Forensic Science International: Genetics 41 (2019) 107–119

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Forensic Science International: Genetics

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

Review article

Forensic molecular biomarkers for mixture analysis T

⁎
Fabio Oldoni , Daniele Podini
Department of Forensic Sciences, The George Washington University, 2100 Foxhall Road NW, Washington, DC 20007, United States

A R T I C LE I N FO A B S T R A C T

Keywords: The deconvolution of DNA mixtures has gathered the attention of forensic DNA scientists for over two decades.
Forensic mixture profiling To enhance mixture deconvolution capabilities, a new generation of sensitive DNA-typing approaches has been
Genetic markers recently proposed. In this review, we describe novel, forensically relevant multi-SNP loci (i.e., microhaplotypes
Autosomal STR or microhaps), compound markers (i.e., DIP-STRs, SNP-STRs and DIP-SNPs) and lineage markers (i.e., rapidly
Y-STR
mutating Y chromosome STRs) that improve the deconvolution of two and more than two-person mixtures typed
Rapidly mutating Y-STR
using conventional STR, binary and non-binary loci. We explore the features and applications of these emerging
Indel/DIP
SNP molecular biomarkers with respect to their ability to forensically detect same-or-opposite sex donors. Finally, we
Microhaplotype discuss the impact of initial massively parallel sequencing (MPS) investigations of STR, microhaplotype and
Compound markers SNP/indel assays for DNA mixture profiling.
DIP-STR
SNP-STR
DIP-SNP
Capillary electrophoresis
Massively parallel sequencing

1. Introduction deconvolution capabilities of mixed-DNA source samples. In this review

1.1. Forensic context: challenges of mixture detection and deconvolution
The development of DNA-typing tools for mixture deconvolution
has been extensively documented by the forensic DNA community [1].
Crime scene samples recovered from body fluids such as blood, saliva,
or semen, tissues and contact-type traces originated following criminal
disputes (e.g., sexual and physical assault, and murder) can generate
DNA profiles of two or more same-or-opposite sex donors [2].
In an attempt to enhance mixture deconvolution capabilities of
conventional autosomal and Y chromosome short tandem repeat (STR)
polymorphism analysis, a new generation of highly sensitive supple-
mental DNA typing approaches has been recently introduced (Fig. 1).
Notably, compound biomarkers including insertion and deletion poly-
morphism closely linked to STR (DIP-STR), single nucleotide poly-
morphism closely linked to STR (SNP-STR) and insertion and deletion
polymorphism closely linked to single nucleotide polymorphism (DIP-
SNP) were specifically developed for resolving imbalanced two-source
mixtures and rapidly mutating STRs on the Y chromosome (Y-STRs) for
discriminating closely related males. By taking advantage of massively
parallel sequencing (MPS) technology, emerging microhaplotypes also
display similarly promising characters to further enhance the
we provide a detailed description of the features of these novel nuclear
biomarkers and their applications, and also discuss the impact of initial
MPS evaluations of STR, microhaplotype and SNP/indel assays for
mixture detection and deconvolution.
2. Capillary electrophoresis (CE)-DNA mixture analysis
2.1. The “gold standard” autosomal STR marker
Short tandem repeat (STR) polymorphisms have been the mainstay
genetic markers for forensic DNA analysis [2] (Fig. 2). Since they are
highly polymorphic and discriminative among individuals, they were
adopted as reference loci for the Combined DNA Index System (CODIS)
and also facilitated the worldwide implementation of the crime Na-
tional DNA Databases (NDNADs) [3]. A variety of commercial and non-
commercial capillary electrophoresis (CE)-based autosomal STR am-
plification kits have been developed and used for human identification,
paternity/maternity/kinship relationship, missing-persons identifica-
tion, familial searching and mixture deconvolution [4–20]. Albeit STRs
are useful for addressing forensically relevant DNA-oriented questions,
they display serious limitations for the deconvolution of complex mix-
tures (i.e., more than two person mixed samples) such that only a

⁎
Corresponding author.
E-mail address: fabio.oldoni@yahoo.it (F. Oldoni).

