Genemapper id x software v1

DNA sample of 1 ng was used to conduct the multiplex PCR of 27 loci according to the following specification. First, we prepared 10 μL PCR cocktail comprising 5 μL STRtyper-27 comp Master Mix, 2.5 μL STRtyper-27 comp Primer Mix, 2.5 μL ddH₂O, and 1 ng DNA sample. Second, PCR was conducted on the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, United States ) under reaction conditions of initial denaturation at 95°C for 5 min; 28 cycles of 94°C for 10s, 61°C for 60s, and 70°C for 30 s; and 60°C for 15 min. Third, we mixed 1 μL amplified product/STRtyper-27 comp Allelic Ladder Mix with 8.75 μL deionized HiDi Formamide and 0.25 μL ILS-500 (HEALTH Gene Technologies) and then denatured the mixture at 95°C for 3 min, followed by chilling at 4°C for 3 min. Finally, the mixture was electrophoresed and separated by the 3500xL Genetic Analyzer (Thermo Fisher Scientific). STR typing of each locus was determined by the GeneMapper® ID-X Software v1.5 (Thermo Fisher Scientific) in comparison with the allelic ladder.

STR loci and the amelogenin sex-determining marker were amplified using the COrDIS plus kit (Gordiz, Moscow, Russia) (detecting amelogenin, D5S818, D21S11, D7S820, CSF1PO, D2S1338, D3S1358, vWA, D8S1179, D16S539, TPOX, TH01, D19S433, D18S51, FGA, and D13S317) according to the manufacturer’s instructions in a GeneAmp^®PCR system 9700 (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoretic analysis was performed using a 3730/3130xl DNA Analyzer (Thermo Fisher Scientific, Waltham, MA, USA). After electrophoresis, the data were analyzed with Gene Mapper^® ID-X Software v1.5 [22 (link)] (Thermo Fisher Scientific, Waltham, MA, USA) to categorize peaks by size in relation to an internal standard allelic ladder.

Amplification was performed in a single multiplex PCR system using 7.5 µL Fast Reaction Mix 2.0, 2.5 µL Primer Mix, 0.5 ng genomic DNA and appropriate amount of water for a final reaction volume of 25 µL. PCR was performed on the GeneAmp 9700 PCR system (Thermo Fisher) with a Gold-plated Silver 96 Well Block in the “Max Mode”, including 3 cycles at 98 °C for 30 s, 64 °C for 55 s and 72 °C for 5 s; 27 cycles at 96 °C for 10 s, 61 °C for 55 s and 72 °C for 5 s; a final extension at 68 °C for 2 min and then held at 60 °C for 2 min.
Amplified products were analyzed by adding 1 µL of each PCR product to 12 µL of a 1:24 mixture of BTO_550 size standard (60, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 425, 450, 475, 500, 525, and 550 bp) (QIAGEN) and Hi-Di formamide (Thermo Fisher) for CE detection. The mixture was then denatured by heating to 95 °C for 3 min and cooling to 4 °C for 3 min. Samples were injected at 1.6 kV for 33 s and electrophoresed at 13 kV for 1 550 s using a run temperature of 60 °C, the BT6 filter set and POP4 polymer (Thermo Fisher) on the 3500xL Genetic Analyzer (Thermo Fisher). Genotyping data were collected and analyzed by the GeneMapper^®ID-X Software v1.5 (Thermo Fisher). Peak detection default threshold of the analysis software, above 100 Relative Fluorescence Units (RFUs), was applied for analysis.