielding visible bands. Before sequencing, the purified pool was quality checked using an Agilent 2100 BioAnalyzer and Agilent DNA High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA). The purified pool was stored at -20C and then sequenced by Wright Labs (Huntingdon, PA, USA) using an Illumina MiSeq v2 chemistry with paired-end 250 base pair reads.