title: 16S (V4) marker-gene survey of integrated fixed-film activated sludge wastewater treatment biofilm: WWTP D, Train 3, Aerobic basin
design: DNA was extracted from approximately 2 mg dry mass biofilm by dissecting carrier media and adding directly to the bead lysing tube of a Mo Bio Powersoil DNA extraction kit, and PCR amplified using the 515FB-806RB primer set (Walters, et al. 2015. mSystems https://doi.org/10.1128/mSystems.00009-15) targeting the V4 variable region of the 16S rRNA gene for bacteria and archaea.  A two-step library preparation was used including the incorporation of illumina overhang adapters onto locus-specific PCR primers, followed by a second round of PCR to add sequencing adapters and sample barcodes using an Illumina Nextera XT Index Kit.