Hi di formamide master mix

Fragment analysis of 27 Y-STR loci (DYS576, DYS389I, DYS635, DYS389II, DYS627, DYS460, DYS458, DYS19, YGATAH4, DYS448, DYS391, DYS456, DYS390, DYS438, DYS392, D YS518, DYS570, DYS437, DYS385, DYS449, DYS393, DYS439, DYS481, DYF387S1, DYS533) was performed using the Yfler Plus Amplification Kit (ThermoFisher Scientific, USA) on a SimpliAmp Thermal Cycler (ThermoFisher Scientific, USA). PCR products were separated by electrophoresis using LIZ600 size standard v2 (ThermoFisher Scientific, USA) in a Hi-Di Formamide Master Mix (ThermoFisher Scientific, USA) on an 8 capillary Applied Biosystems 3500 genetic analyzer (ThermoFisher Scientific, USA). ThermoFisher Scientific’s GeneMapper IDx v.1.6 software was used to examine the electropherograms. Samples with non-standard patterns, off-ladder and microvariant alleles were repeated. Haplotypes were used to determine haplogroups with the Nevgen Y-DNA haplogroup prediction tool [47 ]. Subsequently, genotyping was performed according to 23 Y-SNPs candidate for haplogroups (M174, M35, F1756, M48, F1918, M407, M285, P287, M69, M253, M438, M267, M172, M178, P43, P31, M122, M346, M198, M478, M269, M124, M70) on a QuantStudio5 instrument (ThermoFisher Scientific, USA) using TaqMan assays (ThermoFisher Scientific, USA).

DNA was obtained from 1.2-mm diameter discs punched from blood-spots using the easyPunch STARlet system (Hamilton). A total of 27 Y-STRs were directly amplified with the Yfiler Plus Amplification Kit using 28 PCR cycles according to manufacturer’s recommendations on a Veriti 96-well Thermal Cycler (Thermo Fisher Scientific). The PCR products (1 µl) were separated by capillary electrophoresis in an ABI 3500xl Genetic Analyzer along with LIZ600 size standard v2 in a Hi-Di Formamide master mix (all Thermo Fisher Scientific). GeneMapper ID-X Software v1.4 was used for genotype assignment using the allelic ladders provided with the Yfiler Plus kit following ISFG recommendations (Gusmão et al. 2017 (link)). Samples displaying non-standard patterns, off-ladder and microvariant alleles were repeated. The resulting profiles were submitted to the Y-chromosomal Haplotype Reference Database (YHRD) (Roewer 2017 ) under the following accession numbers: Capital Governorate YA004556, Northern Governorate YA004557, Southern Governorate YA004558 and Muharraq Governorate YA004559.