3730xl genetic analyzer

Genomic DNA was extracted from the blood samples using the DP-318 Kit (Tiangen Biotechnology, Beijing, China), and the DNA extraction protocol for the saliva samples was adapted from the high-salt DNA extraction method (Quinque et al. 2006 (link)). The samples were typed as the most recent Y-chromosome phylogenetic tree (ISOGG 2017 ). The selected samples belonged to several subclades of haplogroup Q.
Binary markers were hierarchically genotyped by SNaPshot (ABI SNaPshot Multiplex Kit, Carlsbad, CA, USA) and fluorescent allele-specific PCR. The PCR products were electrophoresed on a 3730xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA). Seventeen Y-chromosomal STRs (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385a, DYS385b, DYS438, DYS439, DYS437, DYS448, DYS456, DYS458, DYS635 and YGATAH4) were amplified using the AmpFlSTR Yfiler PCR amplification kit (Applied Biosystems). The amplified products were separated and identified using a 3730xl Genetic Analyzer (Applied Biosystems) according to the protocol recommended by the manufacturer. The data were analyzed using a Gene-Mapper ID v3.2 (Applied Biosystems). In the analyses, DYS389II was calculated by subtracting the DYS389I allele size.

Bacterial DNA from pure cultures were extracted using the DNeasy PowerLyzer Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. The purity and concentration of the DNA samples were measured with the nanophotometer (Implen, Westlake Village, CA, USA) and the samples were stored at −20 °C. The 16S rRNA gene from the bacteria were amplified with the 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACCTTGTTACGACTT-3′) primers in a reaction volume of 25 μL containing 0.2 mM dNTPS, 10 ng/μL of the forward and reverse primers, 1 × PCR buffer, 2 mM MgSO₄, 1 μL of template DNA, and 2 U of Taq DNA polymerase (GenScript, Piscataway, NJ, USA). The PCR conditions were: 35 cycles of temperature cycling of 95 °C for 45 s, 56 °C for 30 s, and 72 °C for 45 s, and a final extension at 72 °C for 7 min. The resulting amplicons were visualized by electrophoresis using 1% agarose gel stained with Ethidium Bromide. The PCR product was cleaned with ExoSAP-IT (USB, Cleveland, OH, USA) and bidirectional sequencing was performed with the 8F and the 1492R primers using the 3730XL Genetic Analyzer (Life Technologies, Applied Biosystems).