METHODS
Samples were collected from five affected animals, which had symptoms suggestive of Lumpy Skin Disease during an ongoing outbreak of the disease in Rajasthan. Nasal swabs or skin scabs or both were collected by trained veterinary professionals. DNA extraction from the collected specimens was performed in biosafety level 2 laboratory standards using the automated nucleic acid extraction protocol (Genolution, Korea). Sequencing-ready libraries were constructed by adopting the Illumina tagmentation technique with minor modifications to the original protocol (Bhoyar et al., 2021). After quality checks, the synthesised libraries were sequenced on the NovaSeq 6000 platform (Illumina, USA) with 150X2 base-paired end reads.
The raw FASTQ files for each sample generated by the sequencer were trimmed using Trimmomatic (v0.39) to remove adapters and bases below the quality score of Q30 (Bolger et al., 2014). The quality controlled reads for individual samples were aligned to the Lumpy skin disease virus NI-2490 genome (NC_003027.1) using hisat2 (v2.1) to generate a reference-based mapping for the isolates (Tulman et al., 2001; Kim et al., 2015). Variant calling and consensus sequence generation were performed using VarScan (v2.4.4) and seqtk (v1.3) respectively (Koboldt et al., 2009). Only variants having a variant allele frequency by read count greater than 50% were considered. Variant calls for all individual samples were annotated using a custom database file created using ANNOVAR (Wang et al., 2010). For comparative analysis, 57 LSDV FASTA sequences deposited in GenBank with nucleotide completeness >90% were downloaded (Sayers et al., 2019). Variant calling and annotation for the downloaded sequences were performed using snp-sites and ANNOVAR respectively.
Phylogenetic analysis of the samples was done using the 57 genomes from GenBank and the genome NC_003027.1 as the root. A multiple sequence alignment file was generated using MAFFT (v7.505) and the phylogenetic tree was constructed using iqtree (v2.2.0.3) (Katoh and Toh, 2008), (Nguyen et al., 2015).