https://doi.org/10.1016/j.fsigen.2019.04.003
Received 21 November 2018; Received in revised form 6 March 2019; Accepted 17 April 2019
Available online 30 April 2019
1872-4973/ © 2019 Elsevier B.V. All rights reserved.
F. Oldoni and D. Podini
Fig. 1. Forensic molecular biomarkers for detection and deconvolution of two
and more than two-person DNA mixtures.
limited range of mixture contribution ratios can be resolved routinely
[21]. Mixture interpretation can also be challenging due to the low
sensitivity level of STR loci, which enable the detection of the minor
donor in the presence of between ˜10- and 19-fold excess (i.e., 5–10% of
the total STR profile) of the major donor (Table 1). DNA background
masks the alleles of the minor donor at higher mixture ratios [22–24].
The presence of additional donor(s) from the major or minor DNA
fraction adds an extra level of complexity to the mixtures, and thus
seriously impacts the downstream interpretation of STR profiles.
Moreover, it is plausible that obtaining DNA results can be hindered by
the low quantity of DNA available and also the restricted multiplex
capability of CE detection systems that accommodate extensive allele
size ranges. Massively parallel sequencing (MPS) technology, formerly
referred to as next-generation sequencing (NGS), can help overcome
some of the aforementioned (STR) mixture constraints and enhance the
analytical capabilities of challenging mixed samples (refer to para-
graphs 5.1–5.2) [25–31].
2.2. Alternative markers: mini-STRs, indels and SNPs
Environmental degradation can drastically reduce the size of longer
(STR) alleles and make interpretation of mixed DNA profiles difficult
[2]. To address this, alternative genetic markers were developed, in-
cluding the valuable and complementary mini-STR markers, char-
acterized by short PCR amplicons (< 150 bp). Primer-binding sites can
also be moved closer to the repeat region of interest to improve the
success rate of amplifying degraded samples [32–35]. Similarly, short
binary or di-allelic (i.e., simple nucleotide polymorphism) markers can
be used to analyse degraded samples [36]. These markers are char-
acterized by the presence of two and sometimes three or more allelic
variants and include small-scale insertion/deletion polymorphisms
(indels or DIPs) [37] and single nucleotide polymorphisms (SNPs)
[38–41]. Binary markers characterized on autosomal and sex chromo-
somes display desirable forensic features including the absence of a
stutter peak, lower mutation rates than STRs and short amplicon size.
These features are of the utmost importance for addressing the analysis
of degraded samples [33,42–44], human identification [45–49], pa-
ternity and relationship-deficient cases [50–54], inference of biogeo-
graphic ancestry [55–59], and prediction of visible human phenotypic
traits (exclusively by SNP typing) [60,61].
Indels are loci with an average allele (i.e., insertion or deletion) size
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Forensic Science International: Genetics 41 (2019) 107–119
range of 1–5 bases and less often up to ˜25 bases (Fig. 2). These are of
particular interest in forensics because they display higher sensitivity
than binary SNPs for the detection of two-component mixtures because
they can be analysed through a direct PCR-to-CE typing approach.
Recently, Liu et al [62] proposed an allele-specific indel-based method
that requires two separate primer-specific reactions by real-time PCR
that can be used for exclusion of non-related donors in imbalanced two-
person mixtures. This proof-of-concept method uses a difference Ct
value for two real-time PCR reactions to determine the corresponding
indel genotypes of each individual even though it requires final typing
confirmation by CE fragment separation. The authors evaluated the
performance of 14 loci, which were found to be highly polymorphic in
the Chinese Han population, with a variable level of specificity reported
across different loci. The ratio of mixture detection ranged from 1:50 to
1:100 for five indels and from 1:500 to 1:1000 for the remaining loci
using ˜1–10 ng of input DNA. These results indicated that the sensitivity
of this method allows the detection of imbalanced two-person mixtures
similar to Y-STRs and emerging compound markers, irrespective of the
sex of donors (Table 1). However, further marker development and
analysis of trace evidence are encouraged to evaluate the effectiveness
of this new supplemental indel-based typing method for mixture de-
convolution [62].
Additionally, SNPs are short loci resulting from base substitutions
(i.e., transitions or transversions) that allow allele detection of small
PCR amplicon sizes (down to ˜50 bases) (Fig. 2) using single-base ex-
tension (SBE) assays (SNaPShot™). Due to their binary nature and low
level of mixture resolution, they are not considered optimal candidates
for mixture analysis as it is much more challenging to resolve into their
individual contributors using CE fragment analysis. Nonetheless, tri-
and tetra-allelic SNPs were identified and advocated, as they can pro-
vide valuable information on mixture deconvolution. Two initial mix-
ture investigations of tri and tetra-allelic SNPs revealed the possibility
to detect a mixture via additional alleles and disrupted peak-height
ratios. Simulations of a series of mixed samples at ˜0.05–50 ng input
DNA enabled the identification of some tri-allelic SNPs, which further
confirmed the presence of a second donor down to a 1:8 ratio similar to
standard STR analysis (Table 1) [63,64].
2.3. Factors influencing the interpretation of mixed (STR) DNA profiles
The interpretation of CE-DNA profiles can be influenced by nu-
