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.