title: CFAV whole genome sequencing
design: Total RNA was extracted from individual mosquitoes using a Qiagen RNeasy mini kit following the DNase I treatment according to the manufacturers protocol. cDNA was synthesized with SuperScript IV VILO Master Mix (Invitrogen) using 15 l of total RNA.  Equal volumes of cDNA were used in the initial single-plex PCR using Q5 high-fidelity DNA polymerase (M0491S, New England Biolab) to generate amplicons. A total of 35 sets of overlapping primers covering the whole genome of CFAV were used for PCR reaction. 10 L of each amplicon generated from the 35 primers per CFAV-infected mosquito were pooled prior to library preparation. Purified pooled amplicon was used for library preparation using Illumina DNA Prep (M) Tagmentation Kit according to manufacturers instructions followed by quality check by Bioanalyzer DNA 1000 kit (Agilent). An equimolar pool of individual libraries was prepared for sequencing, generating paired-end 300 nt reads using the MiSeq V3 600 cycle kits (Illumina, USA).