merous factors including allele drop-out/drop-in, allele sharing among
contributors, pull-up of signal from dye colour, biological artefact of
PCR amplification (stutter), and split peak caused by incomplete ade-
nylation [2,21,65–67]. Low levels of template DNA corresponding to
approximately 15 diploid copies of cells/genomes (˜100 pg of DNA) can
result in the failure to detect heterozygous alleles, and stochastic effects
can cause the disruption of STR profile inter-locus balance [68–73]. In
addition, the failure to detect a single allele, referred to as allele drop-
out, can lead to false homozygosity while the presence of additional
alleles or allele drop-in can be the result of contamination event(s).
Stutter peak is a PCR by-product typically one repeat unit shorter than
the true allele arising during PCR due to DNA strand-slippage that can
influence the interpretation of STR data [74]. This generally results in a
higher signal (peak) for di- and tri-nucleotides than tetra- and penta-
nucleotide STR repeat motifs. Such an artefact can severely impact the
deconvolution of mixed DNA profile(s) and affect their downstream
interpretation [75] when peaks from the minor donor are in the stutter
range position of alleles from the major donor [2,76,77]. In addition,
mixed samples characterized by the presence of more than two con-
tributors can make the interpretation of the resulting profiles more
difficult. To assist DNA analysts in the interpretation of complex pro-
files multiple probabilistic genotyping software were developed.
F. Oldoni and D. Podini
Fig. 2. Schematic representation of the structure and features of conventional and novel forensic biomarkers for mixture DNA profiling.
2.4. Probabilistic genotyping software models for assisting in mixture
interpretation
Initial approaches for interpretation of forensic DNA profiles in-
cluded binary methods, which assigned a value of zero (exclusion of
genotype) or one (inclusion of genotype) to the probability of evidence
given the observed genotypes. These approaches used quantitative
(peak height) or qualitative (not peak heights) information and also a
series of thresholds (analytical and stochastic, peak height ratio, and
mixture proportion) to define the possible combinations of genotypes
from donors contributing to a mixed profile. However, their utilization
was restricted by the inability to handle multi-source low-level DNA
profiles and make full use of peak heights information [78].
Complex mixtures have become more frequent given the increased
sensitivity of typing technologies [79], which fostered the development
of new probabilistic genotyping (PG) approaches that enable the in-
corporation of the probabilities of allele/locus dropout/dropin, pre-
ferential amplification of smaller alleles, DNA degradation, and PCR
artefacts such as stutter into the likelihood ratio (LR) statistic. Prob-
abilistic genotyping generally relates to the use of bio-statistical mod-
elling and theory to calculate a likelihood ratio and predict the geno-
types of the different contributors to a DNA profile from the evidence
[78,80].
These software programs can be categorized into two main groups
according to the specific mathematical algorithm used: semi-continuous
or fully continuous. The first, also defined as discrete software models,
can factor in the probability for allele/locus dropout and dropin;
however, they are unable to incorporate quantitative information (peak
heights) and model artefacts (stutter). The second type of software uses
a continuous distribution, which allows the modelling of allele/stutter
peaks since it enables the incorporation of the peak heights behaviour
given the all-possible genotype combinations for each contributor.
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Forensic Science International: Genetics 41 (2019) 107–119
Overall, commercial [81–83] and open-source [84–87] probabilistic
genotyping software packages reduce the subjectivity in the inter-
pretative analysis of complex low-level and high-order mixed profiles
enhancing overall consistency in the conclusions. However, a feature
deserving further development is the estimation of the number of
contributors to a mixture, which is a parameter assigned by the analyst
in almost all current probabilistic software programs in use [88].
A growing number of forensic laboratories worldwide have recently
started implementation of probabilistic genotyping in casework. Such a
trend is likely to increase in the upcoming years in light of the possi-
bility to also analyse Y-chromosome STR, bi-allelic [89] and multi-al-
lelic SNP MPS mixture data.
3. Y-STR profiling
3.1. Standard and rapidly mutating (RM) Y-STR markers for mixture
analysis
Beside autosomal STR markers, STR polymorphisms located on the
X- and Y-chromosome generate forensically informative haplotypes
[90]. While X-STRs are considerably less useful for mixture analysis
[91,92], Y-STRs play a pivotal role in male detection [2,93–96]. These
are of great utility for the investigation of sexual and other biological
mixed samples with one (or more) male donor(s) mixed into a female
background. Though the discriminatory power of Y-STR analysis is
significantly lower than autosomal STRs, these markers are highly
sensitive down to ˜0.1 ng input DNA and specific at > 12,800:1 female
major:male minor and < 19:1 male major:male minor mixture ratios
[97,98] (Table 1). Since the first Y-STR casework applications in the
early 1990s [99–102], different commercial and non-commercial Y-STR
kits have been developed and successfully applied to solve challenging
cases [97,103–108]. Y-STRs are helpful for the identification of
F. Oldoni and D. Podini Forensic Science International: Genetics 41 (2019) 107–119

