Ion Sequencing of Amplified Honey DNA

Sequencing of the obtained amplicons was carried out following the protocol already described [41 (link)] with a few modifications. Briefly, PCR products obtained from each honey DNA sample using csd primers were purified with ExoSAP-IT^® (USB Corporation, Cleveland, OH, USA) and then sequenced using an Ion S5-Ion Chef System (Thermo Fisher Scientific Inc., Waltham, MA, USA). A total of 12 libraries were produced by end-repair and ligation of the DNA fragments with a specific barcode using the Ion Xpress^TM Plus Fragment Library and Ion Xpress™ Barcode Adapter 1–32 kits (Thermo Fisher Scientific Inc.). Each library was quantified with the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific Inc.) by qPCR with the QuantStudio™ 7 Pro Real-Time PCR System (Thermo Fisher Scientific Inc.). Libraries were first clonally amplified by emulsion PCR and sequenced following the manufacturer’s instructions using the Ion 510™ and Ion 520™ and Ion 530™ Kit-Chef after having pooled them for sequencing in one Ion 520 chip (Thermo Fisher Scientific Inc.).