Analytical Biochemistry 490 (2015) 1e6

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Differentiating between monozygotic twins through next-generation

mitochondrial genome sequencing
Zheng Wang a, b, Ruxin Zhu a, Suhua Zhang a, b, Yingnan Bian a, Daru Lu b, Chengtao Li a, *
a
Shanghai Key Laboratory of Forensic Medicine, Institute of Forensic Science, Ministry of Justice, P. R. China, Shanghai 200063, China
b
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, China

a r t i c l e i n f o a b s t r a c t

Article history: Monozygotic (MZ) twins, considered to be genetically identical, cannot be distinguished from one
Received 13 June 2015 another by standard forensic DNA testing. A recent study employed whole genome sequencing to
Received in revised form identify extremely rare mutations and reported that mutation analysis could be used to differentiate
2 August 2015
between MZ twins. Compared with nuclear DNA, mitochondrial DNA (mtDNA) has higher mutation
Accepted 19 August 2015
Available online 29 August 2015
rates; therefore, minor differences theoretically exist in MZ twins' mitochondrial genome (mtGenome).
However, conventional Sanger-type sequencing (STS) is neither amenable to, nor feasible for, the
detection of low-level sequence variants. The recent introduction of massively parallel sequencing (MPS)
Keywords:
Forensic genetics
has the capability to sequence many targeted regions of multiple samples simultaneously with desirable
Mitochondrial genome (mtGenome) depth of coverage. Thus, the aim of this study was to assess whether full mtGenome sequencing analysis
Monozygotic twins can be used to differentiate between MZ twins. Ten sets of MZ twins provided blood samples that un-
Massively parallel sequencing (MPS derwent extraction, quantification, mtDNA enrichment, library preparation, and ultra-deep sequencing.
Point heteroplasmies were observed in eight sets of MZ twins, and a single nucleotide variant (nt15301)
was detected in five sets of MZ twins. Thus, this study demonstrates that ultra-deep mtGenome
sequencing could be used to differentiate between MZ twins.
© 2015 Elsevier Inc. All rights reserved.

Today, individual identification in forensic casework is mainly polymorphisms (SNPs) for the differentiation between MZ twins.
based on short tandem repeat (STR) polymorphisms, and a set of Compared with nuclear DNA, mitochondrial DNA (mtDNA), an
19e24 highly polymorphic STR markers is often genotyped [1,2]. extranuclear genome, exhibits higher mutation rates due to the
However, monozygotic (MZ) twins are considered to be genetically presence of fewer DNA repair mechanisms [8,9]. The 10-fold higher
identical in that they have identical STR profiles [3]. Thus, they mutation rate, relative to nuclear DNA, helps to introduce more
cannot be differentiated using STR profiling. This inability causes variability in mitochondrial genome (mtGenome); thus, it has been
problems in distinguishing one from another in criminal cases with suggested that significant variability presents between individuals
an MZ twin as a suspect. Recently, small epigenetic differences, and that all individuals would display mtDNA heteroplasmy
especially DNA methylation pattern, between twins have been [10e12]. Although the use of full mtGenome data would no doubt
described [4e6]. However, DNA methylation analysis generally increase the information content and increase its utility in practical
needs bisulfite or enzymatic digestion treatment, which is very forensic casework [13e16], conventional Sanger-type sequencing
labor-intensive and complex and has risk for DNA damage. (STS) is not suitable for the analysis of the full mtGenome and not
In 2014, Weber-Lehmann and coworkers [7] employed whole possible to clearly identify minor heteroplasmic variants.
genome sequencing to search potential single nucleotide The recent introduction of massively parallel sequencing (MPS),
also termed next-generation sequencing, has revolutionized
genomic studies; many targeted regions' sequence information of
Abbreviations used: STR, short tandem repeat; MZ, monozygotic; SNP, single multiple samples can be obtained much more quickly and cost-
nucleotide polymorphism; mtDNA, mitochondrial DNA; mtGenome, mitochondrial efficiently [17]. MPS technology has the potential to increase both
genome; STS, Sanger-type sequencing; MPS, massively parallel sequencing; PCR, sample throughput and overall process efficiency, thereby holding
polymerase chain reaction; rCRS, revised Cambridge Reference Sequence; IGV,
great potential for efforts to expand mtDNA typing beyond current
Integrative Genomics Viewer; PHP, point heteroplasmy.
* Corresponding author. Fax: þ86 21 52352959 capabilities. Next-generation mtGenome sequencing is currently
E-mail address: [email protected] (C. Li). being used in forensic applications, including mtDNA validation

