Samples for DNA extraction were collected from the groundwater and the outflows of each reactor. The community DNA was extracted using a freeze-grinding method (Zhou et al. 1996, "DNA recovery from soils of diverse composition.", AEM 62(2):316-322) . 16S rDNA was first amplified using standard methods (Caproasso et al. 2010, "Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.", doi:10.1073/pnas.1000080107). Reactions were performed in triplicate for each sample. The triplicate products from the first step PCR were combined, purified and aliquoted into three new PCR tubes (15 ? each). A second round of PCR was performed using phasing primers with Illumina adapters, spacers, target primers, and barcodes on the reverse primers using standard methods (Wu et al. 2015 "Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis." , doi:10.1186/s12866-015-0450-4 ) with triplicates for each sample.
SUB1405039
305T02