Nextseq 500 550 mid output reagent cartridge v2 300 cycle

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.

To characterize the genome constellation of the novel EHDV, six whole blood samples with the lowest rRT-PCR Ct values were selected for Illumina genome sequencing. Total RNA was treated and purified, as previously described, and then used for the assessment of SISPA [26 (link)]. The PCR products were purified by ExpinTM PCR SV (GeneAll Biotechnology CO., Seoul, Korea) and then quantified using the Qubit DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Library preparation was performed using Illumina^® DNA Prep, (M) Tagmentation (96 Samples, IPB) (Illumina Inc., San Diego, CA, USA) according to the manufacturers’ protocol. Sequencing was performed 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 trimming of raw reads data using FastQC v0.11.5 and Trimmomatic v0.36, respectively, host depletion was performed by Bowtie2 [27 (link)]. The remaining reads were used for the de novo assembly using SPAdes 3.5 [28 (link)] and the consensus sequences of each genome segment were blasted against the NCBI database to identify related EHDV sequences.