Amplicon-Seq of Cebuella pygmaea
Primer pairs for suitable STR candidates were designed using PrimerPlex (v. 2.76, Premier Biosoft, San Francisco, United States). PCRs were carried out with the Multiplex PCR Kit (Qiagen, Hilden, Germany) on a Biometra TGradient Thermocycler (Analytik Jena, Jena, Germany). A temperature gradient was performed and the optimal TA was visually determined after an agarose gel electrophoresis (1.5%, 100V, 60min). The PCR protocol was slightly adapted from the manufacturer's protocol (95C for 15min, (95C for 30sec, TAC for 1,5min, and 72C for 1,5min) 35 cycles, 72C for 10min, 4C for ). Before library preparation, PCR products were purified with AMPure XP (Beckman Coulter, Indianapolis, United States). Libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, United States). Samples were sequenced on an Illumina MiSeq (Illumina, San Diego, United States).