Forensic Science International: Genetics 6 (2012) 208–218

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Forensic Science International: Genetics

journal homepage: www.elsevier.com/locate/fsig

A new future of forensic Y-chromosome analysis: Rapidly mutating Y-STRs for

differentiating male relatives and paternal lineages
Kaye N. Ballantyne a,1,2, Victoria Keerl a,1,3, Andreas Wollstein a,b, Ying Choi a, Sofia B. Zuniga c,
Arwin Ralf a, Mark Vermeulen a, Peter de Knijff c, Manfred Kayser a,*
a
Department of Forensic Molecular Biology, Erasmus MC University Medical Center Rotterdam, 3000 CA Rotterdam, The Netherlands
b
Cologne Center for Genomics, University of Cologne, D-50674 Cologne, Germany
c
Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

A R T I C L E I N F O A B S T R A C T

Article history: The panels of 9–17 Y-chromosomal short tandem repeats (Y-STRs) currently used in forensic genetics
Received 23 January 2011 have adequate resolution of different paternal lineages in many human populations, but have lower
Received in revised form 8 April 2011 abilities to separate paternal lineages in populations expressing low Y-chromosome diversity. Moreover,
Accepted 27 April 2011
current Y-STR sets usually fail to differentiate between related males who belong to the same paternal
lineage and, as a consequence, conclusions cannot be drawn on the individual level as is desirable for
Keywords: forensic interpretations. Recently, we identified a new panel of rapidly mutating (RM) Y-STRs, composed
Y-chromosome
of 13 markers with mutation rates above 1  102, whereas most Y-STRs, including all currently used in
Y-STRs
Rapidly mutating Y-STRs (RM Y-STRs)
forensics, have mutation rates in the order of 1  103 or lower. In the present study, we demonstrate in
Lineage differentiation 604 unrelated males sampled from 51 worldwide populations (HGDP-CEPH) that the RM Y-STRs provide
Relative differentiation substantially higher haplotype diversity and haplotype discrimination capacity (with only 3 haplotypes
Haplotype resolution shared between 8 of the 604 worldwide males), than obtained with the largest set of 17 currently used Y-
STRs (Yfiler) in the same samples (33 haplotypes shared between 85 males). Hence, RM Y-STRs yield
high-resolution paternal lineage differentiation and provide a considerable improvement compared to
Yfiler. We also find in this worldwide dataset substantially less genetic population substructure within
and between geographic regions with RM Y-STRs than with Yfiler Y-STRs. Furthermore, with the present
study we provide enhanced data evidence that the RM Y-STR panel is extremely successful in
differentiating between closely and distantly related males. Among 305 male relatives, paternally
connected by 1–20 meiotic transfers in 127 independent pedigrees, we show that 66% were separated by
mutation events with the RM Y-STR panel whereas only 15% were with Yfiler; hence, RM Y-STRs provide
a statistically significant 4.4-fold increase of average male relative differentiation relative to Yfiler. The
RM Y-STR panel is powerful enough to separate closely related males; nearly 50% of the father and sons,
and 60% of brothers could be distinguished with RM Y-STRs, whereas only 7.7% and 8%, respectively, with
Yfiler. Thus, by introducing RM Y-STRs to the forensic genetic community we provide important
solutions to several of the current limitations of Y chromosome analysis in forensic genetics.
ß 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction biology [1–3]. Most commonly, Y-STRs are used to unambiguously

Y-chromosome short tandem repeats or microsatellites (Y-
STRs) have assumed a valuable role within forensic molecular
* Corresponding author at: Erasmus MC University Medical Center Rotterdam,
Department of Forensic Molecular Biology, P.O. Box 2040, 3000 CA Rotterdam, The
Netherlands. Tel.: +31 10 7038073; fax: +31 10 7044575.
E-mail address: m.kayser@erasmusmc.nl (M. Kayser).
1
These authors contributed equally to this work.
2
Current address: Forensic Services Department, Victoria Police, 31 Forensic
Drive, Macleod 3085, Victoria, Australia.
3
Current address: Institute of Anthropology, Department of Biology, Johannes
Gutenberg-University, Mainz, Germany.
resolve the male component of DNA mixtures when a high female
background is present [4,5], or to reconstruct paternal relation-
ships between male individuals [6–8]. For these applications the
currently available Y-STR panels, including several commercial
kits of up to 17 markers such as Yfiler (Applied Biosystems), have
proven to be effective tools with numerous case reports and
hundreds of population data reports to be found in the literature.
Furthermore, large and growing reference databases now exist for
estimating Y-STR haplotype frequencies among globally
dispersed human populations (e.g. http://www.yhrd.org or
http://usystrdatabase.org/).
As valuable as the currently available Y-STR sets have been,
there are some limitations to their use in forensic investigations. In

