First Identification of Human Adenovirus Subtype 21a in China With MinION and Illumina Sequencers

Ye, Fuqiang; Han, Yifang; Zhu, Juanjuan; Li, Peng; Zhang, Qi; Lin, Yanfeng; Wang, Taiwu; Lv, Heng; Wang, Changjun; Wang, Chunhui; Zhang, Jinhai

To validate and correct the genomic discrepancies between MinION and Illumina sequencing, we designed seven pairs of PCR primers covering these discrepant sites via Primer Premier 5.0 software. The PCR reaction was carried out in a total volume of 50 µL that contained 5 units Taq DNA polymerase (TakaRa, Dalian, China), 0.2 µM of each sense and antisense primer, 2.0 mM MgCl 2 , 200 µM dNTP, and 2 µL of template (2 ng/µL). Thermal cycling conditions involved an initial denaturation step at 95 • C for 5 min, followed by 30 cycles of 95 • C for 40 s, 40 • C for 40 s, and 72 • C for 3 min, and a final extension step at 72 • C for 10 min. Polymerase chain reaction-amplified products were electrophoresed on a 1% agarose-gel, stained with ethidium bromide, and visualized under UV light to compare their lengths with DNA markers. Polymerase chain reaction products approximately covering their target regions were then sent out for Sanger sequencing (Sangon, Shanghai, China). The primers were listed as follows: