NextSeq 500 paired end sequencing; Illumina NextSeq 500 sequencing
Nucleic acids extraction was performed using the NucliSens Extraction Reagents (bioMérieux, France) to obtain 100µL of nucleic acids in elution buffer. An additional RNA purification was performed using the RNA Clean & Concentrator TM-5 kit (Zymo Research, Irvine USA) (Strubbia et al., 2019b) to remove endonucleases. Random hexamers and the SuperScript II reverse-transcription kit (Invitrogen) were used to synthetize cDNA from 15µL of purified RNA. The second DNA strand was obtained using the reaction buffer from NEBNext Ultra RNA Library prep (New England Biolabs), followed by a physical fragmentation (Ultrasonicator M220, Covaris). Next steps of the library preparation (end repair, adaptor ligation and cleaning using AMPure beads) were performed using the KAPA Hyper Prep kit (Roche), according to the manufacturer's instructions. At the end of the process, a pre-capture PCR (15 cycles) was performed to reach the minimum recommended DNA concentration (5 ng/µL per library). VirCapSeq hybridization to enrich libraries in viral sequences were performed using the probes and protocol designed and published by Briese and colleagues (Briese et al., 2015) on pools of maximum 10 libraries, using the HyperCap Target Enrichment Kit. Finally, a post-capture PCR was performed to obtain a minimal recommended DNA concentration of 10 ng/µL. PCRs were done using a PTC-100 (MJ Research Ink) or a SimpliAmp (Applied Biosystems) thermocycler.