1872-4973/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigen.2011.04.017
 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218
general, although the diversity of Yfiler haplotypes is good for
outbred populations, usually at 0.995 and higher [8–10], the ability
to discriminate between individuals is considerably less than that
of the autosomal STR sets used in forensics. Although this is mainly
due to the uniparental inheritance and lack of recombination of
male-specific Y-chromosome markers in general, the particular Y-
STRs currently in forensic use also contribute to the incomplete
haplotype resolution. For instance, the 9 Y-STRs defining the
minimal haplotype were ascertained at a time when only very few
male-specific Y-STRs were known [11], and have lower resolution
power than others found in later years. Although in recent years
this core set has been expanded with additional Y-STR markers
[8,12], there remains potential for substantial improvement.
Furthermore, in populations which have encountered size
contraction followed by rapid expansion in their recent history
[13–16], or have specific cultural practices such as patrilocal
residence [17–20], the current Y-STR panels provide limited
resolution due to the overall reduced Y-chromosome diversity in
such inbred populations. A number of studies have demonstrated
that Y-STR haplotype diversity, as measured with current Y-STR
sets, can be increased and male lineage differentiation can be
improved by adding carefully ascertained additional Y-STRs [21–
29]. For instance, in a previous worldwide study we demonstrated
that the male lineage resolution achieved with different sets of
currently applied Y-STRs can be improved with additional simple
single-copy Y-STRs, although complete resolution could still not be
achieved on a global level [29]. However, full haplotype resolution
may be considered as the ultimate goal for Y-STR analysis in
forensic biology.
The most conspicuous weakness however of current Y-
chromosome analysis for forensic purposes is the inability to
exclude close or distant patrilineal relatives of the suspect from
having deposited the biological material instead of the suspect
himself. As such, the currently used Y-STRs are of little help in
those cases where close or distant male relatives may be involved
because of their relatively low mutation rates of only a few
mutations per thousand generations per locus [30]. However, the
success of familial DNA searching using autosomal STR profiles
suggests that relative involvement in crime may not be a rare
occurrence [31]. While familial searching may sometimes be useful
for cases where reliable autosomal STR profiles can be obtained,
when these are unavailable or only available as partial profiles,
which often is the case in materials from sexual assault i.e.
scenarios where Y-STRs are usually employed, the high number of
potential relatives within a database or suspect pool may hinder
the investigation of a case. Notably, the limitation of the currently
applied Y-STRs in their inability to differentiate paternally related
males is evident not only in those particular forensic cases with an
a priori hypothesis of male relative involvement (such as familial
rape), but moreover applies to all forensic cases where current Y-
STR analysis has not yielded an exclusion. This is because the
current sets of Y-STRs do not usually allow a differentiation to be
made between a profile match caused by correct identification of
the suspect and a match caused by different males that are
paternally related with the suspect [32]. Clearly, applying the
benefits of Y-chromosome DNA analysis for male DNA analysis in
male–female mixed stains while keeping the ability to make
conclusions on the individual level would greatly improve forensic
analysis especially in cases of sexual assault.
Recently, we investigated the mutation rates of 186 Y-STRs (for
many markers for the first time), by analysing up to 2000 DNA-
confirmed European father-son pairs for each marker, representing
the largest study of its kind to date [33]. The majority (93%) of the
186 Y-STRs, including all markers currently used in forensic
genetics, displayed mutation rates between 1  104 and 1  103
(95% credible interval 1.4  105 to 1.57  102). However, 13 Y-
209
STRs showed substantially higher than the average mutation rates,
varying between 1.19  102 and 7.73  102 (95% CI 7.05  103
to 9.09  102). We have therefore termed these 13 markers
‘rapidly mutating (RM) Y-STRs’. With this previous study we
additionally provided theoretical as well as preliminary empirical
evidence for the suitability of these RM Y-STRs to differentiate
paternally related males [33].
In the present study, we provide the first empirical evidence of
the ability of the 13 RM Y-STRs for improving paternal lineage
resolution by analysing a worldwide set of 604 individuals from 51
populations (HGDP-CEPH). Furthermore, we provide enhanced
empirical evidence for the ability of these 13 RM Y-STRs to
differentiate closely and distantly related males. To illustrate the
values of the RM Y-STRs, we compare the results with those from
Yfiler, the largest Y-STR set commercially available thus far.
2. Materials and methods
2.1. DNA samples
The power of the RM Y-STRs to differentiate between unrelated
individuals (of separate paternal lineages) was tested with the
Human Genome Diversity Panel (HGDP [34]), provided by the
Centre d’Etude du Polymorphisme Humain (CEPH). The complete
set (including relatives, but excluding duplicates and atypical
samples) comprises 668 males from 59 populations in 8
geographic regions. To measure the lineage differentiation in the
absence of related individuals, a reduced H952 set was used, in
which all known first- and second-degree relatives were removed
[35]. This resulted in a final set of 604 unrelated males from 51
populations in 8 geographic regions: 81 from sub-Saharan Africa (6
populations), 20 from North Africa (1 population), 50 from the
Middle East (3 populations), 162 from South/West Asia (8
populations), 17 from Oceania (2 populations), 83 from Europe
(8 populations), 22 from the Americas (5 populations), and 169
from East Asia (18 populations).
The ability for the RM Y-STRs to differentiate between male
relatives was tested using 305 individuals from 127 separate
pedigrees or small families (156 pairs of relatives, measuring only
independent meioses). Samples originated from the Greifswald,
Kiel and Berlin areas of Germany, the Leuven area of Belgium, and
the Warsaw area of Poland, as well as Canada and Central Germany
as described previously [29,33,36]. In addition, all known first- and
second-degree relatives from the HGDP-CEPH panel that were
excluded from the diversity analysis described above were
included in the analysis of male relatives. The familial relationships
between samples within the HGDP-CEPH samples had previously
been identified from 783 autosomal STRs, with the nature of
familial relationship (i.e., father–son, cousins, uncle–nephew, etc.)
being calculated based on allele sharing [35]. In addition, potential
relationships between HGDP-CEPH individuals displaying match-
ing Y-STR haplotypes were investigated further using nearest
neighbour clustering analysis of the level of allele sharing of 783
autosomal STRs between putative relative pairs in SPSS v15.0 (SPSS
Inc.).
2.2. Multiplex Y-STR genotyping
The 13 RM Y-STRs were amplified in 3 multiplex PCR reactions:
RM1 (amplifying DYF399S1, DYF387S1, DYS570 and DYS576), RM2
(DYS518, DYS526a + b, DYS626 and DYS627), and RM3 (DYF403-
