Capture-hybridisation of RSV: paediatric nasopharyngeal swabs
Total nucleic acid was extracted from 200 L of swab suspension in Viral Transport Medium, using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics, Australia) and eluted at a volume of 50 L, according to manufacturers protocol. Using Superscript IV (Thermo Fisher Scientific) and Klenow Fragment (NEB, MA, USA) with random hexamers, the RNA portion was converted into double stranded cDNA. Hybridization-capture sequencing of viral nucleic acid was then performed following the Twist NGS Workflow (Twist Biosciences, USA). Briefly, sequencing libraries were constructed and target enrichment was performed using the Twist Respiratory Virus Research Panel (103067; Twist Biosciences, San Francisco, CA), which consists of 41,047 probes (120-mers) targeting 29 human respiratory viruses. A total of 308 libraries were combined by equal mass into 14 capture pools (16- to 24-plex). Pools were hybridized to probes overnight for 16hrs following the Twist Target Enrichment Protocol. Post-capture library pools were PCR amplified for 16 cycles, and then bead purified. Sequencing was performed in-house on an Illumina MiSeq (Illumina, USA) with the MiSeq Reagent Kit v2 (300 cycles).