Forensic Science International: Genetics 15 (2015) 2126

Contents lists available at ScienceDirect

Forensic Science International: Genetics

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

Helena, the hidden beauty: Resolving the most common West Eurasian
mtDNA control region haplotype by massively parallel sequencing an
Italian population sample
Martin Bodner a, Alessandra Iuvaro a,b, Christina Strobl a, Simone Nagl a, Gabriela Huber a,
Susi Pelotti b, Davide Pettener c, Donata Luiselli c,*, Walther Parson a,d,**
a

Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria

Department of Medical and Surgical Sciences, Institute of Legal Medicine, University of Bologna, Bologna, Italy
c
Department of Biological, Geological and Environmental Science, Laboratory of Molecular Anthropology, University of Bologna, Bologna, Italy
d
Penn State Eberly College of Science, University Park, PA, USA
b

A R T I C L E I N F O

A B S T R A C T

Keywords:
Massively parallel sequencing
mtDNA
Forensics
Most common haplotype
Power of discrimination
mtDNA haplogroup H

The analysis of mitochondrial (mt)DNA is a powerful tool in forensic genetics when nuclear markers fail
to give results or maternal relatedness is investigated. The mtDNA control region (CR) contains highly
condensed variation and is therefore routinely typed. Some samples exhibit an identical haplotype in
this restricted range. Thus, they convey only weak evidence in forensic queries and limited phylogenetic
information. However, a CR match does not imply that also the mtDNA coding regions are identical or
samples belong to the same phylogenetic lineage. This is especially the case for the most frequent West
Eurasian CR haplotype 263G 315.1C 16519C, which is observed in various clades within haplogroup H
and occurs at a frequency of 34% in many European populations.
In this study, we investigated the power of massively parallel complete mtGenome sequencing in
29 Italian samples displaying the most common West Eurasian CR haplotype and found an unexpected
high diversity. Twenty-eight different haplotypes falling into 19 described sub-clades of haplogroup H
were revealed in the samples with identical CR sequences. This study demonstrates the benet of
complete mtGenome sequencing for forensic applications to enforce maximum discrimination, more
comprehensive heteroplasmy detection, as well as highest phylogenetic resolution.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Forensic DNA analyses are routinely performed by determining
the genetic ngerprint, i.e. the alleles of polymorphic nuclear
microsatellite markers that display high diversity, stability,
and Mendelian inheritance, and thus allow identication,

Abbreviations: codR, coding region of the mtDNA; CR, control region of the mtDNA;
EDNAP, European DNA Proling Group; EMPOP, EDNAP mtDNA population
database; MPS, massively parallel sequencing; MRCA, most recent common
ancestor; mtDNA, mitochondrial DNA; np(s), nucleotide position(s).
* Corresponding author. Tel.: +39 0512094194; fax: +39 0512094191.
** Corresponding author at: Institute of Legal Medicine, Innsbruck Medical
University, Innsbruck, Austria. Tel.: +43 512900370651; fax: +43 512900373640.
E-mail addresses: [email protected] (D. Luiselli),
[email protected] (W. Parson).
http://dx.doi.org/10.1016/j.fsigen.2014.09.012
1872-4973/ 2014 Elsevier Ireland Ltd. All rights reserved.

individualization and pedigree reconstruction [1]. These markers however do not regularly yield results from compromised
samples containing degraded or low quantities of nuclear DNA. The
haploid, maternally inherited mitochondrial (mt)DNA has become a
vital niche in analyzing those samples due to its abundance and
stability as multi-copy circular molecule protected in organelles. As
a lineage marker, it can be used to exclude identity or corroborate
(even distant) maternal relatedness [26].
The outcome of (forensic) mtDNA investigations, besides
precise base calling and the availability of high-quality databases
[710], mainly depends on the amount of information generated
from the individual sample [11,12]. Because of nancial, technical
and legal restrictions, the current standard is to sequence
(hypervariable parts of) the 1.1 kbp non-coding control region
(CR) of the 16.5 kbp mitochondrial genome (mtGenome), that
contains densely concentrated variation due to a higher mutation

