Powerplex y23 system

Twenty-three Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATAH4, DYS481, DYS533, DYS549, DYS570, DYS576 and DYS643) were amplified in all 619 samples using the PowerPlex Y23 system (PPY23, Promega Corporation, Madison, WI), following the manufacturer’s instructions, separated on an ABI3130xL and analysed using GeneMapper v 4.0 software (both Applied Biosystems).

The proportions of autosomal and Y chromosome STR loci were amplified using Globalfiler Express (Applied Biosystem, USA) and PowerPlex Y23 System (Promega Corporation, Madison USA) on GeneAmp PCR System 9700 Thermal Cycler (Life Technologies, Foster City, CA). Standard reaction mixture and thermal cycling conditions recommended by the manufacturers were followed. Amplified products were injected at 1.2 kV, 24-s and separated by capillary electrophoresis containing POP-4 Polymer (Life Technologies, CA, USA) on the 3500xL Genetic Analyzer (Applied Biosystems, USA). The capillary electrophoresis results were determined by GeneMapper ID-X version 1.4 software (Life Technologies, USA) with 250 relative fluorescence unit (RFU) set as the peak detection threshold for STR allele calls. The threshold was set during RMP DNA Lab internal validation for both kits. These values are considered as both analytical and stochastic threshold to avoid false allele calling.

In total, 23 Y-STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) were typed in all samples using the PowerPlex Y23 system (Promega) according to manufacturer’s instructions. TMRCA was calculated using 21 STRs (omitting the bilocal DYS385ab), 17 STRs (additionally omitting the RM-YSTRs DYS570, DYS576 and the complex STRs DYS389II, DYS448), or 13 STRs (additionally omitting the non-Yfiler loci DYS481, DYS533, DYS549, DYS643). Dating methods were rho, implemented within the program NETWORK 4.612 (Bandelt et al. 1999 (link)), and ASD (Goldstein et al. 1995a (link), 1995b (link)). Further details are given in supplementary material, Supplementary Material online.

In total, 42 Y-STR loci were genotyped for all samples as described in previous studies [53] (link), [54] (link). However, instead of PowerPlex Y the recent developed PowerPlex Y23 System (Promega Corporation, Madison, WI, USA) was used. All 42-Y-STR haplotypes were submitted to Whit Atheys Haplogroup Predictor [55] to obtain probabilities for the inferred haplogroups. Based on these results, the samples were assigned to specific Y-SNP assays according to previously published protocols [53] (link), [56] (link) to confirm the main haplogroup and to assign the sub-haplogroup to the most accurate level of the latest published Y-chromosomal tree [57] (link), [58] (link).
GenAlEx version 6.5 [59] , [60] (link) was used to calculate the differences between all observed haplotypes based on the 42 Y-STR loci. Next, the median joining haplotype network for all the samples belonging to haplogroup I2a* (I-P37.2*) was constructed based on 26 single-copy Y-STRs by NETWORK version 4.5.1.0 [61] (link) (http://www.fluxus-engineering.com) together with all I2 (I-P215) samples already observed in the autochthonous Belgian population by Larmuseau et al. [53] (link), [54] (link), [56] (link). The network analysis used the weighting scheme described by Qamar et al. [62] (link) due to different mutation rates among the markers based on Ballantyne et al. [23] (link).

The samples were genotyped for 23 Y-STR loci using the PowerPlex^® Y23 System (Promega Corporation, Madison, WI, USA). Amplification was performed according to the manufacturer’s recommended protocol, and PCR products were separated and detected on an ABI3500 Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Genotyping was performed using the GeneMapper™ Software v5.0 (Applied Biosystems), and alleles were assigned based on the allelic ladder provided with the kit.

A total of 33 Y-STR markers were amplified by combining 22 Y-STR markers of the PowerPlex^® Y23 System (Promega Corporation) and 11 Y-STR markers of an adapted RM-Yplex assay (13 Y-STRs in total) based on Alghafri et al. [26 (link)]. The modified primer mix can be found in Supplementary File S1. Instead of the DYS518 marker that was used by Alfghafri et al. [26 (link)], the marker DYS464 was added to the multiplex PCR. This multi-copy Y-STR marker with at least four copies on the Y chromosome was selected for its outstanding diversity [27 ,28 (link)]. The primer sequence for DYS464 was taken from Redd et al. [29 (link)] and labelled with ATTO 550. PCR amplification was performed in a final volume of 15 µL per reaction: 7.5 µL Platinum™ Multiplex PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 3.3 µL primer mix according to Supplementary File S1, 3.2 µL PCR grade water, and 1 µL DNA template (1 ng/µL). To confirm a duplication observed within the marker Y-GATA-H4 in one sample we performed an additional PCR using the Yfiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific) with this sample and the sample of the associated son. CE ana­lysis was carried out on the 3500 Genetic Analyzer (Applied Biosystems, Forster City, CA, USA) and data were analysed using the GeneMapper IDX version 1.4 software (Thermo Fisher Scientific).

A set of 23 Y-STRs (short tandem repeats) was analyzed for 13 unrelated male p.R337H carriers and four first-degree male relatives in the main cohort of 38 individuals using the PowerPlex Y23 System (Promega) as described by the manufacturer. Y-STR haplotypes were also determined for Brazilian male individuals harboring the p.R337H allele (validation cohort) that was paternally inherited (n = 41). Alleles were separated and detected on an ABI 3500 sequencer (ThermoFisher Scientific) and sizes were assigned to the different fragments using GeneMapper v 6.0 (ThermoFisher Scientific). The alleles were named according to the number of repeated units, based on the sequenced allelic ladder following International Society for Forensic Genetics recommendations.¹⁵ (link)^,16 The classification of Y chromosome haplogroup was done using the Haplogroup Predictor program FTDNA 2.0 (http://www.hprg.com/hapest5/index.html) and Y chromosome Haplotype Reference Database (https://yhrd.org/search).