Next generation sequencing:
The genomic DNA extracted from the enriched virus pellet was tested for
the presence ASFV genome by real time PCR. The quality and quantity of
extracted nucleic acid was measured by nanospectrophotometer (Eppendorf,
Germany).The complete genome sequencing of ASFV in the extracted DNA was
performed by next generation sequencing commercially (Eurofins India
Pvt. Ltd, Bengaluru, India). A sequencing library was constructed for
each sample using the Nextera XT DNA library preparation kit (Illumina,
San Diego, CA, USA) and 2 x 150 NextSeq500paired-end sequencing was
performed on NextSeq500 sequencing system (Illumina, USA).Adapter and
reads of quality less than Phred score 30 were trimmed by Trimmomatic
v0.38 software (Bolger et al., 2014). De-novo assembly of adapter
trimmed raw reads was carried out by using SPAdes 3.15.2 software
(Bankevich et al., 2013).Blastn was performed to identify the closest
available ASFV sequence using scaffold contigs of size less than 200 Kb.
The trimmed reads were mapped against the closely matched ASFV genome
(MN393476.1_Wuhan/2019-1) to generate the whole genome sequence of the
Indian isolates using HISAT-2 software (Kimet al., 2015). Putative genes
were annotated from assembled sequences using Genome Annotation Transfer
Utility Software (Tcherepanov et al., 2006) and the complete genome
sequences were submitted to Genbank with accession numbers OL692743 and
OL692744.