title: ITS2 marker-gene survey of integrated fixed-film activated sludge wastewater treatment biofilm: WWTP D, Train 3, Anoxic 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 an equimolar mixture of six forward primers (ITS3tagmix) and the ITS4ngs reverse primer (Tedersoo, et al. 2014. Science 346, 10521053. https://doi.org/10.1126/science.1256688) targeting the ITS2 region of the nuclear ribosomal RNA gene for fungi.  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.