http://dx.doi.org/10.1016/j.ab.2015.08.024
0003-2697/© 2015 Elsevier Inc. All rights reserved.
2 Z. Wang et al. / Analytical Biochemistry 490 (2015) 1e6

[18e20], larger mtGenome reference data establishment [21,22], Sequencing and data analysis
and investigation of heteroplasmy occurrence [11,12]. In this study,
we explored the use of MPS technology to identify minor differ- Sequencing was performed on the Illumina HiSeq 2000 system
ences in mtGenomes between MZ twins. The Illumina HiSeq 2000 with chemistry version 3.0 and using the 2  100-bp paired-end
Sequencing System uses a reversible terminator-based sequencing read mode according to the manufacturer's recommendations.
by synthesis method capable of producing a massive parallel The initial data analysis was carried directly on the HiSeq 2000
sequencing environment. For mtDNA sequencing in the forensic system during the run. The FASTQ files comprising the sequence
context, mtGenome is amplified, fragmented, and modified with information were mapped to the revised Cambridge Reference
adaptors and dual indexes, and it is pooled (for multiplexing), Sequence (rCRS; GenBank ID: NC_012920.1) [24] using Bur-
generates DNA cluster by bridge polymerase chain reaction (PCR), rowseWheeler Alignment software (BWA version 0.6.2) [25]. Using
and is sequenced. Consequently, the aim of this study was to the SAMtools software package version 1.2 [26], the mapping re-
evaluate whether next-generation mtGenome sequencing analysis sults were filtered by applying a mapping quality threshold of 20.
will allow for the differentiation between MZ twins. By multi-sample calling function (mpileup file), a single BAM file
containing good-quality unique mapping reads was obtained for
Materials and methods each of the twins. VarScan2 software [27] was used to identify
variants, and Variant Call Format (VCF) files were generated. The
Sample preparation analysis in this study used the manual settings for minimum base
call quality (Q30), min coverage 500, min reads2 (minor compo-
Human blood samples were collected with the approval of the nent) 200, and min var freq (minor component frequency) 0.05.
ethics committee of the Institute of Forensic Science, Ministry of The variant nucleotides from the reference were annotated by base
Justice, P. R. China. Informed written consent was obtained from 10 difference and verified by manually viewing BAM files in an Inte-
sets of MZ twins (25e58 years old). Whole blood samples were grative Genomics Viewer (IGV) [28].
collected by venipuncture without anticoagulation treatment. DNA
was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Hil- Conventional sanger sequencing
den, Germany) according to the manufacturer's instructions. The
quantity of extracted DNA was estimated using the Quantifiler Hu- PCR amplification for STS was performed using two pairs of
man DNA Quantification Kit (Thermo Fisher, Foster City, CA, USA) on primers (F-L16047: 50 -CATGGGGAAGCAGATTTG-30 and R-H16464:
the Applied Biosystems 7500 Real-Time PCR System following the 50 -TTAGCTACCCCCAAGTGT-3ʹ; F-L29: 50 -GGT CTATCACCCTATTAAC-
manufacturer's recommendations. Samples were normalized to CAC-30 and R-H408: 50 -CTGTTAAAACTGCATACCGCCA-30 ) to amplify
1 ng/ml and stored at 20  C until mtDNA enrichment. hypervariable regions. Two independent reactions were performed,
The monozygosity of the twins was confirmed by typing all each in a total volume of 50 ml containing 5 ml of 10  PCR buffer,
individuals with the GoldenEye 20A Kit (PeopleSpot, Beijing, China) 5 ml of 25 mM MgCl2, 2 ml of 10 mM deoxynucleoside triphosphate
