EHDV-8 Genome Sequencing from Deer and Culicoides

To characterize the genome constellation of EHDV-8 in deer and in Culicoides pools, five samples were selected for Illumina sequencing, three from deer and two from Culicoides (Table 1). Total RNA was treated and purified, as previously described, and then used for the assessment of SISPA protocol [24 (link)]. The PCR products were purified using ExpinTM PCR SV (GeneAll Biotechnology CO., Seoul, Korea) and then quantified using Fluorstar Omega (BMG LABTECH, Weston Parkway, Cary, NC, USA). Library preparation was performed using Illumina^® DNA Prep, (M) Tagmentation (96 samples, IPB) (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Sequencing was carried out on the NextSeq500 platform (Illumina Inc., San Diego, CA, USA) using the NextSeq 500/550 Mid Output Reagent Cartridge v2 (300 cycle) (Illumina Inc., San Diego, CA, USA) and standard 150 bp paired-end reads. After quality check and the trimming of raw reads data using FastQC v0.11.5 and Trimmomatic v0.36, respectively, host depletion was performed using Bowtie2 [25 (link)]. Obtained reads for each sample were mapped against the reference EHDV-8/17 TUN2021 sequence (acc. nos. OP381190-99). Consensus sequences were shared with the Genbank database and analyzed along with eight additional whole genome sequences of EHDV-8 obtained from infected blood collected from cattle in Tunisia in 2021 and 2022.