Supplementary information

Typing of Y chromosome SNPs

To classify males into known Y-chromosome haplogroups using the latest Y Chromosome Consortium nomenclature 1we typed a range of informative Y-chromosome SNPs using the SNaPshot method (Applied Biosystems,Foster City, California), 65 in total2-10.We performed a stepwise analysis using three different custom made multiplex primer mixes for different hierarchical levels in the phylogeny (Figure S1):

  1. All males were screened in multiplex for a core set of 26Y-chromosomal SNPs (‘Y-kit-Basis’) in order to allocate them to one of the main Y-haplogroup. All males were allocated to haplogroup E.
  2. Then,allmales were screened in multiplex for a set of 19 SNPs (‘Y-kit-E’) in order to allocate them to the main Y haplogroup E subgroups. All males were allocated to haplogroup E1b1a.
  3. Finally,all males were screened for an additional set of 20 SNPs (‘Y-kit-E1b1a’) in order to further subdivide haplogroup E1b1a.

All relevant information on the markers, primers and their reaction conditions can be found in Table S1. The phylogenetic tree of all markers typed in this study, depicting the stepwise procedure, is shown in Figure S1.

Reaction details

The sequence of each locus was obtained using nucleotide BLAST on the human genome database11. Accession codes can be found in Table S1.Primer sequences for M45, M173, M175, M213 andSRY1532 were taken from literature 5.PCR-primers for the other markers were designed with the Primer 3.0 program v.0.2 12using default settings and a fragment size of 70 to 150 base pairs. Such lengths increase sensitivity and facilitate multiplex PCR and subsequent minisequencing reactions. Primer length ranged from 20 to 29 nucleotides. Primers with five or more bases at the 3’ end complementary to part of another primer were discarded or redesigned to avoid primer-dimer formation. Amplicon sequences were checked with BLAST to avoid sequence homology with other sequences in the genome. Primers for SNP detection by minisequencing were designed with the 3’ end base corresponding to the last base before the SNP-position. Primers were designed using Assay Design Software version 1.0.6 (Biotage, Uppsala, Sweden) and primers with four or more bases at the 3’ end complementary to part of another primer were discarded or redesigned if possible to avoid nonspecific primer-extension. To be able to distinguish markers in multiplex using capillary array electrophoresis, the primers were synthesized with different lengths. This was achieved by adding a piece of a ‘neutral’ sequence or a poly-C tail, as described by Sanchez et al.13.

Each primerpair was tested in a monoplex PCR in a 12.5 µl reaction volume containing 0.5 ng template DNA from a selection of samples (including a female control), 1 x PCR buffer containing 1.5mM MgCl2(Applied Biosystems), 100 µM of each dNTP (GE Healthcare Europe GmbH) 0.4 µM of each primer (desalted, Biolegio bv, Nijmegen, the Netherlands) and 0.6 units of AmpliTaq Gold® DNA polymerase (Applied Biosystems).

In a multiplex PCR, 0.5 ng template DNA was amplified in a 12.5 µl reaction volume containing 1 x PCR buffercontaining 1.5mM MgCl2, 200 µM of each dNTP, 0.1 µM of each primer,and 2.5 units of AmpliTaq Gold® DNA polymerase.ExtraMgCl2 wasadded to a total concentration (including PCR buffer) ranging from 3-8 mM to determine the optimal concentration for every multiplex. Then, primerconcentrations of the PCR and minisequencing were adjusted (0.03-0.4µM) to achieve a more balanced intensity for all markers. All reactions were performed in a GeneAmp 9700 thermal cycler (Applied Biosystems) with the following settings: pre-denaturation 94°C for 10 min followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C, and a final extension for 5 min at 72°C. In order to eliminate the excess of primers and dNTPs, 2 µl ExoSAP-IT® reagent (USB Corporation, Cleveland, Ohio) was added to the PCR product and incubated at 37°C for 30 min. The ExoSAP-IT® reagent was inactivated by incubation at 80°C for 15 min.

Minisequencing reactions were performed in a 5 µl reaction volume using 1 µl purified PCR product, 2.5 µl of SNaPshot reaction (Applied Biosystems) and 0.02-0.25 µM of the primers. Mini-sequencing reactions were performed in a GeneAmp 9700 thermal cycler (Applied Biosystems) using the following program: pre-denaturation at 96°C for 2 min, followed by 25 cycles for 10 s at 96°C, 5 s at 50°C and 30 s at 60°C. After the minisequencing reaction, 1.25 µl SAP® -reagent (USB Corporation) was added and the sample was incubated at 37°C for 1 hour to remove the 5’ phosphoryl groups of the unincorporated fluorescent ddNTPs. SAP® was inactivated by incubation at 75°C for 15 min. 2 µl of the purified minisequencing product was analyzed using an ABI3100 automated DNA sequencer with a 36 cm capillary array containing POP-4 polymer (Applied Biosystems) using default settings. GeneScan-120 LIZ® (Applied Biosystems) was added as internal size standard. The data were analyzed using GeneMapper® Analysis software v. 2.0 (Applied Biosystems). After background subtraction and colour separation, peaks were sorted into bins according to sizes by comparison to the internal size standard.

Literature cited

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11BLAST, < (

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13Sanchez, J. J. et al. Multiplex PCR and minisequencing of SNPs--a model with 35 Y chromosome SNPs. Forensic Sci. Int.137, 74-84 (2003).