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M. Bodner et al. / Forensic Science International: Genetics 15 (2015) 2126

rate compared to the remaining segment, the coding region (codR)

[7,13,14]. CR data enable coarse discrimination of numerous
maternal lineages [15] and may yield non-identity (exclusion) of
donors and their maternal relatives, respectively, in many forensic
cases [16,17]. However, identical CR haplotypes are found on
different haplogroup backgrounds across the mtDNA phylogeny, as
several lineages are poorly dened in the CR [18]. Consequently, a
shared CR haplotype (non-exclusion) does not necessarily imply
that two mtDNAs are identical in their codR, or even belong to the
same lineage.
For these reasons, the current partial analyses of the mtDNA
molecule greatly restrict and possibly bias (forensic) interpretation, and are particularly problematic for populations with
extremely few CR lineages (e.g., Ref. [19]). For example, when
working with samples of West Eurasian (sometimes referred to as
Caucasian) maternal background, it is very likely to encounter
diverse clades of haplogroup H that encompasses 40% in many
European populations [2022]. The currently available CR data
convey a rather uniform picture of distribution, while pivotal
studies at higher levels of phylogenetic resolution have demonstrated a cluster of >100 distinct radiating lineages within this
haplogroup, with signicant differences in dispersal and frequency
(e.g., Refs. [20,21,2327]). These investigations have so far mostly
focused on only few or non-random samples, involved limited
codR sequencing, contained no (detailed) information on the
donors geographic origin or were derived from small regions
[10]. Only about one fth of the H lineages can be distinguished
within the CR, but markers are often homoplasic [14,15,18,23]
(Fig. 1).
Correspondingly, many West Eurasian individuals exhibit
identical CR haplotypes clustering within haplogroup R0 (the
CR-MRCA of haplogroup H) only for the lack of further sequence data.
This is particularly the case for the most common West Eurasian CR
haplotype 16519C 263G 315.1C (relative to the revised Cambridge
Reference Sequence, rCRS [28]), observed in numerous sub-clades of
haplogroup H [15] with a frequency of 34% in any western Eurasian
population [22]. It can be anticipated that codR analysis would reveal a
high number of different lineages (cf. [12,29]).
Different strategies have been applied to access codR information in forensic casework [14,30] but circumvent laborious
complete mtGenome Sanger-type sequencing [6,31,32]. These
have included sequencing only short segments comprising
variation considered relevant [33,34] or, more commonly, targeting distinct markers of a few principal clades in a (usually
minisequencing) multiplex assay in order to determine the main
haplogroups on macro-region (e.g., Refs. [35,36]) or even global

Fig. 1. Schematic phylotree of haplogroup H from entire mtGenome sequencing.

The root haplogroup H haplotype (at the bottom) is the origin of ve (for the 100)
radiating lineages that give rise to further sub-branches. Four explicative derived H
haplotypes are shown at tips of the tree. If only the mtDNA CR (continuous
segments of the circles) were sequenced, all haplotypes in this gure would be
identical and would fall into a single H* lineage. Their individual or lineage
diagnostic codR variation, indicated by asterisks in the dotted segments of the
circular genomes, would not be detected.

