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.