Table 1
Summary features of autosomal STR, Y-STR and rapidly mutating Y-STR, SNP and indel/DIP, microhaplotypes, DIP-STR, SNP-STR, DIP-SNP markers used for forensic
DNA mixture profiling on CE and MPS platforms.
Autosomal marker Lineage marker

Type of marker CODIS Binary/non- Binary Multi-SNP Compound Multi-allelic

binary STR
Locus STR SNP Indel/DIP Microhaplotype DIP-SNP DIP-STR SNP-STR Y-STR RM Y-STR

Sensitivity (ng)
˜ 0.05 – 0.1 ˜ 0.05 – 50 ˜ 0.05 – 10 ˜ 0.05* ˜ 0.1 – 10 ˜ 0.03 – 0.1 ˜ 0.05 – 10 ˜ 0.0625 ˜ 0.0625

Specificity
< 19:1 ˜ 8:1 ˜ 50:1 – > 19:1* ˜ 50:1 – 1000:1 ˜1000:1 ˜ 19:1 – > 10,000:1 > 10,000:1
1000:1 1000:1 (F:M) (F:M)
< 19:1 (M:M) < 19:1 (M:M)

Advantage
Mutation rate ↑ ↓ ↓ ↓ ↓ DIP, SNP ↓ DIP ↑ STR ↓ SNP ↑ ↑ ↗
STR
Two-person mixture ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
More than two-person ✓ ✕ ✕ ✓ ✕ ✕ ✕ ✓ ✓
mixture
Power of discrimination ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓

Disadvantage
Stutter ✓ ✕ ✕ ✕ ✕ ✓ ✓ ✓ ✓
Core marker ✓ ✕ ✕ ✕ ✕ ✕ ✕ ✓ ✓
Commercially available kit ✓ ✓ (binary ✓ ✕ ✕ ✕ ✕ ✓ ✓
(s) only)
Reference database ✓ ✕ ✕ ✕ ✕ ✕ ✕ ✓ ✕

Technology used
CE ✓ ✓ ✓ ✕ ✓ ✓ ✓ ✓ ✓
MPS ✓ ✓ ✓ ✓ ✕** ✕** ✕** ✓ ✓

Other applications
Identification ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
Paternity/kinship testing ✓ ✓ ✓ ✓ ✕** ✕** ✕** ✓ ✓
Prenatal paternity testing ✓ ✓ ✕ ✓ ✓ ✓ ✓ ✓ (1/2 X) ✓ (1/2 X)
Familial searching ✓ – – – – – – ✓ –
Missing-person ✓ ✓ ✓ ✓ – – – ✓ ✓
Degraded sample ✓ (CE mini- ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
STR/MPS STR)
Ancestry inference ✕ ✓ ✓ ✓ ✕** ✓ ✕** ✓ –

F: female; M: male; ↑: high; ↓:low; ↗: improved discriminating power; - nd; 1/2 X: only if foetus is male.
* Ongoing evaluation.
** Potential implementation.

individuals from the same paternal lineage of an unknown male culprit
and unless a mutation occurs, individuals within the same paternal
lineage will share the same Y-STR haplotype. To date, Y-STR profiling
has been applied for the analysis of imbalanced mixtures as well as in
paternity and kinship testing, paternal lineage identification and fa-
milial searching, and paternal biogeographic ancestry inference.
On top of Y-STRs, the recently developed rapidly mutating (RM) Y-
chromosomal STRs offer the opportunity to improve the identification
and discrimination of single individuals descending from same paternal
lineage both closely and distantly related [105,109]. Since rapidly
mutating Y-STRs are characterized by higher mutation rate than stan-
dard Y-STRs they significantly improve differentiation among male
relatives within the same paternal lineage [110]. A rapidly mutating Y-
STR multiplex assay was developed to simultaneously amplify the first
set of 13 loci [111,112], two and seven of which were also included in
the PowerPlex® Fusion 6C System kit [113] and Yfiler® Plus PCR Am-
plification kit [106], respectively. A comprehensive pedigree study on
the endogamous Pakistan population [114] and additional worldwide
populations [115–125] were further conducted to gather global hap-
lotype diversity datasets useful for differentiation among male relatives,
estimation of the discrimination power and statistical weight of a ra-
pidly mutating Y-STR haplotype match observed in an evidence sample.
To further increase the differentiation among male relatives, an
expanded set of rapidly mutating Y-STRs has been recently developed
[126] and shown to perform similarly to the first established markers.
Altogether the rapidly mutating Y-STR sets are as sensitive and specific
as standard Y-STRs, and enable the improved differentiation among
male close relatives, compared to results obtained using only 13 loci.
Overall, rapidly mutating Y-STR is a powerful test used for mixed
samples (Table 1) and the analysis of compromised skeleton remains
(i.e., bones and teeth) [127]. However, further development of geo-
graphically exhaustive Y-reference databases is essential for expanding
the probability of a Y-haplotype match [128].
4. Compound biomarker profiling
4.1. Compound biomarkers for solving imbalanced two-person mixtures
The development of a specific PCR amplification method targeting
unique genomic regions (i.e., alleles) of the minor donor represents an
alternative typing approach to untangle the detection and deconvolu-
tion of mixtures. To overcome the constraints associated with the
analysis of autosomal STRs (refer to section 2.1), a new category of
forensic molecular biomarkers named compound markers has been
recently introduced to specifically target imbalanced mixtures of two
DNA contributors.
Initially, Hall et al. developed the analytical DIP-STR (i.e., deletion
insertion polymorphism-short tandem repeat) profiling approach [129].
This new marker relies on pairing a DIP (i.e., target sequence) with a
closely linked STR locus, which provides additional informational