level [3739], or to dissect identical samples or a specic lineage

(for haplogroup H, e.g., Refs. [24,40,41] (compared in Ref. [42]),
[43,44]). The number of markers that can be included in such an
assay is limited; therefore, a selection toward those considered
more frequent is usually made. In any approach, countless SNPs are
left undetermined and consequently a somewhat rough resolution
is yielded only complete mtGenome sequencing would reveal
untargeted private or phylogenetic variation in total.
This loss in discrimination power cannot be compensated even
when highly uctuating individualizing markers are included in
some assays (cf. [42]). Emerging benchtop high-throughput
massively parallel (MPS; or next generation) sequencing solutions,
that are very promising in terms of sample throughput, speed,
amount of data generated and costs per sample, now make
obtaining complete mtDNA information relatively easy. In this
pilot study, we investigated the power of entire mtGenome MPS on
an identical most common West Eurasian mtDNA CR haplotype
sample set, carefully considering methodological challenges
[45,46] and taking advantage of insights from systematic data
reviews during recent complete mtGenome etalon (i.e. carefully
selected high quality reference, cf. [47]) dataset generation by both
Sanger-type sequencing [6,48] and MPS [49].
2. Materials and methods
2.1. DNA samples
An earlier population study [44] included 884 randomly
selected individuals representing eight macro-areas of Italy that
donated blood samples after informed consent. DNA was extracted
using a modied salting out method [50], sequenced for mtDNA CR
and typed for 22 codR SNPs to determine the main West Eurasian
haplogroups. The samples found to belong to haplogroup R0 were
further subjected to a SNP multiplex analysis designed to resolve
17 distinct haplogroup H lineages [44]. In this study, we used a
subset of 29 of these samples harboring the most common West
Eurasian mtDNA CR haplotype 16519C 263G 315.1C (relative to
the rCRS) [28]. Three samples were re-extracted using the QIAamp
DNA Mini kit (QIAgen, Hilden, Germany). The donors originated
from provinces throughout Italy, including Sicily and Sardinia
(Fig. 2 and Table S1).
2.2. Complete mtDNA sequence data generation and interpretation
After simultaneous verication of integrity and quantitation of
mtDNA in a modular real-time quantitative assay [51], we
amplied two overlapping 8.5 kb fragments covering the entire
mitochondrial genome [31] and performed MPS on the Ion Torrent
Personal Genome Machine (PGM) using Ion PGM Sequencing
200 Kit v.2 chemistry on an Ion 316 chip. Raw data were inspected
twice using independent software, mirroring the gold standard in
Sanger-type sequencing [7]. The threshold for heteroplasmic
mixtures was dened 10% (of total coverage). We used Sangertype sequencing to clarify discordant positions. Sequences were
aligned to the rCRS [28]. According to a basic forensic principle, we
compared the MPS results to those from Sanger-type sequencing
[44]. We assigned the samples to mtDNA haplogroups according to
Phylotree [15], build 16, aided by the EMMA software package [52]
and calculated genetic parameters as described in Ref. [53]. We
followed strict quality control protocols according to a preceding
validation study of the PGM in forensic mtDNA sequencing [46].
3. Results
Entire mtGenome sequencing revealed an extremely high
degree of variation in the samples that shared the CR motif 16519C

M. Bodner et al. / Forensic Science International: Genetics 15 (2015) 2126

Fig. 2. Origin of the 29 Italian samples included in this study. Circles represent
individual samples and are assigned to their province of origin. The color codes
distinguish between samples of haplogroups H1, H3, and H7 and those falling into
other H clades (see text for details). (For interpretation of the references to color in
this gure legend, the reader is referred to the web version of the article.)

263G 315.1C. In total, 28 different haplotypes were discerned

within the set of 29 samples analyzed in this study. Two
(seemingly) unrelated donors from the same province ([44],
Table S1), DB525 and DB559, revealed identical mtGenomes. The
haplotype diversity using entire mtGenome information reached
99.8%, corresponding to a random match probability of 0.037
(Table 1). The average MPS coverage along the mtGenomes ranged
between 377 and 1154 (median: 687; mean: 697).
The MPS results were fully concordant with the partially
available Sanger-type sequencing results [44], with the exception
of a low-level point heteroplasmy at np 16311 in sample DB1766
that had not been visible after Sanger-type sequencing, which
speaks for an advantageous detection of (heteroplasmic) mixtures
with MPS. The 29 complete mtGenomes are reported in Table S1,
available from GenBank (accession numbers KM252727
KM252755) and will be uploaded onto the EDNAP mtDNA
population database (EMPOP V3) [22]. They classied into
19 reported haplogroup H sub-clades ([15], build 16), 17 of which
were unique in the dataset. Haplogroups H1 and H3 comprised the
largest number of mtDNAs (13 and ve, respectively) among the

Table 1
Comparison of the diversity parameters in the 29 Italian samples using different
sequence ranges.
mtDNA range

Haplotypes
Unique haplotypes
Haplogroupsb
Unique haplogroupsb
RMPc
Haplotype diversity
a
b
c

CR

CR + 39 codR SNPsa

Complete mtGenome

1
0
1
0
1.000
0.0%

6
2
6
2
0.296
72.9%

28
27
20
18
0.037
99.8%

Thereof 17 specic for haplogroup H clades [44].