S1a + b, DYF404S1, DYS449, DYS547 and DYS612). The 10 ml PCR
reactions contained 1 AmpliTaq Gold PCR buffer, MgCl2 (RM1
2.27 mM, RM2 1.5 mM, RM3 2.0 mM), 250 mM dNTPs, and
AmpliTaq Gold DNA polymerase (RM1 0.25 U, RM2 0.35 U and
RM3 0.5 U). Concentrations for each PCR primer were: RM1 –
210 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218
DYF387S1, DYS570 and DYS576 0.09 mM, and DYF399S1 0.36 mM;
RM2 – DYS518 0.5 mM, DYS526a + b 0.35 mM, DYS626 0.2 mM and
DYS627 0.15 mM; RM3 – DYF403S1a + b and DYS547 0.6 mM,
DYF404S1 and DYS449 0.1 mM, and DYS612 0.2 mM. Primer
sequences for the RM Y-STR loci can be found elsewhere [33]. To
ensure successful amplification, 1–2 ng of DNA was used for each
multiplex amplification. All thermal cycling was conducted on
GeneAmp 9700 thermal cyclers (Applied Biosystems). RM1 was
amplified with a 70–50 8C touchdown protocol (95 8C for 10 min,
20 cycles of 94 8C for 30 s, 70–1 8C every cycle for 45 s and 72 8C for
1 min, followed by 15 cycles of 94 8C for 30 s, 50 8C for 45 s and
72 8C for 1 min, with 45 min at 60 8C). RM2 and RM3 operated with
65–55 8C touchdown protocols (RM2: 95 8C for 10 min, 10 cycles of
94 8C for 30 s, 65–1 8C every cycle for 30 s and 72 8C for 45 s,
followed by 25 cycles of 94 8C for 30 s, 50 8C for 30 s and 72 8C for
45 s, with 45 min at 60 8C; RM3: 95 8C for 10 min, 10 cycles of 94 8C
for 30 s, 65–1 8C every cycle for 45 s and 72 8C for 1 min, followed
by 25 cycles of 94 8C for 30 s, 50 8C for 45 s and 72 8C for 1 min, with
30 min at 72 8C).
AmpFlSTR Yfiler (Applied Biosystems) PCR amplifications were
performed as recommended by the manufacturer, although using
half volume (12.5 ml) reactions.
2.3. Allelic detection and genotyping
For fragment length analysis, a 1 ml aliquot of PCR product was
added to 9.7 ml HiDi formamide (Applied Biosystems) and 0.3 ml of
ILS-600 for RM1-3 (Promega Corporation) or LIZ-500 (Applied
Biosystems) for Yfiler. A 10 s, 3 kV injection was used on an
ABI3130xl Genetic Analyser (Applied Biosystems) with a 36 cm
capillary and POP-7 polymer. All genotyping was performed with
GeneMapper v3.7 (Applied Biosystems), with a threshold of 50 RFU
for peak calling, and custom kit and bin sets for each RM Y-STR
multiplex.
2.4. Availability of the data
Individual RM Y-STR and Yfiler Y-STR haplotypes as used in this
study are made publicly available via the HGDP-CEPH Genome
Diversity Panel Database (http://www.cephb.fr/en/hgdp/) and are
included in the Human Genome Diversity Genotype Database.
2.5. Statistical analysis
P 2
Haplotype diversities were calculated as ðn=n  1Þð1  fi Þ,
where n is the number of samples, and fi the frequency of the ith
haplotype. Discrimination capacity (number of different haplo-
types observed in a given population) was calculated as the
(number of different haplotypes/total number of haplotypes).
The contribution of each Y-STR to the haplotype diversity or
discrimination capacity was computed using a hill-climbing
approach, where the marker with the highest variability was
selected first, followed by the marker providing the highest
increase in diversity/discrimination of the newly formed
haplotype in the set, etc. To reduce computing time, individuals
that had been distinguished by the chosen marker(s) were
excluded from the next calculation. Both the haplotype
diversities and the hill-climbing selection were implemented
in Matlab v7.7.0.471 R2008b (Mathworks, Natick, MA, USA). FST
values were computed using analysis of molecular variance
(AMOVA) in Arlequin v3.5 [37]. Pairwise allelic comparisons
were performed with an in-house developed macro in Microsoft
Excel 2007, and were calculated as the number of alleles which
differ between all possible pairs of samples. All significance
calculations (ANOVAs, t-tests, etc.) were performed in SPSS
v15.0.
3. Results
3.1. RM Y-STR description
Of the 13 RM Y-STRs introduced recently [33], nine are single
copy and four are multi-copy markers, the latter with between 2
and 4 copies per each marker. Detailed information regarding the
repeat structure, genomic position, observed allele ranges and
mutation rates of the 13 RM Y-STRs can be found in Table 1.
3.2. Discrimination capacity and haplotype diversity
Among the 604 worldwide HGDP-CEPH males investigated, 595
unique haplotypes were seen with the RM Y-STR set (98.3%
discrimination capacity), and 511 with the Yfiler set (90.4%
discrimination capacity), representing a statistically significant 8%
increase in discrimination capacity with RM Y-STRs relative to
Yfiler on a global scale (paired t-test, t = 4.334, p = 0.002). Six of
the eight worldwide regions (sub-Saharan Africa, North Africa,
Middle East, South/West Asia, Oceania and Europe) were fully
resolved with the RM Y-STRs, whereas only North Africa was with
Yfiler (Fig. 1). Large increases in discrimination capacity were seen
with the RM Y-STRs across worldwide geographic regions. The
greatest increase was found in the Middle East, from 84%
discrimination with Yfiler to 100% with RM Y-STRs, other
geographic regions saw increases of 11% (sub-Saharan Africa
and South/West Asia), 8% (East Asia), 6% (Oceania), 5% (South
America) and 1% (Europe) with RM Y-STRs relative to Yfiler.
With the RM Y-STRs only three haplotypes were shared among
eight males in the entire data set of 604 males, compared to 33
Yfiler haplotypes shared among 85 males. The three shared RM Y-
STR haplotypes, which are all shared within populations respec-
tively, concern one pair of individuals from the Kalash population
of South Asia (00313 and 00315) and two sets of triplets, one from
the Yakut population of East Asia (00952, 00961 and 00965), and
the other from the Karitiana population of South America (00998,
01015 and 01019). Notably, the 604 males used already excluded
male relatives of the HGDP-CEPH set, up to a second degree of
relationship as identified with over 700 autosomal STRs in all
populations except for South Americans [35]. The two trios had
previously displayed matching haplotypes with 67 Y-STRs [29],
whereas the pair 00313 and 00315 was not included in our
previous study due to amplification failure at too many markers.
Nearest neighbour analysis of the degree of allele sharing of over
700 autosomal STRs, obtained from the HGDP-CEPH Genotype
Database (v2.0, www.cephb.fr/en/hgdp/), showed that individuals
00998 and 01019 (but not 01015) from Karitiana are likely to share
a second degree relationship, such as grandparent/grandchild, first
cousin or half sibling (data not shown). Excluding these related
individuals from the analysis resulted in 0.954 haplotype
resolution for South America with RM Y-STRs and 0.905 with
Yfiler. Inspection of the HGDP-CEPH autosomal STR dataset
revealed that all other pairs of individuals that share RM Y-STR
haplotypes showed autosomal STR allele sharing levels consistent
with third degree or greater relationships, or being unrelated (data
not shown).
In addition to increased discrimination capacity, the RM Y-STRs
showed a substantial increase in haplotype diversity relative to
Yfiler, as may be expected. Although the difference in haplotype
diversity between the two marker sets was not statistically
significant (paired t-test, t = 2.074, p = 0.072), the pattern
observed for haplotype diversity was similar to that seen with
discrimination capacity between the two sets of markers (Fig. 2).
As for discrimination capacity, the largest increase of haplotype
diversity with the RM Y-STRs compared to Yfiler was observed in
the Middle East (1.55%), followed by Oceania (0.74%), sub-Saharan
Table 1
RM Y-STR marker information.

