Twist comprehensive viral research panel

The extracted RNA was converted into double-stranded copy (c)DNA using the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA), NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs), and Random Primer 6 (random hexanucleotides; New England Biolabs). The obtained double-stranded (ds)DNA and DNA samples were subsequently converted to Illumina TruSeq-compatible libraries using Twist Library Preparation Enzymatic Fragmentation Kit (Twist Biosciences, CA, USA) and Twist Universal Adapter System-TruSeq Compatible (Twist Biosciences). We performed a single hybridization capture for each sample and enrichment using the Twist Comprehensive Viral Research Panel (Twist Biosciences). All libraries were converted into libraries for the DNBSEQ using the MGIEasy Universal Library Conversion Kit (App-A). Sequencing was performed using the DNBSEQ-G400RS High-throughput Sequencing Kit (MGI Tech, Tokyo, Japan) in 100-base paired-end mode. Each read was subjected to KRAKEN2 analysis against the PlusPFP database containing archaea, bacteria, viral, plasmid, human, UniVec_core, protozoa, fungi, and plant^{33 (link),34 (link)}.

RNA extracts were converted to cDNA using Protoscript II First Strand cDNA Synthesis Kit (New England Biolabs Inc.), NEBNext Ultra II Non-Directional RNA Second Strand Module (New England Biolabs Inc.), and Random Primer 6 (New England Biolabs Inc.). A total of 25 ng of the cDNA and DNA mix was used for library construction using Twist Library Preparation EF 2.0 Kit and Twist Universal Adaptor System (Twist Biosciences). The libraries were pooled, a maximum of 16 samples per pool, at equal mass to a total 1,500 ng per pool. The Twist Comprehensive Viral Research Panel (Twist Biosciences) was used to hybridize the probes at 70 °C for 16 h. The post-capture pool was further PCR amplified for 12 cycles and final libraries were sequenced on Illumina NovaSeq 6000 SP flow cell, to generate 2×150 bp paired-end reads. Following sequencing, raw data files in binary base call (BCL) format were converted into FASTQs and demultiplexed based on the dual-index barcodes using the Illumina ‘bcl2fastq’ software.