title: Amplicon sequencing of Yellow fever virus (YFV) 17D-204 (STAMARIL(R), Sanofi Pasteur)
design: Viral RNAs were extracted from around 1.4x10E9 YFV-17D genomes using Trizol (Ambion, TRIzol  Reagent), were re-suspended in 20 L of RNAse-free water and treated with DNAse with the DNA-free kit (Ambion) before being stored at -80C. Synthesis of cDNAs was carried out with the Maxima H Minus First Strand kit (Thermo Fisher Scientific) from 250 ng of viral RNA. Three fragments of the viral genome were amplified by 25 rounds of PCR using the Phusion High-Fidelity DNA Polymerase kit (NEB). The PCR products were purified with the NucleoSpin Gel kit and PCR Clean Up (Macherey-Nagel), resuspended RNAse-free water and stored at -20C. Libraries were prepared after pooling 400 ng of three overlapping amplicons, which have a size between 3725 and 3891 pb. The PCR products were fragmented randomly with the NEBNext dsDNA fragmentase kit (NEB) and then purified with the AMPure XP Beads kit (Beckman Coulter, Inc.). The Illumina sequencing library were prepared with the NEBNext Ultra DNA Library Prep kit (NEB) by selecting 400 bp fragments. NEBNext Multiplex Oligos for Illumina primers (NEB) were used. Purification was performed with the AMPure XP Beads kit and quantification using the Qubit  dsDNA BR Assay kit (Thermo Fisher Scientific). Samples from the library, diluted to 4 nM, were sequenced on a NextSeq 500 sequencer (Illumina) machine with the NextSeq 500 Mid Output Kit v2 kit (150 cycles) (Illumina), to generate single-end reads of 150 nt.