WGS of human respiratory syncytial virus: 2024RSV_86
Total nucleic acids were extracted from nasopharyngwal suspended in 200 L of Viral Transport Medium using the MagNA Pure 96 DNA and Viral NA Small Vol Kit (Roche Diagnostics, Australia) as per the manufacturers protocol and eluted at a volume of 50 L. cDNA was synthesised as per the SuperScript IV First-strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lithuania) protocol. Tiled RSV-A and RSV-B amplicons were generated from cDNA using the ARTIC RSV Primer Scheme protocol, described by Maloney et al 2025, with the Q5 High-Fidelity 2X Mastermix (Biolabs, USA). Cleanup was performed using DNA purification beads (Twist Bioscience, USA) as per the manufacturers protocol. The dsDNA concentration (ng/L) was quantified using the PicoGreen dsDNA Kit (Thermo Fisher Scientific, USA) as per the manufacturers protocol. RSV amplicons were prepared for WGS using the Illumina DNA Library Prep Kit (Illumina, USA) as per the manufacturers protocol, with a DNA input of 100  500 ng per sample. Libraries were sequenced in-house on an Illumina MiSeq (paired-end, 2x150 bp), using the MiSeq Reagent Kit v2 (Illumina, USA).