Sample prep comparison for wastewater sequencing
This 16S library was generated from the filtrate fraction of a wastewater sample filtered through a 3-m membrane, replicate 3. For taxonomic profiling of the prokaryotic community, the 16S rRNA gene was amplified using primers F341 (5'-CCTACGGGNBGCASCAG-3') and R806 (5'-GGACTACHVGGGTWTCTAAT-3'). Libraries were prepared once per sample with the Quick-16S NGS Library Prep Kit and Quick-16S Primer Set V3-V4 (cat# D6405-2-400; Zymo Research, Irvine, CA) using real-time PCR assays, quantified by quantitative fluorescence PCR, and pooled at equalized final concentrations. Libraries were cleaned with the Select-a-Size DNA Clean & Concentrator (Zymo Research, CA, USA), then quantified with TapeStation (Agilent Technologies, CA, USA) and Qubit (Thermo Fisher Scientific, WA, USA). The ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, CA, USA) served as a positive control for each library prep; blank extraction and blank library-prep controls were included as negative controls. Libraries were sequenced on an Illumina NextSeq 1000 to generate 2  301 bp reads.