Bigdye 3.1 sequencing kit

In preparation of the sequencing, DNA was isolated from serum samples of the PCV3-positive donor pigs using the NucleoSpin Virus Kit (Macherey-Nagel, Berlin, Germany). To maximize the amount of viral DNA, rolling circle amplification (RCA) was performed prior to amplification of the PCR products for sequencing. For RCA the TempliPhi amplification Kit (GE Healthcare, Chalfont St Giles, Buckinghamshire, United Kingdom) was used, according to the manufacturer’s protocol and applying 1 µL of isolated DNA. Afterwards, three partially overlapping amplicons were produced using a high fidelity PfuUltra II Fusion HS DNA polymerase (Agilent Technologies, Santa Clara, California, USA) and three different primer-sets (Sequencing 2, 5 and 3 from Table 1). For amplification, the following thermal profile was used: 95 °C for 2 min and 40 cycles of 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 1 min; and a final extension step of 72 °C for 3 min. PCR products were verified by agarose gel electrophoresis and sequenced using the same sequencing primer sets and the BigDye 3.1 Sequencing Kit (Thermo Fischer Scientific, Waltham, MA, USA).

The mitochondrial cytochrome B (cytB) gene was targeted for amplification as described in Lyman et al. 1999 [61 (link)], to produce a 415 bp fragment with no insertions or deletions. PCR amplification products were visualized in 2% agarose gels, stained with ethidium bromide, and successful amplicons were purified for cycle sequencing using a MultiScreen®HTS Vacuum Manifold (Millipore, USA). DNA cycle sequencing reactions were performed for both strands with a BigDye 3.1 sequencing kit (ThermoFisher), following standard manufacturer protocols, and sequenced in an automated DNA sequencer (ABI 3130, Applied Biosystems). Sequences were manually assembled and aligned with SeqmanPro (DNASTAR, Inc) and BioEdit 7.2.0 [62 ].

Genomic DNA from whole-blood samples was isolated using standard methods. Exon 4 of the APOE gene was amplified by polymerase chain reaction and purified by ExoSap-IT (USB), as previously described [32 (link)]. Amplified fragments were sequenced by the Sanger method using the BigDye 3.1 sequencing kit (Applied Biosystems, Waltham, MA, USA) in an automated ABI 3500xL sequencer (Applied Biosystems). DNA sequences were analyzed using Variant Reporter software (Applied Biosystems).