Marker Repeat type Repeat motif Chromosomal Chromosome Allele range Mutation rate
(variable motif in bold type) location position (size in bp)

K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218

DYF387S1 Tetra, complex, (AAAG)3(GTAG)1(GAAG)4N16 Yq11.2 28.0 Mb 28–38 (241–281) 1.59  102 (1.08  102 to 2.24  102)
multi (2) (GAAG)9 (AAAG)13 Yq11.23 25.9 Mb
DYS399S1 Tetra, complex, (GAAA)3N7–8(GAAA)10–23 Yq11.223 25.1 Mb 10–23 (261–313) 7.73  102 (6.51  102 to 9.09  102)
multi (3) Yq11.23 26.77 Mb
Yq11.2 27.2 Mb
DYS403S1a+b Tetra, complex, A: (TTCT)10–17N2–3(TTCT)3–17/B: Yp11.2 6.2 Mb A: 12–39 A: 3.10  102 (2.30  102 to 4.07  102)
multi (3 + 1) (TTCT)12N2 Yp11.2 9.65 Mb (310–438) B: 1.19  102 (7.05  103 to 1.86  102)
(TTCT)8(TTCC)9(TTCT)14N2(TTCT)3 Yp11.2 9.52 Mb B: 40–59
Yp11.2 (b) 6.3 Mb (b) (414–490)
DYF404S1 Tetra, complex, (TTTC)10–20N42(TTTC)3 Yq11.23 25.95 Mb 10–20 (171–211) 1.25  102 (7.92  103 to 1.84  102)
multi (2) Yq12 28.0 Mb
DYS449 Tetra, complex (TTCT)13–19N22(TTCT)3N12(TTCT)13–19 Yp11.2 8.28 Mb 24–37 (309–361) 1.22  102 (7.54  103 to 1.85  102)
DYS518 Tetra, complex (AAAG)3(GAAG)1(AAAG)14–22(GGAG)1 Yq11.221 17.32 Mb 23–35 (243–291) 1.84  102 (1.25  102 to 2.60  102)
(AAAG)4N6(AAAG)11–19N27(AAGG)4
DYS526a+b Tetra, complex, (CCCT)3N20(CTTT)11–17(CCTT)6–10N113 Yp11.2 3.64 Mb A: 10–17 1.25  102 (7.88  103 to 1.87  102)
multi (1 + 1) (CCTT)10–17 (138–166)
B: 29–42
(345–397)
DYS547 Tetra, complex (CCTT)9–13T(CTTC)4–5N56(TTTC)10–22N10 Yq11.221 18.87 Mb 36–48 (410–458) 2.36  102 (1.70  102 to 3.18  102)
(CCTT)4(TCTC)1(TTTC)9–16N14(TTTC)3
DYS570 Tetra, simple (TTTC)14–24 Yp11.2 6.86 Mb 10–21 (246–286) 1.24  102 (7.52  103 to 1.91  102)
DYS576 Tetra, simple (AAAG)13–22 Yp11.2 7.05 Mb 13–23 (170–210) 1.43  102 (9.41  103 to 2.07  102)
DYS612 Tri, complex (CCT)5(CTT)1(TCT)4(CCT)1(TCT)19–31 Yq11.221 15.75 Mb 14–31 (187–255) 1.45  102 (9.61  103 to 2.09  102)
DYS626 Tetra, complex (GAAA)14–23N24(GAAA)3N6 Yq11.223 24.41 Mb 11–23 (221–269) 1.22  102 (7.70  103 to 1.82  102)
(GAAA)5 (AAA)1
(GAAA)2–3(GAAG)1(GAAA)3
DYS627 Tetra, complex (AGAA)3N16(AGAG)3(AAAG)12–24N81 Yp11.2 8.65 Mb 10–24 (301–372) 1.23  102 (7.80  103 to 1.81  102)
(AAGG)3

211
212 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218

Fig. 1. Discrimination capacity achieved with RM Y-STR and Yfiler Y-STR sets in eight geographic regions, and globally from the HGDP-CEPH. The RM Y-STR set displayed
significantly greater discrimination power in 7 of the 8 regions, and an 8% increase globally, relative to the Yfiler Y-STR set.

Fig. 2. Haplotype diversity obtained with RM Y-STR and Yfiler Y-STR sets in eight geographic regions, and globally from the HGDP-CEPH. The RM Y-STR set displayed
substantially greater haplotype diversity in 7 of the 8 regions, and an increase from 0.994 to 0.9996 globally, relative to the Yfiler Y-STR set.

Africa (0.34%), East Asia (0.25%), South/West Asia (0.2%) and
Europe (0.03%). Only two regions showed no increase in haplotype
diversity with the RM Y-STRs compared to the Yfiler set – North
Africa (both sets with the maximal diversity of 1), and South
America (both at 0.9870). Globally, the haplotype diversity
increased from 0.9994 with Yfiler to 0.99996 with the RM Y-STRs.
Considerably lower numbers of markers were required from the
RM Y-STR set to achieve maximal discrimination capacity and
maximal haplotype diversity within each geographic region
covered by the HGDP-CEPH samples relative to Yfiler (Table 2).
It should however be noted that multicopy Y-STRs were regarded
as single Y-STR markers within this analysis, as the individual loci
cannot be separately genotyped with the currently developed
multiplexes. By doing so, the Yfiler set contained 15 Y-STR markers
(instead of 17 loci, with DYS385a + b and DYS389I + II being
considered as two markers, rather than four), and the RM Y-STR set
13 markers (instead of 20 loci). Despite the RM Y-STR set having
two fewer markers to select from in the hill-climbing approach
used, consistently lower numbers of RM Y-STRs than Yfiler markers
were required for maximal discrimination and diversity estimates,
again illustrating that RM Y-STRs are more informative than Yfiler
Y-STRs. In four geographic regions (sub-Saharan and North Africa,
Europe and East Asia), only two RM Y-STR markers, DYF399S1 and
DYF403S1, were sufficient to resolve all individuals, while on the
global level seven RM Y-STRs were needed to achieve the maximal
discrimination and diversity, compared to 12 with Yfiler for
maximal discrimination and maximal diversity (Table 2). The list of
markers from each set required for maximal discrimination and
diversity within each geographic region and globally can be found
in Table 3. When all RM and Yfiler Y-STRs are combined in a single
set of 28 markers for ascertaining the most informative subsets,
RM Y-STRs consistently show higher power than any of the Yfiler
markers and were selected preferentially (data not shown), as may
be expected based on mutation rate differences between RM Y-
STRs and Yfiler Y-STRs. The superior discrimination power of the
RM Y-STRs resolved 98.7% of all individuals, leaving three Yfiler loci
to discriminate the remaining 0.5% of individuals able to be
discriminated based on all 28 Y-STRs.
3.3. Genetic population substructure
Significant population substructuring is seen between geo-
graphic regions with the Yfiler haplotypes (Table 4). Although the
pairwise FST values between geographic regions (as well as the
regional FST values) were low, every region expressed statistically
significant differences from all others, with the exception of North
Africa (only represented by a single population in our dataset). In
contrast, the RM Y-STRs showed even lower and mostly non-
 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218 213

Table 2
Minimum number of markers required to achieve maximum discrimination capacity and haplotype diversity for RM Y-STRs and Yfiler Y-STRs in the HGDP-CEPH. Increased
diversity estimates were seen in every category with the RM Y-STRs compared to the Yfiler set, including an order of magnitude increase in the global haplotype diversity.
Substantially lower numbers of Y-STRs were required from the RM Y-STR set compared to the Yfiler set to achieve maximal diversity estimates.

Sub-Saharan North Africa Middle East South/West Oceania Europe Native East Asia Global
Africa Asia America

Yfiler
Maximal discrimination capacity 6 7 7 9 3 9 5 8 13
Maximal haplotype diversity 6 7 7 9 3 10 5 8 12
RM Y-STRs
Maximal discrimination capacity 2 2 4 3 2 2 2 3 7
Maximal haplotype diversity 2 2 5 5 2 2 2 4 7

significant pairwise FST values, indicating that the level of
population substructure between regions detected with RM Y-
STRs is considerably lower than that detected with Yfiler Y-STRs.
Only South Americans compared to sub-Saharan Africans, Middle
Easterns, South/West Asians, and Europeans, as well as East Asians
compared to South/West Asians and South Americans showed
statistically significant differences with RM Y-STRs (Table 4).
Global FST estimated from RM Y-STRs was extremely low at 0.0009,
4 times lower than obtained Yfiler at 0.00375. AMOVA revealed
that with RM Y-STRs only 0.09% of the total variation observed was
expressed among populations, with 99.91% within populations,
compared to 0.37% among and 99.63% within populations for
Yfiler.
3.4. Allelic comparisons
Pairwise comparisons between individuals drawn from the
HGDP-CEPH panel show that the RM Y-STRs displayed higher
numbers of allelic differences than Yfiler, both in absolute and in
relative terms (Fig. 3). The 222,000 allelic comparisons performed
gave an average number of allelic differences between pairs of
11.81 with Yfiler (69.4% of possible alleles), and 17.99 with the RM

Table 3
Most informative markers for maximising haplotype diversity and discrimination capacity for RM Y-STRs and Yfiler Y-STRs in the HGDP-CEPH. Markers are listed in
descending order, with the first listed contributing the most to the parameter being measured. Any Y-STR not listed did not provide any additional information to those
already listed.

Sub-Saharan Africa North Africa Middle East South/West Asia Oceania Europe Native America East Asia Global