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F. Oldoni and D. Podini
content, and facilitates the detection of a specific minor donor when
mixed with a foreign DNA in an imbalanced two-person mixture
[130,131]. This compound marker is characterized by a given haplo-
type including DIP and STR alleles targeted with highly specific PCR
primers (Fig. 2). The full marker region is amplified using a forward
primer that overlaps upstream of the deleted/inserted sequence and a
reverse primer that anneals downstream of the STR region. The forward
primer is specific for the DNA sequence of interest (insertion or dele-
tion) and covers either the insertion (long or “L” allele) or deletion
(short or “S” allele) allele sequence by targeting unique DIP alleles of
the minor donor. The reverse primer is not allele specific and, along
with the forward primer, enables full STR region coverage and gen-
eration of a polymorphic haplotype, which is produced by the specific
amplification of S/L DIP alleles of the minor donor in the presence of up
to 1000-fold excess of the major donor (Table 1). The specificity in
primer design and allele amplification renders this novel marker ap-
proximately one hundred times more sensitive than standard autosomal
STR loci. A direct PCR-to-CE typing approach is used for the amplifi-
cation of these markers that can be either informative or uninformative
according to the specific S/L allele DIP mismatch between major and
minor donor. Readers are referred to [130,132] for a detailed descrip-
tion of different case scenarios.
As with DIP-STR, the SNP-STR marker type was also developed to
aid deconvolution of imbalanced two-person mixtures. These markers
are known for sequence-specific amplification and are characterized by
the presence of a bi-allelic SNP closely linked to a polymorphic STR
locus [133–136]. SNP-STRs enable the detection of different subtypes of
STR alleles based on the linked SNP allele observed in the flanking STR
region. To prevent common PCR bias in mixture analyses, Zhang et al.
[137] proposed an amplification refractory mutation system (ARMS)
PCR method for the amplification of the DNA sample of interest with a
specific target SNP [138] (Fig. 2). This is an unconventional amplifi-
cation strategy where sequence-specific PCR primers are designed to
discriminate among DNA templates that differ by a single nucleotide.
These loci are PCR amplified using two forward allele-specific primers
labelled with different fluorescence and one reverse primer positioned
flanking the core-STRs linked to the target SNP of interest. The primers
enable the amplification of selected sequences whose 3′ends is com-
plementary to the SNP allele of interest by the addition of a deliberate
mismatch at the 3′ends of the primers. The generated SNP-STR fluor-
escently labelled amplicons are genotyped using the same techniques as
for STR profiling. However, the ARMS primers for these markers can
target the unique SNP allele of the minor donor when the genotype of
the major donor is different at the SNP location.
More recently, Liu et al [139] have proposed a third type of mole-
cular compound biomarker named DIP-SNP, which combines a DIP
locus of 3–7 bp and a downstream SNP locus within a short expanse of
DNA amplified using allele-specific primers. This is specifically de-
signed to target unique alleles of the minor donor similar to DIP-STR
and SNP-STR primers. In particular, the 3′end of the primers is designed
to pair with the deletion (S-primer) or insertion (L-primer) of the up-
stream DIP locus while the reverse primer (R-primer) anneals to a
shared base position downstream of the SNP locus (Fig. 2). Two PCR
amplifications are performed before minisequencing with the SNaP-
shot™ assay: one for the L-reaction containing L- and R-primers and one
for the S-reaction containing S- and R-primers. The genotyping of the
mixed sample is the result of the typing combination of L- and S-reac-
tions.
In essence, the allele-specific amplification process characterizing
compound biomarkers is an alternative PCR method that uses primer
pairs specifically tailored to the detection of the unique alleles of the
minor donor. Consequently, these markers suffer from the requirement
of using reference DNA samples to select and target informative loci
from the expected minor donors [130]. However, in common forensic
practice the reference samples from alleged perpetrators are often un-
available for DNA testing. Provided that these are available to the
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Forensic Science International: Genetics 41 (2019) 107–119
forensic DNA analyst, a blind interpretation of DNA profiles must also
be performed to comply with standard DNA testing procedures and
avoid post hoc rationalization of results. The use of this molecular ap-
proach for typing of imbalanced mixtures of two donors, along with
