Gridion with the r9.4.1 flow cells

Viral RNA was extracted using the High Pure Viral Nucleic Acid kit (Roche, Germany). For library preparation and NGS performance, two platforms were used: Illumina and Oxford Nanopore Technologies. For Illumina platform, the Nextera DNA Flex kit (Illumina, USA) and the Respiratory Virus Oligo Panel kit (Illumina, USA) were used for library preparation and hybridization. After clean‐up, quality control evaluation was done using Qubit (Thermo Fisher, USA). At last, sequencing was performed utilizing NextSeq 550 machine (Illumina, USA).
For Nanopore platform, PCR tiling of SARS‐CoV‐2 virus with rapid barcoding and Midnight RT‐ PCR Expansion (SQK‐RBK110.96 and EXP‐MRT001) (Version: MRT_9127_v110_revM_14Jul2021) was used. The library was prepared by the Rapid Barcoding Sequencing (SQK‐RBK004) kit (Version: RBK_9054_v2_revX_14Aug2019) from Oxford Nanopore Technologies®, UK. The library concentration was measured using the Qubit HS dsDNA assay kit (Thermo Fisher, USA). Finally, the sequencing was done by the GridION with the R9.4.1 flow cells (Oxford Nanopore Technologies®, UK).

The ARTIC Network SARS-CoV-2 tiled amplicon approach was followed for the amplicon generation and library preparation of the SARS-CoV-2 genome isolated from the patients [24 ]. For the sequencing, we followed the protocol from Oxford Nanopore Technologies® (version PTCN_9103_v109_revG_13Jul2020) with slight modifications. The reverse transcription PCR was done using the NEBNext® ARTIC SARS-CoV-2 Companion kit (Oxford Nanopore Technologies®, UK) from New England Biolabs, USA, with the ARTIC Network SARS-CoV-2 V3 primers. Next, the sequencing library was prepared using the Native Barcoding Expansion 96 (EXP-NBD196) and Ligation Sequencing (SQK-LSK109) kits both from Oxford Nanopore Technologies®, UK. The library concentration was measured using the Qubit HS dsDNA assay kit (Thermo Fisher, USA). The sequencing was carried out on the GridION with the R9.4.1 flow cells (Oxford Nanopore Technologies®, UK). The MinKNOW software (version 21.10.8) was used for operating the device and “high-accuracy” base-calling was selected. The barcode settings were set as follows: barcodes on both ends, a minimum barcoding alignment score of 60 and a mid-read barcoding alignment score of 50. Additionally, the barcodes were trimmed off after base-calling.