2.2 SARS-CoV-2 nucleic acid isolation, amplification and sequencing

Naso- and oropharyngeal swabs were collected and placed into a conical tube containing 2 ml of viral transport medium (VTM, Thermo Fisher Scientific, Waltham, MA). Viral DNA and RNA were extracted with the QIAamp MiniElute Virus Spin Kit (QIAGEN, Chatsworth, CA) according to manufacturer's instructions. All cDNAs were synthesized in duplicate using the SuperScript™ III First-Strand Synthesis System (Thermo Fisher Scientific). The SARS-CoV-2 complete genome amplification was based on an openly available protocol developed by the ARTIC network (https://artic.network/ncov-2019, accessed March 26, 2020) using the V.3 multiplex primers scheme and Platinum Taq DNA Polymerase High Fidelity (Thermo Fisher Scientific). Positive PCR products were purified with the ReliaPrep™ DNA Clean-Up and Concentration System (Promega, Madison, WI). Genomic libraries were constructed with the Nextera XT DNA Sample Preparation kit (Illumina Inc., San Diego, CA) according to the manufacturer’s protocol, pooled with 1 per cent denatured PhiX DNA (sequencing control) and sequenced in a MiSeq platform (2 × 251 cycles paired-end run; Illumina). New PCR reactions using combinations of the primers described above were carried out to cover regions with low coverage for each sample. Positive products were purified and sequenced by Sanger using the BigDye Terminator kit (Thermo Fisher Scientific) in an automated 3130XL Genetic Analyzer (Thermo Fisher Scientific). Sequences were edited and assembled with SeqMan v.7.0.0 (DNAStar Inc., Madison, WI).