Variation of the non-recombining part of the human Y-chromosome (NRY) was identified by means of 44 NRY-SNPs in total. Twenty four NRY-SNPs were genotyped in all samples (including: SRY 1532, M91, M168, M145, M174, 12f2, M96, M213, M201, M69, M52, M170, M172, M9, M20, M106, M214, Tat, M175, M45, MEH2, M207, M269, and M124). Aiming to maximise continental differentiation of haplogroup origins we additionally genotyped 20 NRY-SNPs on subsets of samples based on the results from the 24 SNP analyses. M3 was genotyped on samples with the derived allele of MEH2, M242 among samples of haplogroup P(xQ1a, R), and eighteen additional SNPs among samples identified as belonging to haplogroup E (M33, P2, M2, M154, M191, M215, M35, M78, V12, M224, V32, V13, V22, M81, M123, M281, V6, and M75). A single multiplex SNaPshot assay using the principle of primer extension was designed for a core set of 24 NRY-SNPs. Primer sequences for M45, M52, M170, M172, M173, M175, M213, 12f2 and SRY1532 were taken from the literature (Sanchez et al., 2003). For the remaining NRY markers, reference sequences for each locus were obtained from the BLAST human genome database (http://www.ncbi.nlm.nih.gov/blast/) and PCR-primers were designed for fragments ranging from 70 to 225 bp in length using Primer 3 v.0.2 (http://frodo.wi.mit.edu) with default settings. Lengths of designed primers ranged from 19 to 27 nucleotides, primers with five or more bases at the 3′ end complementary to part of another primer in the multiplex were discarded or redesigned to avoid primer-dimer formation. Amplicon sequences were checked with BLAST for sequence homology in the human genome (all primer information can be found in Supporting Table S3). Extension primers were designed using Assay Design Software Version 1.0.6 (Biotage, Uppsala, Sweden). Primers with four or more bases at the 3′ end complementary to part of another primer in the multiplex were discarded or redesigned to avoid non-specific primer-extension. To achieve different fragment length differences the multiplex primer lengths were altered by adding a piece of a “neutral” sequence or a poly-C tail as described by Sanchez et al. (2003). Each primer pair was first validated in a singleplex PCR containing 0.5 ng template DNA from a selection of samples (including a female control), 1 × PCR buffer containing 1.5 mM MgCl2 (Applied Biosystems, Foster City, CA, USA), 100 μM of each dNTP (GE, the Netherlands)) 0.4 μM of each desalted primer (Biolegio Nijmegen, the Netherlands) and 0.6 units of AmpliTaq Gold® DNA polymerase (Applied Biosystems). In the final multiplex PCR, 0.5 ng template DNA was amplified in a 12.5 μl reaction volume containing 1 × PCR buffer, 6.5 mM total MgCl2, 200 μM of each dNTP and 2.5 units of AmpliTaq Gold® DNA polymerase. During multiplex validation primer concentrations were adjusted (0.1–0.4 μM) to achieve optimally balanced signal intensity for all markers. All initial PCRs were performed in a GeneAmp 9700 thermal cycler (Applied Biosystems) with an initial denaturation at 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. To eliminate excess primers and dNTPs, 2 μl ExoSAP-IT® (USB, Affymetrix, Cleveland, USA) was added and incubated at 37°C for 30 min, followed by a final enzyme inactivation at 80°C for 15 min. Extension reactions were performed in a 5 μl reaction volume using 1 μl purified PCR product, 2.5 μl of SNaPshot multiplex Ready Reaction Mix (Applied Biosystems) and 0.4 μM primer (HPLC or PAAGE purified). During multiplex validation primer concentrations were optimized (0.06–0.5 μM) for balanced signal intensity. All reactions were performed using a GeneAmp 9700 thermal cycler with a initial denaturation at 96°C for 2 min, followed by 25 cycles of 10 s at 96°C, 5 s at 50°C and 30 s at 60°C. To eliminate unincorporated ddNTPs 1.25 μl SAP® -reagent (USB) was added incubated at 37°C for 1 hour. SAP was inactivated by incubation at 75°C for 15 min. 2 μl of the SAP-treated extension product was analysed with an ABI3100 Genetic Analyzer using a 36 cm capillary array, polymer POP4 and Genescan 120 LIZ as internal size standard. Data were analyzed using GeneMapper ID v3.2.1 software (Applied Biosystems). After background subtraction and colour separation, peaks were sorted into bins according to sizes by comparison to the internal size standard. An Excel-sheet was used to transfer exported allele tables and for automatic haplogroup assignments.