Yfiler
Haplotype DYS385 DYS385 DYS385 DYS385 DYS385 DYS385 DYS385 DYS385 DYS385
diversity DYS389 DYS439 DYS389 DYS389 DYS389 DYS389 DYS389 DYS389 DYS389
DYS458 DYS390 (DYS635 DYS635 DYS635 DYS635 (DYS458 DYS458 DYS458
DYS19 DYS456 or DYS393) DYS390 DYS390 or DYS448) DYS392 (DYS635
(DYS393 (DYS458 (DYS448 DYS458 DYS458 (DYS392, DYS635 or DYS390)
or DYS439) or DYS635) or DYS390) DYS439 DYS448 DYS438, DYS19 DYS19
DYS635 DYS389 DYS439 (DYS19, (DYS456 DYS390 (DYS448, DYS448
DYS448 DYS19 DYS393, or DYS438) or DYS439) DYS439, DYS439
DYS438, (DYS439 DYS456 DYS393 DYS393
or Y-GATA-H4) or DYS19) or DYS390) DYS392
(DYS448, (DYS437 or DYS456 (DYS438
DYS392, Y-GATA-H4) or YGATAH4)
or DYS437) DYS392 DYS456
(DYS456 (DYS437
or DYS391) or DYS391)
Discrimination DYS385 DYS458 DYS385 DYS385 DYS385 DYS385 DYS385 DYS385 DYS385
capacity DYS389 DYS635 DYS458 DYS389 DYS389 DYS389 DYS389 DYS389 DYS389
DYS19 DYS385 DYS448 DYS458 DYS635 DYS448 DYS391 DYS458 DYS458
DYS393 DYS390 DYS389 DYS390 DYS635 DYS438 DYS456 DYS439
DYS456 DYS456 DYS19 DYS439 DYS390 DYS437 DYS439 DYS19
DYS635 DYS389 DYS437 DYS635 DYS391 DYS448 DYS448
DYS19 DYS635 DYS19 DYS437 DYS392 DYS390
DYS456 DYS456 DYS635
DYS448 DYS19 DYS456
DYS392
DYS438
DYS393
RM Y-STRs
Haplotype DYF403S1 DYF399S1 DYF399S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1
diversity DYF399S1 DYF403S1 DYF403S1 DYF399S1 DYS612 DYF399S1 DYS547 DYF399S1 DYF399S1
DYS526 DYS526 DYS526 DYS526
DYS627 DYS627 DYF387S1 DYF387S1
DYS518 DYS612 DYS518
DYS547
DYS612
Discrimination DYF403S1 DYF399S1 DYF399S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1 DYF403S1
capacity DYF399S1 DYF403S1 DYF403S1 DYF399S1 DYS612 DYF399S1 DYS547 DYF399S1 DYF399S1
DYS526 DYS612 DYS612
DYS518 DYS547
DYS526
DYS518
DYS627
214 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218

Table 4
Pairwise FST values of regional groups achieved with RM Y-STR (below the diagonal), and Yfiler Y-STR sets (above the diagonal) from the HGDP-CEPH. Significant comparisons
(p < 0.05) are shown in italics.

Sub-Saharan North Middle South/West Oceania Europe Native East Asia Population
Africa Africa East Asia America specific FST

Sub-Saharan Africa 1.1  103 8.8  103 2.0  103 4.6  103 1.2  103 7.4  103 2.5  103 3.7  103
North Africa 1.6  104 8.0  103 9.6  104 3.6  103 1.5  104 6.5  103 1.4  103 4.2  103
Middle East 1.6  104 <1.0  106 8.6  103 1.2  102 7.8  103 1.4  102 9.0  103 4.0  103
South/West Asia 2.3  104 0.8  105 0.8  105 4.4  103 1.1  103 7.2  103 2.3  103 3.6  103
Oceania 1.6  104 <1.0  106 <1.0  106 0.8  105 3.7  103 1.0  102 4.9  103 4.5  103
Europe 1.5  104 <1.0  106 <1.0  106 0.8  105 <1.0  106 6.4  103 1.5  103 3.7  103
Native America 8.5  103 8.7  103 8.4  103 8.3  103 8.8  103 8.3  103 7.6  103 4.4  103
East Asia 3.3  104 1.8  104 1.8  104 1.9  104 1.8  104 1.8  104 8.4  103 3.6  103
Population specific FST 8.8  104 1.4  103 9.9  104 7.9  104 1.6  103 8.7  104 1.6  103 7.9  104

Fig. 3. Distribution of the number of allelic differences between pairs of individuals in the HGDP-CEPH for RM Y-STR and Yfiler Y-STR sets.

Y-STRs (85.7%). The two sets have significantly different distribu-
tions (KS Z = 1.667, p = 0.008), with the RM Y-STR significantly
more likely to generate greater allelic differences than the Yfiler set
(Moses Extreme Reaction, N = 22, p = 0.003). This also translates to
a higher relative number of locus differences with the RM Y-STRs
with 11.66 of the 13 loci (89.7%) differing. As each allele within the
Yfiler set is regarded as a separate locus, the number of locus
differences remains the same at 11.81.
3.5. Differentiating male relatives
The high mutation rates of the RM Y-STRs also prompted the
hypothesis that even closely related males may be differentiable
with this set, given that a single mutation at a single locus may be
sufficient to separate related individuals. We had previously found
an average differentiation rate between individuals related by 1–
20 generations of 70%. Combining our previous with the new data
resulted in a total of 156 pairs of male relatives of various bio-
geographic ancestry, related by 1–20 generations, genotyped with
the RM Y-STRs, and the results were compared with those from the
commercial Yfiler set (Fig. 4).
Results from the Yfiler set can be considered as the maximum of
male relative differentiation achievable with currently available Y-
chromosome analysis, with only 15% of male relatives differenti-
ated across all levels of relationship (1–20 generations) in our
sample set. However, in forensic casework, the closer relationships
such as father–son, brothers and cousins are more often considered
relevant (and known to investigators), than distant relationships of
10+ generations. For these close relationships, Yfiler performed
especially poorly – fathers and sons were differentiated in only
7.7% of cases, brothers in 8%, and cousins (4 meioses separation) in
25% of cases. By contrast, the RM Y-STR set was able to differentiate
66% of all male relatives in the dataset, 4.4 times more than Yfiler
and highly statistically significant (F = 27.31, p = 0.00002). Notably,
this included 48.7% of fathers and sons (more than 6 times more
than Yfiler, F = 14.53, p = 0.0003), 60% of brothers (7.5 times more
than Yfiler, F = 30.41, p = 0.0000003), and 75% of cousins (3 times
more than Yfiler, F = 16.03, p = 0.0003). All pairs that were
separated by 9 or more meioses were distinguished by at least 1
mutation with RM Y-STRs, whereas for Yfiler only 47% of these
pairs were distinguished. There was a clear trend for increasing
differentiation with increasing numbers of meioses (Fig. 4),
although the relationship is somewhat masked by differing sample
numbers between the categories, which shall be investigated
further.
As could be expected, the number of mutations between each
pair increased in both Y-STR sets as the number of meioses
increased (Fig. 5), although this effect was stronger for the RM Y-
STRs than Yfiler (RM Y-STRs partial r2 = 0.544, p = 2.51  1013,
Yfiler partial r2 = 0.264, 0.0009). Given the greater number of
mutations observed in the RM Y-STR set, there were also higher
average numbers of mutations observed with each additional
meiosis between pairs. With 1 meiotic separation, pairs displayed
an average of 0.1 mutations with Yfiler, but 0.65 with RM Y-STRs.
This increased to 0.7 and 2.0 respectively at 10 meioses, and up to 4
mutations with the RM Y-STRs with 20 meioses.
 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218 215

Fig. 4. Male relative differentiation using RM Y-STR and Yfiler Y-STR sets. Genotyping of 156 pairs of relatives from 127 male pedigrees of various bio-geographic ancestries
with both Y-STR sets showed that the RM Y-STR set gives significantly greater discrimination between relatives than the Yfiler Y-STR set. Error bars represent 95% binomial
confidence intervals.

