Vahts universal v8 rna seq library prep kit

The RNA library for next-generation sequencing (NGS) was constructed with VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme Biotech, China) by using a total of 10⁹ copies of SARS-CoV-2 RNA; the copy number was predetermined with a COVID-19 Multiplex 1-Step RTqPCR Kit (Topgen Biotech, Taiwan). In brief, the RNA was pretreated with divalent cations at 94°C for 8 min to obtain small RNA pieces with a length of 150–200 nucleotides. Next, the small pieces of RNA were reverse-transcribed following the manufacturer’s procedures to construct a paired-end cDNA library with an average insert size of approximately 150 bp. The cDNAs were ligated to barcode sequencing adapters, and the quality of the cDNA library was analyzed using a MultiNA MCE-202 (Shimadzu, Japan) with a DNA 2500 Kit (Shimadzu, Japan). NGS of the paired-end cDNA library was performed using a NovaSeq 6000 Sequencing System (Illumina, United States) following the manufacturer’s standard protocol. Approximately twenty million paired-end reads (∼150 bp per read) were produced per cDNA library using a paired-end RNA-seq approach (Illumina, United States). The adapter sequences were trimmed from the sequence reads and filtered by using fastp (v 0.19.5) (33 (link)) with a quality value (QV) ≥ 20; the read lengths were filtered by Filter FASTQ (v1.1.5) (34 (link)) with a cut-off ≥145 bp.

RNA-Seq libraries were constructed using the VAHTS Universal V8 RNA-Seq Library Prep Kit (Vazyme). The libraries were sequenced by Beijing Dia-up Biotech Ltd. (Beijing, China) on a Nova-Seq platform in paired-end 150 bp mode. The generated raw reads underwent further processing, involving the removal of reads containing adapters, poly-N, and those with low sequencing quality. The clean paired-end reads were then mapped to the reference sequences (WUXV strain SXWX1813-2 (GenBank accession: L segment, MN454526; M segment, MN454527; S segment, MN454528)) using CLC Genomics Workbench v20.0.4 to generate a consensus sequence for each segment.