Cost-effective Detection of SNPs and Structural Variations in Full-length Genes of Wheat and Sunflower Using Multiplex PCR and Rapid Nanopore Kit
Multiplex amplicons were purified using 1 Agencourt AMPure XP Beads (Beck-man Coulter, Pasadena, CA, USA) in accordance with the manufacturers instruc-tions. The purified amplicon concentration and integrity were estimated using a NanoDrop One UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Qubit (Qubit dsDNA BR Assay Kits, Thermo Fisher Scientific, Waltham, MA, USA), respec-tively, and checked by gel electrophoresis. The PCR products were equalized in con-centration for Nanopore sequencing. For Rapid barcoding kit, each sample was mixed with 9l of the multiplexing PCR product (50 ng) and 1 l of one rapid barcode. The mixture was incubated at 30C for 2 min followed by incubation at 80C for 2 min. All barcoded DNA samples were pooled, and 800 l of pooled DNA was mixed with an equal volume of AMPure XP Beads (Beckman Coulter, Pasadena, CA, USA). After 5 min of incubation at room temperature on a Hula mixer, the barcoded DNA was cleaned twice with 80% ethanol and eluted with 15 l of Elution Buffer (EB). Incubation was performed at 37 for 30 min.