According to Ref. [15], build 16. H* is considered a haplogroup.
Random match probability.

23

detected lineages: H1* (7/29 = 24.1%), H3* (4/29 = 13.8%), and

singletons (3.4%) of H1ax, H1e*, H1e1a, H1j3, H1q*, H1q2, H3ar,
H7b*, H7b1, H10a, H18b, H58, H59a, H65, H75, H84 and H86. One
sample could not be assigned to a distinct H lineage (Fig. 3).
In addition to the cytosine insertion after np 315 and the
transition at np 16519 (compared to the rCRS [28]) present in all
haplotypes but never listed in Phylotree [15], 23 of the samples
carried up to four additional, so-called private polymorphisms
that possibly highlight yet undescribed lineages (Table S1). Two
substitutions, T15115G (DB604) and C11588T (DB1368), were
previously unobserved. The only recurrent pattern within our
dataset was H1-709-8108-15470, pointing at a novel sub-clade in
the (seemingly) unrelated samples DB1566 and DB1570 (from the
same province [44], and identical aside from a single heteroplasmic position, Table S1). In four instances, unnamed phylogenetic patterns have been reported earlier: H3-2851-11200 in
DB604 and sample DQ523661 from Sardinia [54]; H1q-885614258 in DB763, EF657644 from Italy [55] and JX153975 [56],
KF161678 and KF162479 [S. Li and M.H. Schierup, unpublished]
from Denmark; H1-5581-14905 in DB2633 and JQ703512 [27];
and nally, the transition at np 4245 we found on an haplogroup
H7b background in DB1544 was present on an H7 background in
EF661013 from Italy [57]. Another phylogeny renement is
highlighted by sample DB1049, where the presence of two
diagnostic polymorphisms, viz. transitions at nps 2851 and
14148, indicated haplogroup H1h2 status, while another two
currently listed markers, the transitions at nps 7013 and 14420
([15], build 16), were missing. In sample DB628, the transition at
np 5460 would support haplogroup H1e on the H1 background.
Based on this single marker, the sample would be misassigned, as
three additional SNPs (including a transversion) suggest H1ax
status.

4. Discussion
4.1. MtDNA forensics at its highest resolution
This study strikingly demonstrates the signicance of complete
mtGenome sequencing in forensic genetic practice: highest
mtDNA resolution allowed almost complete discrimination of
haplotypes identical in their CR by rendering virtually every one
unique in this randomly selected sample. In the previous study that
included 39 codR SNPs in addition to the CR [44], a power of
discrimination of 72.9% had been reached using the same sample
set, compared to 99.8% in this study (Table 1). A signicant genetic
diversity increase when analyzing complete mtGenomes has also
been found in larger, randomly chosen population samples with a
more ample haplogroup spectrum [48,49].
The two matching complete mtDNA sequences (i.e. nonexclusion) signpost the current limitation of mtGenome sequencing in forensic applications: these two samples cannot be excluded
as deriving from the same individual or the same maternal lineage,
respectively. Sound sampling (cf. [58]) and large and reliable
sequence databases [10] are necessary prerequisites for a correct
assessment of population variation to weigh such evidence.
Currently, the forensic community is able to assess the probability
of a given haplotype in its (putative) population by using EMPOP
[22] (or other databases) in the CR range. Necessary structural and
query modications have recently been elaborated [6], and with
the upcoming third version of EMPOP, a complete mtGenome
database for forensic application will be accessible (cf. [6,10]).
More than 20,000 (nearly-) complete worldwide mtGenome
sequences are publicly available (cf. [6,15]), but for now,
comprehensive high-quality investigations on randomly selected
population samples are still scarce (see above) [48,49].

