2.4 Virion enrichment, viral nucleic acid extraction, random
amplification and NGS sequencing – metagenome cDNA and DNA
Virion enrichment from faecal samples was performed using a previously
published protocol (Chong et al., 2019; Conceicao-Neto et al., 2015).
Modifications were introduced to prevent DNA sequencing bias as follows:
after the enrichment of virions and isolation of nucleic acids (Chong et
al., 2019; Conceicao-Neto et al., 2015), nucleic acid extracts were
divided into two aliquots of equal volume. One aliquot was subjected to
DNase treatment (Invitrogen, Thermo Fisher Scientific, USA) to remove
viral DNA, leaving viral RNA (Brussel et al., 2020), while the second,
untreated aliquot represented the viral DNA. The viral RNA underwent
random amplification using the Whole Transcriptome Amplification Kit
(WTA2, Sigma-Aldrich, Merck, USA) and the maximum PCR cycle number (22
cycles) to produce cDNA (Brussel et al., 2020; Chong et al., 2019;
Conceicao-Neto et al., 2015). The viral DNA also underwent random
amplification using the Whole Genome Amplification Kit (WGA2,
Sigma-Aldrich, Merck, USA) following the manufacturer’s instructions.
The products from both viral RNA (cDNA) and DNA random amplification
were purified using the GenElute PCR Clean-Up Kit (Sigma-Aldrich, Merck,
USA). Libraries for sequencing were created and sequenced as previously
described (Brussel et al., 2020) Briefly, cDNA and DNA libraries were
produced using the Nextera XT library preparation kit (Illumina, USA)
and were sequenced on the NovaSeq6000 platform (Illumina, USA, 150bp
paired end) at the AGRF (Melbourne, Australia). Four of the 24 FPV-cases
faecal samples had cDNA or DNA extracts that failed quality control
measurements after library preparation and were excluded from sequencing
(Supplementary Data S1). Additionally, five of the 24 FPV-cases cDNA
libraries were of low quality and therefore sequenced on the NextSeq500
platform (Illumina, USA, 150bp paired end) at the AGRF (Melbourne,
Australia). In total 21 FPV-cases cDNA, 22 FPV-cases DNA, 36 healthy
control cDNA and 36 healthy control DNA libraries were processed and
sequenced.