title: Illumina MiSeq paired end sequencing
design: 2-step PCR
library_construction_protocol: PCR was performed in triplicate (single reactions for colony PCR) and the reaction mix contained 5 uL 5X Phusion HF buffer, 0.5 uL dNTPs (10 nM), 1 uL UniTag1-515f primer (5- GAGCCGTAGCCAGTCTGC-GTGYCAGCMGCCGCGGTAA-3) (10 uM), 1 uL UniTag2-806rB primer (5-GCCGTGACCGTGACATCG-GGACTACNVGGGTWTCTAAT-3) (10 uM), 0.25 uL Phusion Hot Start Polymerase (ThermoFisher ScientificTM), and 1-10 uL of extracted DNA  (20 ng/uL). For colony PCR, 1 uL of template was used. Nuclease-free water (Promega) was added to yield a total reaction volume of 25 uL. The PCR program comprised of initial denaturation at 98�C for 30 sec (10 min for colony PCR), followed by 25 cycles of denaturation at 98�C for 45 sec, annealing at 50�C for 30 sec, elongation at 72�C for 10 sec, and a final extension step at 72�C for 7 min. All colony PCR reactions with a negative result were repeated. In the second step PCR a sample-specific barcode was added.