Next-generation sequencing
Specimen RNA pre-treatment and Amplicon-based protocol.  The ProtoScript II First Strand cDNA synthesis kit (New England Biolabs, USA) was used to obtain single strand cDNA from SARS-CoV-2-positive specimen RNA extract using random hexamers. For the multiplex PCR approach, the general method of multiplex PCR was applied using Version3 of amplicon sets, as described in (https://artic.network/ncov-2019 ). After 40 rounds of amplification, the PCR products were cleaned-up with AMPure XP magnetic beads (Beckman Coulter, USA) and quantified with the Qubit 2.0 fluorometer (ThermoFisher Scientific, USA).
Illumina iSeq100 system sequencing.  The sequencing-ready library was prepared using the Nextera DNA Flex Prep kit (Illumina, USA). The library was qualified on an Agilent Technologies 2100 Bioanalyser using a high-sensitivity DNA chip following the manufacturer’s instructions. The resulting library was sequenced using the iSeq100™ System (Illumina, USA) , generating 2x150 bp read length data.
Illumina bioinformatics workflow.  To remove low quality reads, trim off low-quality and contaminant residues and filter out duplicated reads, fqCleaner v.0.5.0 was used, with Phred quality score of 28. Filtered reads were mapped against SARS-CoV-2 reference (NC_045512) using Burrows-Wheeler Aligner MEM algorithm (BWA-MEM) (v0.7.7). SAMtools were used to sort BAM files, to generate alignment statistics and to obtain consensus sequence.