SARS-CoV-2 Genome Sequencing Protocol

Extraction of the viral genomic RNA was performed using the automated platform NucliSens EasyMag (bioMérieux, Marcy l’Etoile, France) according to the manufacturer’s instructions. Quantification of total RNA was performed with the Qubit fluorimeter and the compatible Qubit RNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA). Amplicon-based NGS libraries were constructed using the QIAseq SARS-CoV-2 Primer Panel (Qiagen) quality-controlled using the Agilent High Sensitivity DNA assay for Bioanalyzer 2100 (Agilent Technologies), equimolarly mixed, and sequenced in a NextSeq 500 System (Illumina). The generated FASTQ files were aligned to the reference SARS-CoV-2 genome from Wuhan, China (GenBank Accession: NC_045512.2, GISAID Accession: EPI_ISL_402123) with the Bowtie2 aligner (23 (link)) using the very-sensitive preset. Consensus FASTA sequences were exported from the generated BAM files using bcftools (24 (link)). Sequences were further filtered and those that were incomplete (sequence length < 29,000 nt) or had low coverage (entries with >5% Ns) were filtered out of the analysis.