title: Microbiological impacts of blending advanced-treated wastewater in pipe loops
design: Library preparation for amplicon sequencing followed the Schloss Lab MiSeq wet-lab protocol for amplification of the V4 region of the 16S rRNA gene and Kantor et al. (2019), with slight modifications. Briefy, the V4 region was amplified using uniquely barcoded 515F and 806R primers with Phusion HotStart II polymerase, HF buffer, and 10 to 25 ng (up to 2 L total) of genomic DNA. Triplicate 25-L reactions were combined. For reactions that failed to amplify, PCR was repeated with 3% dimethyl sulfoxide and 0.2% bovine serum albumin added to the reaction mixture (25 L total volume reactions). At the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, the dual-barcoded libraries were pooled and then sequenced on an Illumina MiSeq, yielding 300 bp paired-end reads.