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M. Bodner et al. / Forensic Science International: Genetics 15 (2015) 2126

Fig. 3. Overview of haplogroups of the most common West Eurasian CR haplotype revealed by complete mtGenome sequencing. The central pie chart indicates the
proportions of the mtDNA lineages found in the Italian sample (n = 29). The three peripheral pie charts depict the proportions of the sub-clades found within the segment they
are assigned to. One haplotype does not fall into a described lineage.

4.2. Helenas many daughters

Already the small Italian sample analyzed in this study revealed
20 different sub-lineages hiding behind an identical CR sequence.
Compared to the previous study [44] and using the same sample set,
six of the H* samples could now be assigned to a specic clade and for
ten additional samples (within haplogroups H1, H3, H7 and H10), our
analyses provided further detail. Haplogroup dispersal and representation was (necessarily) random, but revealed that the highly
prevalent haplogroup H1 [20,44,59] not tested in earlier pan-Italian
mtDNA population studies [60,61] is composed of several sublineages (Fig. 2 and Fig. 3). This pilot study lays the scientic
foundation for an extended follow-up project to analyze the highresolution phylogeny and phylogeography of the most common West
Eurasian CR haplotype in Italy (and beyond). Such data is expected to
allow a molecular dissection into sub-clades of more restricted
geographic distribution a possible forensic investigative lead and
younger ages, which will aid to reconstruct migration history
(cf. [20,23]). Together with insights from other lineages, substructure
and stratication revealed could help to establish currently lacking
population genetic correction factors for forensic statistics [17].

Eurasian lineages (e.g., [29,35,44,62]), they can serve as a tool

ready-to-use for rapid discrimination between divergent lineages
in forensic casework or when increased resolution is desirable for
investigative purposes but complete mtGenome sequencing is not
feasible. Modular marker sets for hierarchical typing, and
geographic sets that take the lineages dispersal into account
can be tailored. It should be noted that mtDNA SNP typing can also
be a successful alternative in case regular sequencing fails [63].
As a nal remark, it remains elusive and beyond the scope of
this study to understand why the most common CR motif is so
widespread among the radiating haplogroup H clades regardless of
the high mutation rate. Contributing factors may include a founder
effect; selective advantages or restrictions favoring an mtDNA
population carrying this particular motif, possibly because of
functional importance of certain non-coding nucleotide variants;
or the high presence of an identical small fraction of the entire
molecule could simply be due to the fact that H is the most
common mtDNA haplogroup with the highest number of subclades among the currently overrepresented Western Eurasian
entire mtGenomes. Any hypothesis would need to be tested
through a comparative analysis of another mtDNA haplogroup on a
completely different population background.

5. Conclusion and outlook

Acknowledgments

This study makes the forensic (mito-)geneticists ultimate

desire come true: to discern the identical by entering the nal
genetic phase of mtDNA resolution, the analysis of entire
mtGenomes. However, MPS appears for now out of reach for
most forensic casework laboratories, despite its clear advantages in
terms of discrimination power, heteroplasmy detection and
phylogenetic assignment, which can act as quality control (cf.
[7,49]). Complete mtGenome Sanger-type sequencing is tedious
[31,32], even though large-scale automation protocols for use in
forensic environment have been presented [6]. Also, legal and
ethical questions of (routine) complete mtGenome sequencing
need to be resolved [14]. For the time being, an extended version of
this study could thus yield specic SNP panels for (forensic) testing
of (Italian) samples exhibiting the most common West Eurasian
mtDNA CR haplotype. Combined with panels covering other West

The authors wish to thank the donors who gave their blood for
research and the two anonymous reviewers for their helpful
comments. This work was supported by the intramural funding
program of the Medical University Innsbruck for young scientists
MUI-START, Project 2013042025, the Theodor Korner Fonds zur
Forderung von Wissenschaft und Kunst, the European Union Seventh
Framework Programme (FP7/2007-2013) under grant agreement
n8 285487 and the Austrian Science Fund (FWF) [P22880-B12].

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fsigen.2014.09.012.

M. Bodner et al. / Forensic Science International: Genetics 15 (2015) 2126

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