Supplementary information
Typing of Y chromosome SNPs
To classify males into known Y-chromosome haplogroups using the latest Y
Chromosome Consortium nomenclature 1 we typed a range of informative Ychromosome SNPs using the SNaPshot method (Applied Biosystems, Foster City,
California), 65 in total 2-10. 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 26 Y-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, all males 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 and SRY1532 were taken from literature 5. PCR-primers for the other
markers were designed with the Primer 3.0 program v.0.2 12 using 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 primer pair 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 buffer containing 1.5mM MgCl2, 200 µM of each dNTP, 0.1 µM of
each primer, and 2.5 units of AmpliTaq Gold® DNA polymerase. Extra MgCl2 was added
to a total concentration (including PCR buffer) ranging from 3-8 mM to determine the
optimal concentration for every multiplex. Then, primer concentrations 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.
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