Pop 4

All males were genotyped using RMplex for 30 Y-STRs with increased mutation rates under the conditions as described previously (Ralf et al. 2021 (link)), using the alternative primer for DYS570 and reducing the total reaction volume to 10 µL. Additionally, the males from Cohort 1 were also typed using Yfiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific) following the manufacturer’s protocols, except for a reduced total reaction volume of 10 µL. All amplifications were performed on a Veriti™ 96-Well Fast Thermal Cycler (Thermo Fisher Scientific). Capillary electrophoreses were performed on a 3500 Series Genetic Analyzer (Thermo Fisher Scientific) equipped with a 36 cm 8-capillary array and using POP-4 (Thermo Fisher Scientific). GeneScan™ 600 LIZ™ dye Size Standard v2.0 (Thermo Fisher Scientific) was used as internal size standard. The interpretation of the electropherograms was performed using GeneMapper^® ID-X Software Version 1.5 (Thermo Fisher Scientific).

The novel 43-InDel panel developed by ourselves were multiplex PCR amplification and genotyped on capillary electrophoresis platform. Unless otherwise stated, PCR reaction system contained 12 μl of Master mixture, 0.4 μl of Primer mixture, 1 μl of DNA template and 6.6 μl of nuclease-free water. PCR were performed on the GeneAmp PCR System 9700 Thermal Cycler (Thermo Fisher Scientific, South San Francisco, CA, United States) under the following conditions: 5 min of initial denaturation at 95°C, followed by 35 cycles of 45 s for 94°C, 60 s for 56°C, and 60 s for 72°C, and then the final extension at 60°C for 60 min. ABI 3500xL Genetic Analyzer (Thermo Fisher Scientific, South San Francisco, CA, United States) was used to separate the PCR products with POP-4 (Thermo Fisher Scientific, South San Francisco, CA, United States). Before the electrophoresis, each loading sample containing 8.5 μl of Hi-Di formamide, 1 μl of PCR product (or allelic ladder) and 0.5 μl of Size Standard Org500 were prepared together. The alleles were genotyped using the GeneMapper ID-X software v1.5 (Thermo Fisher Scientific, South San Francisco, CA, United States). Control DNA 9947A and 9948, and deionized water were used as positive DNA and negative control, respectively.