title: 16S wastewater microcosom: BBD1B_not-dosed
design: Amplicon libraries were prepared for sequencing on the Illumina MiSeq platform following the Illumina 16S Metagenomics Sequencing Library Preparation protocol (Part# 15044223 Rev. B). The locus specific primers, S-D-Bact-0341-b-S-17 (5CCT ACG GGN GGC WGC AG) and S-D-Bact-0785-a-A-21 (5- GAC TAC HVG GGT ATC TAA TCC), targeting the V3 and V4 region of the 16S rRNA gene were used(18). Primers were adapted with a forward (5-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG) and reverse (5-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G) Illumina overhang. PCR amplification was performed on gDNA samples normalised to 3-5 ng/L. PCRs were performed in 25 L reactions containing: 5 L of each primer (1 M), 12.5 L 2 x KAPA HiFi HotStart Ready Mix and 2.5 L of Genomic DNA template. For samples with a gDNA concentration of 1-3 ng/L, the PCR reaction mix was modified (30 L) and contained: 5 L of each 1 M primer, 15 L 2 x KAPA HiFi HotStart Ready Mix and 5 L of gDNA template. PCR cycle settings were as follows: initial denaturation 95 C for 3 min, followed by 25 cycles of denaturation 95 C for 30 s, annealing 55 C for 30 s, extension 72 C for 30 s, followed by a final extension 72 C for 10 min. PCR product was cleaned,indexed before normalisation and pooling. Library DNA concentration was quantified prior to normalisation using a Qubit Flurometer (Invitrogen). Pooled library size and quality was validated using a Tapestation 2200 (Agilent, USA). Based on the acquired library amplicon size and concentration, the library was denatured and diluted to 20 pM. The library was then further diluted to 6-10 pM and spiked with 5-10% PhiX control (Illumina, USA) prior to sequencing on the Illumina MiSeq platform using a MiSeq Reagent Kit v3 (2x300 PE).