Fig. 5. Correlation between the number of mutations observed with RM Y-STR and Yfiler Y-STR sets and the number of meioses separating pairs of relatives. The colour
intensity of each symbol on the graph corresponds to the number of observations at each point, with darker colours representing higher frequencies. Linear regression was
used to find the line of best fit for each data set.

4. Discussion showing high diversity measures within specific population(s)

For use within forensic casework, this novel RM Y-STR panel
has proven abilities to substantially increase the differentiation of
both related and unrelated males, due to the elevated mutation
rates of the markers included. Although alternative panels have
been proposed before to replace or complement the existing set of
17 Y-STRs for increased paternal lineage differentiation [21–29],
none have displayed the ability to increase discrimination
capacity to such a great extent with so few markers as we show
here with the RM Y-STR set. Also, no previous Y-STR set was able to
differentiate between male relatives, as we demonstrate here is
possible with RM Y-STR set. The greater effectiveness of the RM Y-
STR set compared to others such as Yfiler to differentiate male
individuals in general is due to the way the markers were
ascertained. Most previous panels have been comprised of Y-STRs
[23,24,26,27]. However, the diversity displayed by a given marker
or haplotype is strongly dependent on the specific demographic
histories of a population. As such, Y-STRs ascertained within a
particular population may perform exceedingly well within this
source population, but be of limited use when applied to other
population samples not sharing the particular demographic
history features as in the source population. This has already
been noted with the Yfiler markers, which have extremely high
diversities in some populations, and very low diversities in others
[13–20]. Thus, a more universal approach to the ascertainment of
effective Y-STRs is needed. In the present study we chose an
alternative approach of considering mutation rates, which are
determined by the molecular features of the repeat unit in
question [33], instead of diversity measures previously applied as
ascertainment criteria.
216 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218
The RM Y-STRs presented here show substantially higher
worldwide discrimination capacities and haplotype diversities
than the current panel of 17 Yfiler Y-STRs. This is caused by the
higher mutation rate of all of the RM Y-STRs relative to the Yfiler Y-
STRs, generating greater allele ranges for each RM Y-STR marker,
which in turn gives the potential for a greater number of RM Y-STR
haplotypes. In addition, the higher rate of mutation erases any
signal of population history or founder effects with RM Y-STRs (at
least in the global dataset tested here), thus resulting in a more
homogenous distribution of haplotypes across worldwide popula-
tions than was obtained with Yfiler Y-STRs. The lower number of
significant pairwise FST comparisons with the RM Y-STRs relative
to Yfiler, along with the lower within-population variation, also
supports the hypothesis that RM Y-STRs detect considerably less
genetic population substructure than Yfiler Y-STRs. Because of the
very low levels of population substructure detected with RM Y-
STRs in our global data it not only appears that correction for
population substructure in estimating match probabilities may not
be necessary for RM Y-STRs, but also that there may be a less
stringent need to know the source population of the sample donor
a priori to accurately estimate haplotype frequencies. Instead, it
may be possible to use, once established, a truly global RM Y-STR
haplotype database for reliable frequency estimation. However, it
would be advisable to test substantially greater numbers of
samples, from many more global populations to ensure this is the
case before implementation.
Although the increase in discrimination capacity of unrelated
males as achieved by the RM Y-STRs is undoubtedly very useful,
especially in populations with overall reduced Y-chromosome
diversity caused by demographic or cultural effects, it is the strong
ability of the RM Y-STRs to differentiate between male relatives,
close as well as distant, which has the potential to greatly expand
the use of Y-chromosome analysis within forensic biology. It has
long been assumed that the Y-chromosome is identical between
parent and offspring, except in very rare cases of mutation. A recent
study estimating the base substitution mutation rate calculated
that 1 SNP would mutate between a father and his son across the
24 Mb Y-specific euchromatin DNA investigated [38]. While this
was heralded as a method to distinguish male relatives using the Y-
chromosome for forensic science, at present the applied method of
next-generation DNA sequencing does not fulfil the data quality
needs demanded in forensic analysis [39,40]. However, the new
RM Y-STRs provide a high probability of differentiating between
male relatives, close and distant ones, in a manner that is
immediately applicable to forensic samples. Although our RM Y-
STR set does not enable male relative differentiation in 100% of
cases investigated here, the data we present provide strong
evidence that the RM Y-STR set will be useful in many cases to
identify if male relatives were involved or not.
It should be noted that multiple Y-STR mutations were
observed between pairs of male relatives in the present dataset,
with both the Yfiler and the RM Y-STR sets. Between fathers and
sons, the maximum number observed were 2 and 3 mutations
within each set respectively. This increased in the RM Y-STR set
with each additional meiotic transfer to a maximum of 6 mutations
observed in male relatives separated by 11 meiotic transfers,
highlighting how the likelihood of observing multiple mutations
between pairs of relatives is simply a probability function
dependant on the combined mutation rate of the set of markers
tested. The higher mutation rate of the RM Y-STR set results in
more mutations being observed, compared to the lower rate with
the Yfiler set. However, even with the lower mutation rate at the 17
Yfiler Y-STRs, there remains a distinct probability of observing 2 or
more mutations per meiosis as seen in the present dataset, and we
have previously observed 3 mutations at Yfiler Y-STRs between a
father and his son [30]. Thus, the current policy to exclude
paternity when 3 (Y) STR mutations are observed [41] should be
revisited. As such a recommendation strongly depends on the Y-
STR used and their underlying mutation rates, and would be
considerably higher for RM Y-STRs, it would be more advisable to
instead provide a probabilistic estimate of the likelihood of
