Genemapper id x version 1

PCR amplification of the overall loci was carried out in a single PCR system using the reagents and reaction conditions as described in the manufacturer's protocol for the AGCU InDel 50 kit (AGCU, Wuxi, China) and performed in a GeneAmp PCR 9700 Thermal Cycler (Applied Biosystem, Foster City, CA, USA). The 25 µL reaction volume for the multiplex amplification system included 10 µL of reaction mix, 5 µL of primer mix, 1 µL of heat-activated C-Taq DNA polymerase, 1 ng of genomic DNA, and filled to the total volume with sdH 2 O. Thermal cycling conditions were programmed as follows: the initial pre-degeneration step was 95 • C for 2 min, followed by 29 cycles of denaturation at 94 • C for 30 s, annealing at 60 • C for 1 min, extension at 72 • C for 1 min, and a final extension at 60 • C for 30 min. The ABI 3500 xL Genetic Analyzer (Applied Biosystem, Foster City, CA, USA) was used to detect PCR product in a 1 µL volume added to 0.5 µL of AGCU Marker SIZ-500 and 12 µL of deionized formamide. Subsequently, InDel genotyping was performed by GeneMapper ID-X version 1.5 Software (Applied Biosystem, Foster City, CA, USA) with the analytical threshold of peak height at 50 relative fluorescence units (RFU) for allele calling. Positive and negative controls were included to ensure the accuracy of results for InDel genotyping.