Next Generation Sequencing of Hieracium PDS amplicon 1
For the first round PCR, primers were designed to generate two amplicons of 200 bp each. First-round PCR reactions contained 5 M of each primer, 1 x KAPA HiFi Hotstart ReadyMix (Roche) and 10 ng of genomic DNA, in 20 l. The PCR consisted of 35 cycles of 98 C for 20 s, 60 C for 15 s, and 72 C for 15 s. Amplicons were purified using Agencourt AMPure XP (Beckman Coulter). The second round PCR was carried out as the first round, except that Illumina Nextera i5 and i7 indexing primers were used, and 15 cycles were performed. Purified amplicons were pooled together and the library was sequenced using a MiSeq (Illumina) by the AGRF