following the manufacturer's protocol. PCR products were sepa- (dNTP), 5 ml of each primer, 0.4 ml of AmpliTaq Gold Polymerase, and
rated on an ABI 3130xl Genetic Analyzer and evaluated using 1 ng of DNA template. PCR was performed in a GeneAmp PCR
GeneMapper ID version 3.2 software (Thermo Fisher). System 9700 thermal cycler under the following conditions: 94  C
hold for 11 min, 35 cycles of 94  C for 30 s, 55  C for 60 s, 72  C for
Long-range PCR amplification 90 s, hold for 10 min at 72  C, and a final hold at 4  C. Amplified
products were purified with the QIAquick PCR purification kit.
The entire mtGenome was amplified by long-range PCR in two Purified products were sequenced on an ABI 3730xl capillary
separate reactions according to the protocol described by Fendt and sequencer equipped with 50-cm capillaries and POP7 polymer
coworkers [23]. Negative controls (negative amplification control following standard protocols. All sequences were imported into
and reagent blank control) were used as controls for potential Vector NTI Suite 8.0 (Thermo Fisher) and aligned relative to the
contamination. PCR products were purified using the QIAquick PCR hypervariable regions of rCRS (commonly referred to as HV1 and
Purification Kit (Qiagen) and then imaged by agarose gel electro- HV2). STS data were analyzed by at least two independent scien-
phoresis to confirm successful amplification. The quantity of tists, and mtDNA haplotypes were recorded relative to the rCRS
amplicons was determined using the Qubit dsDNA BR Quantifica- (NC_012920.1).
tion Kit with the Qubit 2.0 Fluorometer (Thermo Fisher), and then
equal quantities of two amplicons were pooled to produce 1.0 ng of Results
DNA for library preparation.
The monozygosity of the twins was confirmed using standard
Library preparation forensic STR typing with the GoldenEye 20A kit (data not shown).
DNA obtained from blood samples of the MZ twins was used for
Next-generation sequencing libraries were prepared from blood amplification of the entire mtGenome. Two overlapping PCR frag-
samples of the MZ twins according to the common guidelines for ments were confirmed by agarose gel electrophoresis, and no
shotgun library preparation. Briefly, 20 pooled amplicons were amplification band was observed in the negative control samples.
prepared using the Nextera XT DNA Sample Preparation Kit (Illu- Following library preparation and subsequent whole mtGenome
mina, San Diego, CA, USA) following the manufacturer's recom- sequencing, the Illumina HiSeq system generates approximately
mendations. Subsequently, Nextera XT products were purified 13.63 gigabases (Gb) of Q30 data. Assuming equal coverage across
using Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, the mtGenome, each indexed sample would be expected to have
IN, USA). After cleanup, the libraries were quantified using the more than 40,000  coverage at each base position of the mtGe-
Qubit dsDNA BR kit and evaluated for fragment size using the High nome. In practice, coverage was not dispersed evenly among in-
Sensitivity DNA Kit with the Agilent 2100 Bioanalyzer (Agilent dividuals, and Fig. 1 shows the number of nonredundant and
Technologies, Santa Clara, CA, USA). All samples were normalized to uniquely mapped reads for all MZ twins (corresponding mtGenome
the same concentration, pooled, and denatured according to the coverage ranged from 33,078 to 56,130). The production of the raw
manufacturer's instructions. sequencing data generated required approximately 2 weeks. This
 Z. Wang et al. / Analytical Biochemistry 490 (2015) 1e6 3

Fig.1. Numbers of nonredundant and uniquely mapped reads of ten sets of MZ twins.

