SARS-CoV-2 Sequencing

Complementary DNA synthesis was performed using LunaScript RT Super Mix kit (NewEngland Biolabs, MA, USA) and the whole genome of SARS-CoV-2 was obtained by multiplexed PCR amplification using ItokawaK primer set ver_N4. PCR products were purified and subjected to Illumina library construction using Illumina DNA Prep Tagmentation and Nextera DNA CD indexes (Illumina, San Diego, USA). MiniSeq platform (Illumina) was used for sequencing the indexed libraries using MiniSeq High Output Reagent Kit (150-cycles). Amplified PCR product was observed by gel electrophoresis and purified using PCR purification kit (Qiagen). Amplicon was adjusted for libraries using PacBio Barcoded Universal Primers for Multiplexing Amplicons (PacBio, CA, USA) and sequenced using Binding kit 2.2 (PacBio) by Sequel IIe (PacBio).