16S Coarse mesh control after 4 months
Libraries of 16S rRNA gene amplicons were prepared by duplicate PCR with double-indexed fusion primers [21]. The V4 16S rRNA gene fragment was amplified using modified forward F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 (5-GGACTACHVGGGTWTCTAAT-3) prokaryotic primer. Primers were designed to contain: the Illumina adapters and sequencing primers, a 12 bp barcode sequence, a heterogeneity spacer, and 16S rRNA gene universal primers (Fadrosh et al., 2014). PCRs were performed using OneTaq DNA Polymerase (New England Biolabs, USA). All reactions were run with no-template negative controls. Thermocycling conditions were as follows: initial denaturation at 95 C for 2 min, followed by 30 cycles at 95 C for 30 s, 50 C for 50 s, and 68 C for 90 s with a final elongation at 68 C for 5 min. Amplicons were visualised in a 1.2% tris-acetate agarose gel and DNA bands of approximately 440 bp were gel-purified using the QIAEX II Gel Extraction Kit (Qiagen, Germany). The purified amplicons were then quantified using the Qubit 4.0 Fluorometer, pooled in equimolar amounts and the final pool was run on the Illumina MiSeq platform (Illumina Inc., USA) using 500-cycle v2 chemistry kits.