includes DNA isolation, mtDNA enrichment, library preparation, heterogeneity at nt240; one individual observed only adenine (A)
sequencing, and initial data analysis. The time for all subsequent in this site, but 14.74% guanine (G) was presented in the other in-
analysis steps mainly depended on the available computational IT dividual. This PHP detected by the MPS approach was clearly
infrastructure and manual investigations (including the validation confirmed by the conventional STS method (Fig. 2).
of findings using the STS method); this procedure required around Interestingly, one nucleotide position, nt15301, was observed in
1e2 weeks. different bases (or major component) in five sets of MZ twins
All sequences were aligned relative to the rCRS (NC_012920.1), (Table 1). This means that A nucleotide was detected in one sample,
and detailed differences are presented in Supplementary material 1 and G nucleotide was sequenced in the paired sample. Fig. 3 shows
of the online Supplementary material. The haplotypes (partial the same set of samples but has different sequencing results in
mtGenome, HV1 and HV2) produced using the MPS approach show nt15301. In this position, the major component (88.01%) is A
concordance with haplotypes generated through the conventional nucleotide in one sample, but in the paired sample A nucleotide is
STS method. Consensus haplotypes are listed in Supplementary the minor component (9.40%).
Table 1.
Next, VarScan2 software [27] was used to determine potential Discussion
variants. We assigned twin A to be the “normal sample” and twin B
to be the “matched sample” and vice versa, and we ascertained a set MZ twins have identical genomic DNA sequences, making it
of potential differences between MZ twins. Point heteroplasmy difficult to distinguished one from the other using chromosomal
(PHP) refers to the presence of more than one nucleotide call at a genetic markers commonly used in forensics [3]. In 2014, Weber-
specific position. In this study, the minor component detection Lehmann and coworkers [7] employed whole genome sequencing
threshold was set at 5.0%, meaning that at least to search potential somatic mutations for the differentiation be-
200  corresponding coverage was required. With this threshold, tween MZ twins. In this well-designed and comprehensive study,
we identified a total of 16 bases presenting varying degrees of they introduced MPS technology to forensic MZ twins study and
heterogeneity in eight sets of MZ twins, with the exceptions being demonstrated that five SNPs were present in twin A but not in
MZ 5 and MZ 9 (Table 1). Among these nucleotide positions, nt207, twin B [7]. Human mtDNA, an extranuclear genome, is abundant
nt240, nt16129, nt16183, nt16189, and nt16289 are located in HV 1 relative to nuclear DNA in most human cells. Although mtDNA has
and HV 2, and other 10 positions come from the coding region approximately 16,569 bp, the average mutation rate of mtDNA
(Table 1). Fig. 2 shows one of the PHPs observed in the MPS data exceeds that of nuclear DNA by at least an order of magnitude
visualized by IGV [28]. One set of MZ twins had different degrees of [8,9]. Because of the high mutation rate of the mtGenome and

Table 1
Detailed sequence variant and PHPs of MZ twins' mtGenomes

MZ 1 MZ 2 MZ 3 MZ 4 MZ 5 MZ 6 MZ 7 MZ 8 MZ 9 MZ 10

Variant G15301 G15301 G15301 G15301 G15301

1A: A (88.09%) 4A: G 5A: A (88.01%) 9A: G 10A: G (94.55%)
1B: G 4B: A (84.37%) 5B: G (90.60%) (82.11%) 10B: A (88.59%)
9B: A
(88.08%)
PHP A16183 A10397 A240 G207 G15301 T2302 A6116 T9540
1A: C 2A: G (92.02%) 3A; A (85.26%) 4A: A 6A: A (84.73%) 7A: T 8A: G (81.35%) 10A: C (89.43%)
1B: C (82.72%) 2B: G 3B: A 4B: A (94.93%) 6B: A (68.27%) 7B: T (92.02%), 8B: G (82.65%) 10B: C
T16189 C10400 G15346 T16189 G16129 A16289 G15301 A10398
1A: C 2A: T (92.59%) 3A: A 4A: C (93.47%) 6A: G (93.37%) 7A: A (87.41%) 8A: A (83.14%) 10A: G (90.02%)
1B: C (85.90%) 2B: T 3B: A (55.61%) 4B: C (92.81%) 6B: G (94.29%) 7B: A (74.33%) 8B: A (86.4%) 10B:G
C12705 G11719
2A: T 10A: A
2B: T (93.68%) 10B: A (94.42%)
G15301
2A:G
2B: G (89.3%)
4 Z. Wang et al. / Analytical Biochemistry 490 (2015) 1e6

