2.3.1. Library preparation, sequencing, and genome assembly
The Vero cell-adapted virus (passage-5) was harvested followed by freeze‐thawing three times and partially purified as described elsewhere with slight modifications (Babiuk et al., 2009). The DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Germany) as per the manufacturer’s instructions. The quality of DNA was checked on 0.8% agarose gel for the single intact band and DNA concentration was determined using Qubit® 2.0 Fluorometer. The paired-end library was prepared from 200ng DNA using Truseq Nano DNA Library prep kit and sequenced on Illumina (HiSeq) next generation sequencing (NGS) platform using paired-end sequencing reads with 150-nucleotide (nt) length. The filtered reads obtained were mapped to LSDV-NI2490 (GenBank accession number AF325528.1) with 99.99% coverage using the CLC genomics workbench (v. 20.0). After reference mapping, the consensus was called from the resultant BAM file. In addition, the denovo assembly method was also used in which three major contigs were obtained which were aligned with bowite2 on the complete genome of LSDV-NI2490. The consensus sequence of all contigs was then extracted to obtain the draft of the assembled genome of LSDV-WB/IND/19. All variants were manually validated.