03.Apo3
For each sample, PCRs were performed in triplicate using the Qiagen Multiplex PCR kit, between 10 and 80 ng template DNA, a primer concentration of 0.5 然, and a final reaction volume of 25 無. PCRs were performed as follows: One cycle at 95 。 for 15 min, 27 cycles each at 95 。 for 30 s, 55 。 for 90 s, and 72 。 for 30 s, followed by a final extension step at 72 。 for 10 min. Triplicate PCRs for each sample were pooled and cleaned with the Agencourt AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA), and subsequently underwent an indexing PCR to add Nextera XT barcoded sequencing adapters (Illumina) according to the manufacturer掇 instructions. Indexed PCR products were cleaned using the Invitrogen SequalPrep normalization plate kit (Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer掇 instructions and eluted at normalized concentrations (~4 nM) in 20 _l elution buffer and pooled in equimolar ratios. Pooled samples were quality checked on the BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) for presence of primer dimers. The library was sequenced at 8 pM with 10% phiX on the Illumina MiSeq, 2*300 bp paired-end version 3 chemistry according to the manufacturer掇 specifications.