Fig.2. Sequence heteroplasmy at position 240 in MZ 3B possessing both A and G (minor component) nucleotides compared with the same region (positions 235e245) in MZ 3A
containing only G nucleotides at position 240 (visualized with the IGV Viewer [28]). This observation was confirmed by the STS method and is highlighted with a red arrow. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

mutations accumulating over the lifetime, it has been suggested mtGenome sequencing more cost-effective than whole genome
that significant variability presents between unrelated individuals sequencing. However, the STS method commonly used in forensic
and that all individuals would display mtDNA heteroplasmy mtDNA analysis is not suitable for the sequencing of the full
[10e12]. Relatively small size and higher mutation rate make full mtGenome, and the detection of heteroplasmic variants has been
 Z. Wang et al. / Analytical Biochemistry 490 (2015) 1e6 5

Fig.3. MPS results of nucleotide difference at position 15301. Results for MZ 5A and MZ 5B are visualized with the IGV Viewer [28]. The difference is in the middle of the panel and is
highlighted with a red arrow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

limited to the minor component with frequencies greater than 15% threshold (see Supplementary material 1). One particular set of
[18,29]. twins, MZ 2, showed a remarkably large difference; PHP was
The recent introduction of MPS technology has revolutionized observed at four positions (nt10397, nt10400, nt12705, and
genomic studies; many targeted regions of multiple samples can be nt15301). They were 46 years old at the time of sampling but were
obtained with desirable depth of coverage. To date, several studies not the eldest of the ten sets of twins. The eldest MZ twins, MZ 10
have been conducted using MPS in forensics, including genetic (58 years old), detected PHP at three positions (nt9450, nt10398,
investigations of STR [30e32] and SNP [33,34] and mtDNA valida- and nt11719). The remaining six sets of MZ twins, in their 20se40s,
tion [18e20]. All of these studies indicate that DNA typing by MPS all had two PHPs (nt16183 and nt16189 for MZ 1, nt240 and nt15346
holds promise for forensic applications. Strikingly, the detection of for MZ 3, nt207 and nt16189 for MZ 4, nt15301 and nt16129 for MZ 6,
minor heteroplasmic variants in mtGenome could be down to nt2302 and nt16289 for MZ 7, and nt6116 and nt15301for MZ 8).
approximately 0.5e1% by MPS platforms [18,29]. Thus, the aim of Notably, in two sets of MZ twins, MZ 5 (53 years old) and MZ 9 (51
this study was to evaluate the effectiveness of next-generation years old), PHP was not detected based on the application of a 5.0%
mtGenome sequencing for differentiating between MZ twins. This minor variant threshold for detection. There is seemingly no
was achieved by isolating DNA from blood samples, performing obvious trend in the PHP observation of MZ twins' age.
mtDNA enrichment by two long PCRs, and then sequencing full Interestingly, a single nucleotide variant (nt15301) was
mtGenome on the Illumina HiSeq system. observed in relatively old MZ twins (Table 1 and Supplementary
The deep coverage rates of data generated on the Illumina HiSeq material 1). This polymorphic site is located in the coding region
system in this study is sufficient for detection and reliable reporting of cytochrome b, but the G15301A variant will not cause amino
of minor heteroplasmic variants down to approximately 1.0e5.0% change. In the Human Mitochondrial Genome Database (http://
[18]. In practice, a total of 16 basesd6 in the control region and 10 in www.mtdb.igp.uu.se) [35], 867 A nucleotide (32.1%) was observed
the coding regiondpresenting varying degrees of heterogeneity in a database of 2704 sequences, and in this study five MZ samples
were observed in eight sets of MZ twins with a 5.0% detection were profiled A nucleotide in this polymorphic site. The ages of
6 Z. Wang et al. / Analytical Biochemistry 490 (2015) 1e6

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