Y-SNP typing with NGS

The initial haplogroup determination of 74 Y-SNPs was conducted on the Ion Torrent PGM^TM system (Life Technologies, USA). Experimental steps included library preparation, emulsion PCR and sequencing.

10 ng of genomic DNA was used for library preparation using the Ion AmpliSeq^TM Library Kit 2.0 (Life Technologies, USA) on a ProFlex^TM 96-well PCR System (Thermo Fisher Scientific, USA). The library-PCR system were performed in 20 μl total volume each containing: 4 μl of 5X Ion AmpliSeq^TM HiFi Mix; 10 μl of 2X Ion AmpliSeq^TM Primer Pool; 5 μl of Nuclease-free Water; 1 μl of 10 ng/μl genomic DNA dilution. Thermal cycling consisted of: enzyme activation of 2 min at 99 °C; followed by 19 cycles of 15 sec at 99 °C, 4 min at 60 °C; finally 10 °C for hold. The PCR products were treated with 2 μl FuPa (Life Technologies, USA) to partially digest primer sequences and were continued in the thermal cycler with 10 min at 50 °C; 10 min at 55 °C; 20 min at 60 °C; and finally a 10 °C hold. Considering that multiple libraries would test on a single chip, all libraries were assigned a unique barcode using the Ion Xpress^TM Barcode Adapters (Life Technologies, USA) following the manufacturer recommendations. Then the ligation reaction was performed by adding 4 μl of Switch Solution (Life Technologies, USA); 2 μl of diluted barcode adapter mix prepared before addition; 2 μl of DNA Ligase (Life Technologies, USA) to each digested sample in 30 μl total volume and loaded in the thermal cycler following 30 min at 22 °C; 10 min at 72 °C; and a 10 °C hold. After purifying the unamplified library using Agencourt® AMPure® XP Reagent (Life Technologies, USA), the unamplified Ion AmpliSeq^TM library was eluted in 50 μl of Low TE Buffer (Life Technologies, USA). Library quantification was conducted on the 7500 Real Time PCR System (Life Technologies, USA) with Ion Library TaqMan^TM Quantitation kit (Life Technologies, USA) following 2 min at 95 °C and 40 cycles of 15 sec at 95 °C with the Ion Library Quantitation Kit (Life Technologies, USA). All the libraries were unified into a same concentration and the final library was established by collecting 5 μl dilutions from each dilution library.

The final library was attached to Ion Sphere^TM Particles (ISP). Emulsion PCR (emPCR) was conducted using Ion PGM^TM Hi-Q OT2 Kit on the Ion One Touch^TM 2 System (Life Technologies, USA) following the recommended protocol. The template-positive ISPs were enriched on the Ion One Touch^TM ES complying with the description in the user guide.

Finally, sequencing was performed on the Ion Torrent PGM^TM system (Life Technologies, USA) with the Ion PGM^TM Sequencing 200 kit v2 (Life Technologies) using the Ion 318^TM Chip V2 BC. A final volume of 30 μl was loaded onto the chip, containing sequencing primer, Control Ion Spheres^TM Particles and sequencing polymerase of the Ion PGM^TM sequencing kit (Life Technologies, USA). These runs were set in order to achieve a 500X coverage for each sample. We took Hg19 as the human reference genome. Raw sequencing data were collected as DAT files which were processed on the Torrent_Suite Server (v4.6). VariantCaller (v4.6.0.7) and CoverageAnalysis (v4.6.0.3) plugins. The BAM files were verified using IGV_2.3.59.