observing X number of mutations in a given meiotic transfer,
where the locus-specific mutation rates of the (Y) STRs applied
should be considered (as well as the number of meiotic transfers),
rather than applying a blanket rule to all cases.
The higher number of mutations observed underscores the
ability for the RM Y-STRs to differentiate between male relatives,
but also highlights their inability to be used for proving a link
between male relatives, as is required in paternity and family
testing. The greatly increased number of mutations will sever
haplotypic links within pedigrees, and greatly complicate the
reconstruction of familial relationships using Y-STRs. In these
instances, it would be advisable to use the Yfiler markers, due to
their considerably lower average mutation rate (or any other Y-
STRs with low mutation rate). A prime example of this is a large,
deep-rooted German pedigree previously examined with 68 Y-
STRs [36], and included in our previous RM Y-STR analysis [33]. In
this pedigree from which 20 male relatives separated by 1–25
generations were analysed, we observed 4 mutations in 2 of the
Yfiler Y-STRs [36], but 10 mutations in 6 of the RM Y-STRs, the
latter being sufficient to obscure a familial link, although not to
eradicate it completely.
A potential caveat in the application of RM Y-STRs to forensic
investigation is that the repeat sizes of many of the loci are large,
with up to 59 repeats (DYF403S1b) observed. On one hand such
large repeat numbers are essential to create high mutation rates, as
it is the repeat number that drives Y-STR mutation rates to a large
degree [33]. On the other hand the large repeat number means a
long PCR amplicon size, which may cause problems when
analysing heavily degraded DNA as often confronted with in
forensic analysis. Although larger amplicon sizes were applied here
for most RM Y-STRs, a brief inspection of the DNA sequence
structure of the markers suggests that genotyping assays to yield
PCR amplicon sizes below 200 bp would be designable for at least
10 of the 13 RM Y-STRs. Furthermore, four of the RM Y-STRs are
multicopy markers, which have been traditionally avoided in
forensic analysis for ease of mixture designation (although,
commercial Y-STRs sets such as Yfiler or PowerPlex Y also include
multi-copy Y-STRs). However, the multicopy markers included in
our RM Y-STR panel all have exceptionally large allele ranges and
high diversities, and as such carry low probabilities that two
unrelated individuals would share all alleles, while the nine single
copy markers of the RM Y-STR panel would be adequate for
detecting the presence of a mixture.
The increased mutation rate of each of the RM Y-STRs as
observed recently [33] raises an expectation for an increased
probability of observing mutations in sperm analysis, which may
be important for future forensic applications. Hence, in case
material where very few sperm cells are present, it could be
expected that mutations may create the appearance of additional
RM Y-STR alleles. However, the high diversities of each RM Y-STR
ensures that the probability of two individuals matching at 11 or
12 loci of the 13 is extremely remote (as shown in Fig. 5). Thus it is
expected that potential germline mutations that may be observed
in forensic sperm analysis will be clearly differentiable from
sample mixtures caused by different men contributing to a semen
stain. Nevertheless, sufficient validation testing of such samples
shall be performed in subsequent studies. Related to this issue is
the potentially increased somatic mutability of RM Y-STRs.
Notably, we found evidence of an increased rate (2.59% versus
0.08% considering European populations only) of locus duplica-
tions, or mutations giving additional alleles, with RM Y-STRs in the
 K.N. Ballantyne et al. / Forensic Science International: Genetics 6 (2012) 208–218
cell-line material from the HGDP-CEPH panel compared to our
previous analysis of blood-derived DNA [33]. It is known that the
immortalisation process enhances somatic mutation processes, as
does repeated passaging of cell lines [42], which would account for
the increased rates observed in the cell-line derived DNA samples
from HGDP-CEPH. In any case, RM Y-STR studies comparing
different forensically relevant body parts including hairs within
large numbers of individuals shall be performed in future studies
to investigate in vivo effects of somatic mutability of RM Y-STRs.
Although the RM Y-STRs would ideally become the first choice
for Y-STR testing in forensic casework (excluding family testing)
due to their superior value in paternal lineage differentiation as
well as male relative separation, we appreciate that replacing the
hundred thousand Y-STR profiles within existing Y-STR frequency
databases is not appealing within the short term. Therefore, at least
for the near future it can be envisioned that the RM Y-STR set
would become an adjunct to the current Y-STR kits such as Yfiler.
Whenever a non-exclusion is observed with a current Y-STR kit, the
RM Y-STR set should be applied to reduce the probability of
adventitious matches or male relative involvement. Furthermore,
RM Y-STRs, rather than currently available Y-STR sets, may be
applied in all cases with a hypothesis of related males being
involved (such as family rape). Although adding a subset of the 13
RM Y-STRs, i.e. the most discriminating markers, to the current Y-
STR sets will provide some improvements, we cannot recommend
such a strategy because the maximum benefit for both related and
unrelated male differentiation comes from all 13 RM Y-STRs as we
show here. Obviously, RM Y-STR haplotype frequencies will need
to be established in many populations for estimating match
probabilities needed in those cases where a non-exclusion
constellation is established with RM Y-STRs. However, even before
such data are generated the RM Y-STR set is expected to be useful
in forensic casework in highlighting exclusion constellations.
Ideally, a single sensitive multiplex assay for all 13 RM Y-STRs that
performs well on low amounts of heavily degraded template DNA
should be developed for forensic applications. However, until such
a tool is available, the three multiplex assays introduced here,
allowing the genotyping of all 13 RM Y-STRs, are sufficient for
successful analysis of low nanogram amounts of even moderately
degraded DNA.
Acknowledgments
We thank the HGDP-CEPH donors for their contribution to this
panel and Howard Cann for providing DNAs. This work was funded
in part by the Netherlands Forensic Institute (NFI), and by a grant
from the Netherlands Genomics Initiative (NGI)/Netherlands
Organization for Scientific Research (NWO) within the framework
of the Forensic Genomics Consortium Netherlands (FGCN).
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