constraints associated with the assay customization required for each
specific case scenario, however, tend to seriously lessen their applica-
tion in casework. Nonetheless, these markers are an effective added
value marker system useful to complement the analysis of conventional
STR loci when these fail to produce a DNA profile suitable for com-
parison. This is due to their high sensitivity, specificity, and ability to
target any type of two-person DNA mixture, regardless of the sex of the
individuals. Maternally and paternally related individuals, and also
unrelated male and female donors, can potentially be differentiated in a
mixture accordingly.
4.2. Development and evaluation of marker sets
For DIP-STRs, Castella et al. [130] originally developed a proof-of-
concept set of nine markers, characterized by DIPs closely linked to well
characterized STRs. Though the linked STR loci were known, the for-
ensic utility of this marker-set was severely restricted due to some
disadvantageous features that included long PCR products (with three
loci over 650 bp), di-nucleotide repeat motif in seven of the nine loci,
high stutter peak of di- and tri- nucleotide repeat motif and skewed
allele frequencies of some DIPs, which lead to low probabilities of de-
tecting informative haplotypes [130]. To partially overcome these
limitations and increase the number of informative markers available
for testing, Oldoni et al. [131] developed an additional set of nine
markers selected according to forensic technical standards. This ex-
panded set of markers, spreading across the genome, was characterized
by tri-, tetra- and penta-nucleotide motifs of non-core STR loci that
drastically lowered the susceptibility to stutter peaks. As also noted by
the authors, the nine-loci set included new STRs, which can severely
restrict its global application in casework. However, these nine loci
proved highly polymorphic and informative on a set of 103 Swiss in-
dividuals [131] with a total of six to 15 haplotypes and observed het-
erozygosity > 0.64. When the markers were tested in the Southwest
Chinese Han population [140], five to 16 haplotypes and hetero-
zygosity > 0.50 were also observed. This marker-set including loci of
reduced size between 146 and 271 bp, was sensitivite between 0.03 and
0.1 ng, specific down to a 1:1000 ratio and informative (hetero-
zygosity > 0.25), and also potentially useful for the analysis of cell-free
and degraded two-person mixtures (Table 1).
For SNP-STRs, Wang et al [136] reported an early initial proof-of-
concept set of loci including one locus with a SNP linked to a widely
used STR, another locus characterized by a SNP linked to an Extended
European Standard Set STR and a third to a US core STR locus [141].
These loci showed specificity at a 1:40 ratio and 0.025 ng of DNA input
when amplified in a single multiplex PCR reaction, thus making them
potentially useful for complementing standard STR analysis (Table 1).
Tan et al [142] developed an additional set of eight short loci with
SNPs located closer than 250 bp from the core STR locus displaying
minor allele frequency (MAF) > 0.1 and high levels of polymorphism in
the Chinese Han population. However, these markers were only tested
in singleplex reaction on artificially prepared mixtures at 10 ng input
DNA and with the minor donor (heterozygous for the SNP) in the
presence of 10-, 20-, 50- and 100-fold excess of major donor (homo-
zygous for the SNP). The discrimination power reported was low even
though the minor donor was detected at the 1:100 ratio using all pri-
mers. The combined informativeness value showed approximately a
93% chance to discriminate a mixture using seven loci.
To further expand the number of loci available for testing, Tan et al.
also developed three additional loci [143] that were included in a final
11-locus set. These included four known STRs (i.e., D5S2500,
D11S4463, D6S474, D20S482) from commercial AGCU 17 + 1 multi-
plex kits (Wuxi AGCU ScienTech Incorporation, Wuxi, China).
F. Oldoni and D. Podini
Amplicons of the 11 loci ranged between 141 bp and 313 bp and were
tested on 154 unrelated individuals from a southwest Chinese Han
population. The number of observed haplotypes for all markers ranged
between 9 and 19, the MAF varied from 0.09 to 0.46 and the hetero-
zygosity was > 0.700. On average, the probability of detecting in-
formative genotypes among the 11 loci increased up to 0.297, with a
98% probability of detecting at least one informative allele of the minor
donor. All tested allele-specific primers (positive detection at 0.025 ng)
specifically amplified the minor donor down to a 1:100 ratio in a two-
person mixture using 10 ng of combined DNA. Probative results of an
in-silico mixture case generated an RMP value of 2.9 × 10−8 for the
minor DNA based on the informative alleles. As suggested by the au-
