title: Metagenomics of wastewater: VCS march 2020
design: Wastewater samples were collected and kept at 4C in a sterile container until viral particles from 70 ml of sewage were concentrated by centrifugal ultrafiltration. After a debris removal (15 min, 4500 g), the sample was ultrafiltered with a Centricon Plus-70 device (30KDa) following the manufacturer instructions, obtaining a viral concentrate of 200 l. Previous to library preparation, NA were retrotranscribed to cDNA, tagged and complemented to obtain dsDNA. This viral randomly tagged dsDNA was then amplified (25 cycles) to obtain the sufficient amount of DNA for the preparation of libraries, as previously described [Fernandez-Cassi et al, Sci Total Environ, 2018]. Libraries were prepared in duplicate using KAPA HyperPrep Kit following the instructions provided by the manufacturer (Roche-Kapa Biosystems). Briefly, dsDNA was enzymatically fragmented, indexed with Dual Indexes, amplified and the quality of the fragments and the concentration measured by Qubit 3.0 Fluorometer (Thermo Fisher Scientific). Then, one replicate of the libraries was hybridized with probes designed to capture sequences from vertebrate viral pathogens (VirCapSeq Enrichment Kit, Roche). Two negative controls were also processed, one of them also processed with the enrichment kit. Sequencing was performed on an Illumina MiSeq (2x300bp).