Assays were designed with Assay Designer 4.0 (Agena) and analysis performed using iPlex Gold Genotyping Reagent Set (Agena, San Diego, CA) according to manufacturer’s instructions. Target loci were amplified within the samples by multiplex PCR in 1X PCR buffer containing 3.5 mM MgCl2, 25 mM dNTPs, 500 nM each of forward and reverse amplification primer within the multiplex pool and 2.5 U HotStar Taq. dNTPs and primers were removed by incubation with 0.5 U shrimp alkaline phosphotase (SAP) at 37 °C for 40 minutes. SAP was inactivated by incubation at 87 °C for 5 minutes. Single base extension was carried out in 0.2X iPLEX buffer plus, 1X termination mix (containing mass modified termination nucleotides), 1X iPLEX enzyme and primers at 0.84 μM, 1.04 μM and 1.25 μM as appropriate to the relative mass of each primer. Following thermocycling, clean resin and water was added to the MassExtend reaction products. Samples were incubated in clean resin at room temperature with mixing for 5 minutes and centrifuged at 3200 × g for 5 minutes.
Samples were then dispensed to a SpectraChip using the MassArray Nanodispenser according to manufacturer’s instructions. Spectra chips were loaded into the MassArray analyzer and spectra acquired for each sample. Genotype calls were made using Typer 4.0 (Agena) by mass identification of extended primer peaks.
Samples were then dispensed to a SpectraChip using the MassArray Nanodispenser according to manufacturer’s instructions. Spectra chips were loaded into the MassArray analyzer and spectra acquired for each sample. Genotype calls were made using Typer 4.0 (Agena) by mass identification of extended primer peaks.