title: Targeted-Capture of Phlebovirus arbiaense: sandfly-pool
design: RNA was extracted using the NucleoSpin RNA Virus Kit (Macherey&Nagel, Dren, Germany) following the manufacturers protocol. Libraries of extracted RNA were prepared using the KAPA RNA HyperPrep Kit for Illumina sequencing (Roche Diagnostics, Mannheim, Germany) following the manufacturers protocol. RNA fragmentation was done at 85C for 6 minutes. After cDNA synthesis, adapter ligation and bead-based cleanup, library amplification was done in 12 amplification cycles due to initial RNA concentrations of <5 ng/l, using the 2x KAPA Hifi HotStart Ready Mix and P5/P7 primer mix provided in the kit. Library concentrations were measured using the Qubit Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and fragment sizes were identified using the 4200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA). After normalization, the libraries were grouped into pools of four to five libraries according to their sequence divergence from the bait target. The library pools were enriched using the newly designed myBaits in-solution hybrid capture target enrichment system for phleboviruses as described above, following the manufacturers protocol [57]. Hybridization temperature was set to 60C, and the same temperature was used for bead washing. Amplification of the captured libraries was done in 20 amplification cycles using the 2x KAPA Hifi HotStart Ready Mix and the P5/P7 primer mix provided with the KAPA RNA HyperPrep Kit for Illumina sequencing (Roche Diagnostics, Mannheim, Germany). After amplification, the enriched libraries were purified using Kapa PureBeads from the kit. Pool concentrations were measured using the Qubit Fluorometer (ThermoFisher Scientific Inc.) and then normalized before paired-end sequencing on MiniSeq and MiSeq platforms, with a read length of 150 and 250-300 bp, respectively (Illumina, San Diego, CA, USA).