thors, a larger panel of SNP-STR loci is needed to increase the number
of informative alleles from the minor donor.
In addition, Wei et al. developed a new multiplex set of eight loci
[144], including SNPs linked to commonly used STR loci from com-
mercially available CODIS-based loci kits, NIST miniSTR 26plex,
AmpFlSTR Sinofiler™ (Thermo Fisher Scientific, MA, USA) and Power-
Plex®21 (Promega, Madison, WI, USA). The assay was sensitive down to
50 pg of input DNA. The number of haplotypes observed for the eight
loci, varying from eight to 20, was higher than the number of alleles
observed for the corresponding STR loci when tested on 350 unrelated
individuals of Chinese Han population in Hubei province. Overall the
combined PD (power of discrimination) and PE (power of exclusion)
were 0.9999 and 0.9996, respectively. The multiplex PCR assay was
specific down to 0.05 ng with all SNP-STR haplotypes of minor com-
ponents reported at 1:20 ratio in imbalanced DNA mixtures from two
individuals. The detection ratio of the minor DNA increased down to
1:50, 1:100 and 1:1000 for some SNP-STRs when typed with separate
SNP allele-specific primers. Further application to forensic casework-
like samples would be useful to evaluate the utility of SNP-STRs in
mixture analysis.
The high abundance of DIP and SNP loci across the human genome
also justifies the effort to search and characterize additional informative
compound biomarkers. Liu et al developed a proof-of-concept set of 14
DIP-SNP loci and implemented the assay on the CE detection system.
The short amplicon size ranging between 80 and 300 bp makes these
loci useful for the analysis of degraded DNA samples. [139]. Overall
these markers show high sensitivity in singleplex and multiplex PCR
reactions and enable detection of the minor donor down to 0.1 ng DNA,
though a minimum input amount of 1–10 ng was suggested for optimal
results. This 14-locus set allowed the detection of the minor donor
down to a 50-100-fold excess of the major donor in multiplex and at
1000-fold of the major donor in singleplex reactions (Table 1). Most loci
showed a high level of heterozygosity on a set of 60 Chinese Han in-
dividuals from Shanxi province and balanced distribution of alleles in
all but one marker (rs558727698-rs71436264) for which no insertion
allele was observed in the population tested. The overall combined
power of discrimination for the DIP-SNPs (rs558727698-rs71436264
excluded) was 0.99 and no linkage disequilibrium among loci located
on the same chromosome was observed. In addition, the probability of
markers being informative for the minor donor was < 0.374 for 14 loci
with an average I value of 0.33 for 13 loci (rs558727698-rs71436264
excluded).
4.3. Mixture application in forensic scenarios
Thus far the application of compound biomarkers to forensic-like
and real evidence samples has been evaluated in few studies. For DIP-
STRs, Oldoni et al. designed two specific mixture studies to compare the
performance of DIP-STRs to standard autosomal STR and Y STR ana-
lysis. One aimed to target the minor donor on casework-like trace
biological material transferred through handling (“touch” DNA), re-
gardless of sex [145]. Selected DIP-STRs from the original marker set
were tested in singleplex PCR reaction and showed an improved per-
formance in mixture detection compared to conventional autosomal-
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Forensic Science International: Genetics 41 (2019) 107–119
and Y chromosome STR profiling [145]. The second study tested the
two currently published sets of DIP-STRs on a series of real casework
contact- and intimate-type traces [146]. Evidence DNA samples in-
cluded same and opposite sex individual pairs, low total DNA amounts
and/or high major/minor DNA ratios, and trace samples collected on
suspects that masked the DNA of the victim. Overall, this initial case-
work study indicated that DIP-STR loci can complement the analysis of
autosomal and Y chromosome STRs. Such a technique may provide
additional probative value to the only evidence available in support of
the propositions from prosecution or defence. DIP-STR profiling may
also benefit the analysis of imbalanced mixtures occurring in vivo (i.e.,
DNA micro-chimerism) in a much broader sense. These loci can be
applied as peripheral biomarkers for non-invasive patient care in pre-
natal genetics [147], and also in transplant-induced DNA chimerism.
The latter can originate through the transient circulation of minute
quantities of foetal DNA into the maternal blood during pregnancy
[148,149] or transient of low quantities of donor’s DNA into biological
fluids (e.g., blood and urine) of organ-transplanted patients [150–153].
A recent investigation of 28 DIP-STRs typed from circulating maternal
blood samples collected during early pregnancy of gestational age en-
