2.4 PCV2 Whole-genome sequencing
For PCV2 characterization, the PCV2 PCR positive samples with a ct
< 30 were selected for whole-genome sequencing. The samples
were further selected to cover different profiles of the samples,
including the date of sample collection and the geographical regions of
the farms. PCV2 whole-genome samples were amplified using two pairs of
overlapping primers as previously described (An, Roh, Song, Park, &
Park, 2007; Fenaux, Halbur, Gill, Toth, & Meng, 2000) (Table S1). All
PCR reactions were performed in 50 ul reaction mixtures containing 3 ul
of the extracted DNA, 0.2 uM of each forward and reverse primers
(PCV2-sF1/PCV2_sR1 and PCV2-sF2/PCV2-sR2), and 25 ul of Onetaq® 2x
Master Mix (NEB, MA, USA). The PCR condition consisted of initial
denaturation at 94°C for 30s, followed by 35 cycles of 94°C for 30s,
55°C for 45s, 68°C for 90s, and 68°C for 5min. PCR products were
visualized by 1% Agarose gels containing nucleic acid staining.
Successfully amplified samples were purified using NucleoSpin™ Gel
and PCR Clean-up (MACHEREY-NAGEL, Germany) and submitted for sequencing
by barcode-tagged sequencing platform (Celemic, Seoul, Korea). The
nucleotide sequences were further assembled and validated with SeqMan
and EditSeq software v.5.03 (DNASTAR Inc., Madison, Wisconsin, USA) and
submitted to GenBank.