title: Whole shotgun metagenomics sequencing  from Conwy River freshwater
design: Samples of river water (500 L) were removed from the flasks at the 0 h, 141 h, and 506 h and centrifuged (46,839 g, 10 min), removing the supernatant. Whole genome amplification (WGA) was achieved using the REPLI-g UltraFast Mini kit (Qiagen, Germany) as per the manufacturers protocol. Sample viability and contamination were checked using gel electrophoresis (1.8% agarose gel). Library preparation and Illumina MiSeq sequencing were performed at the Centre of Environmental Biotechnology (CEB), Bangor University, UK. After settings optimization, approximately 1 g of DNA was sheared using Bioruptur Pico sonicator system (Diagenode) by sonicating at 4C with four cycles of 15 sec on and 90 sec off, in order to obtain fragments of 600-800 bp size. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) according to the protocol provided by the manufacturer. Briefly, fragmented DNA was end repaired and ligated to the Illumina adaptor. Adaptor-ligated DNA was amplified with index primers provided in NEBNext Multiplex Oligos Set 1 and Set 2 (New England Biolabs). Size-selection and PCR clean up were performed by gel purification using the QIAEX II Gel extraction kit (Qiagen, Germany). Subsequently, the barcoded libraries were quantified using Qubit dsDNA HS Assay Kit with the Qubit 4 Fluorometer. Samples were then pooled in equimolar amounts and the resulting pool was diluted to a final concentration of 4 nM. The final pool was denatured and sequenced using the MiSeq Reagent Kit v3, 600 Cycles Sequencing kit on the MiSeq System (Illumina).