Next-generation sequencing
For whole-genome sequencing, DNA was extracted from 200 µl of
bone-marrow homogenate using the NucleoMagVet kit (Macherey-Nagel,
Düren, Germany) according to the manufacturers’ protocol. For rapid
generation of sequence information, DNA from the same sample was
sequenced on the Illumina MiSeq platform (San Diego, USA) as described
elsewhere (Wylezich, Papa, Beer, & Hoper, 2018).
The resulting read data was mapped against a custom database including
all ASFV sequences available from International Nucleotide Sequence
Database Collaboration (INSDC) databases as of 15th September 2020 using
Bowtie2 v.2.3.5.1 (Langmead & Salzberg, 2012) with the highest
sensitivity settings followed by de novo assembly of the mapped reads
using SPAdes v.3.13 (Antipov, Korobeynikov, McLean, & Pevzner, 2016)
with default parameters. The resulting whole-genome sequence was aligned
with all other available ASFV whole-genome sequences from
INSDC-databases (as of 21st September 2020) using MAFFT v7.388 (Katoh &
Standley, 2013) in Geneious Prime v.2019.2.3 (Biomatters, Auckland, New
Zealand), annotated on the basis of ASFV Georgia 2007/1 (FR682468.2)
using Geneious, and a phylogenetic tree was constructed with IQTREE v
1.6.5 (Hoang, Chernomor, von Haeseler, Minh, & Vinh, 2018;
Kalyaanamoorthy, Minh, Wong, von Haeseler, & Jermiin, 2017; Nguyen,
Schmidt, von Haeseler, & Minh, 2015). Whole genome sequences from
Poland were kindly provided by N. Mazur and G. Woźniakowski ahead of
publication in INSDC databases and compared to the ASFV Germany 2020/1
whole-genome sequence using MAFFT v.7.388 with default parameters in
Geneious prime.