Viral RNAs were extracted from nasopharyngeal swabs by using QIAamp Viral RNA Mini Kit, followed by purification with Agencourt RNAClean XP beads. Both the concentration and the quality of all isolated RNA samples were measured and checked with the Nanodrop.
Amplicons of whole genome sequences of SARS-CoV-2 were generated with a 50 ng viral RNA template, by using CleanPlex SARS-CoV-2 Research and Surveillance Panel, QIAseq DIRECT SARSCoV-2 Kit and Illumina COVIDSeq Assay following manufacters’ protocol. Libraries were then generated using the Nextera DNA Flex library preparation kit with Illumina index adaptors and sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) with 2 × 150-bp paired-end reads. Raw reads were trimmed for adapters and filtered for quality (Phred score > 28) using Fastp (v0.23.2)52 (link). Reference-based assembly was performed with BWA-mem (v0.7.17)53 (link) aligning against the GenBank reference genome NC_045512.2 (Wuhan, collection date: December 2019).
SNP variants were called with freebayes (v1.3.2)54 and all SNPs having a minimum supporting read frequency of 2% with a depth ≥ 10 were retained.
Synonymous and non-synonymous SNPs characterizing Omicron lineages were defined as high-abundant mutations if characterized by a read frequency ≥ 40%, and low-abundant mutations if characterized by a read frequency between 2 and 40%.
Amplicons of whole genome sequences of SARS-CoV-2 were generated with a 50 ng viral RNA template, by using CleanPlex SARS-CoV-2 Research and Surveillance Panel, QIAseq DIRECT SARSCoV-2 Kit and Illumina COVIDSeq Assay following manufacters’ protocol. Libraries were then generated using the Nextera DNA Flex library preparation kit with Illumina index adaptors and sequenced on a MiSeq instrument (Illumina, San Diego, CA, USA) with 2 × 150-bp paired-end reads. Raw reads were trimmed for adapters and filtered for quality (Phred score > 28) using Fastp (v0.23.2)52 (link). Reference-based assembly was performed with BWA-mem (v0.7.17)53 (link) aligning against the GenBank reference genome NC_045512.2 (Wuhan, collection date: December 2019).
SNP variants were called with freebayes (v1.3.2)54 and all SNPs having a minimum supporting read frequency of 2% with a depth ≥ 10 were retained.
Synonymous and non-synonymous SNPs characterizing Omicron lineages were defined as high-abundant mutations if characterized by a read frequency ≥ 40%, and low-abundant mutations if characterized by a read frequency between 2 and 40%.
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