abled to target sequence-specific paternally inherited foetal alleles
[147]. In addition, the low and fast evolving DIP-STR haplotypes are
likewise promising markers for targeting and predicting the ancestry
inference of a donor in a DNA sample, as recently shown in a pre-
liminary evaluation of a set of 23 DIP-STRs across the worldwide
HGDP-CEPH reference population panel [154].
In general, SNP-STRs are more abundant in the human genome than
DIP-STRs, for which no reference data of DIP-linked STR loci [131] are
available, and can be used for different forensic applications including
paternity testing [155]. As reported in an initial alleged father-son duo,
investigation where one inconsistency was reported at the CSF1PO STR
locus, the characterization of a SNP-CSF1PO locus enabled the attain-
ment of a higher PD value than STR and SNP alone [155]. Similarly, in
a non-invasive prenatal paternity testing investigation [156], six in-
formative SNP-STRs (< 210bp) were screened based on the Expanded
U.S. Core STR loci from maternal and paternal reference profiles. Po-
sitive detection of the cell-free foetus (cff) DNA circulating in maternal
plasma of ten pregnant women at 16 to 22-weeks was confirmed by
informative SNP-STRs. The widespread localization of SNP-STRs and
different mutation rate of SNPs and STRs make these loci also poten-
tially useful for the evolutionary analysis of human [136,157], and non-
human DNA [158,159]. As with other compound loci, DIP-SNPs can
also be used for the analysis of mixtures occurring in vivo (e.g., foetus
free DNA circulating in pregnant women’s plasma).
Overall, these initial investigations of compound biomarkers high-
lighted the need for further marker development in compliance with
current forensic technical standards in order to increase the detection of
informative haplotypes. In addition, more robust application-oriented
studies on real evidence and casework-like samples are needed along
with a rigorous update of the statistical framework for the evaluation of
compound biomarker DNA profiling [160].
5. Emerging MPS strategies for mixture deconvolution
5.1. Impact of MPS STR profiling
Massively parallel sequencing provides extensive high-throughput
data and the ability to rapidly and inexpensively (on a per-base fashion)
interrogate a large battery of forensically relevant markers of up to 400
nucleotides in length, in parallel, and with no problems with alleles
from different loci overloading. Since multiplex assays for MPS are no
longer constrained by the number of available fluorescent labels, pri-
mers can be designed to PCR amplify a panel of STR loci within a si-
milar size range.
Such features make MPS particularly suitable for simultaneously
addressing different forensic questions in a single targeted workflow
F. Oldoni and D. Podini
and may in turn enhance the resolution of mixture deconvolution
analysis by increasing the discrimination power of DNA evidence.
Current MPS platforms allow sequencing of relatively short sequence
reads, which cover most core STR loci except those characterized by
long repetitive stretches [26,27]. Some early MPS investigations at-
tempted to evaluate the resolution limit of conventional STRs by tar-
geting the minor donor in two-source mixtures of known ratios, and
proved that the interpretation of STR sequence data is not as straight-
forward as initially anticipated [74]. This was also due to the un-
desirable presence of stutter peaks of similar height to the minor allele
peaks in STR amplifications, which can be detected in stutter range
position of the parental alleles of the major contributor(s), further
complicating the analysis of STR profiles. The stutter formation is in-
fluenced by different characteristics of a given sequence that comprises
the A/T content of the specific repeat unit and the number of unin-
terrupted repeat units within the specific STR locus [161].
While the generation of stutter peaks is more straightforward for
simple STR loci, additional attention must be given to those STR loci
characterized by compound and complex repeat structure (e.g., D21S11
and D12S391), which are the most polymorphic and informative loci
[74]. Stutter occurrence can be proportional to the length of the longest
uninterrupted stretch (LUS) of repeats occurring within an STR locus
[77,162].
To help determine the characteristics of stutter of sequenced STR
alleles, Hoogenboom et al. developed the Forensic DNA Sequencing
Tools (FDSTools) open-source software solution. [162] This uses a large
empirical dataset to predict (train) stutter and additional PCR or se-
quencing artefacts for each STR allele. Moreover, specific stutter models
can be generated for a given repeat element to predict stutter artefacts
that are not encompassed in the reference set. In particular, Promega
Powerseq™ Auto System data, generated from a set of 450 reference
samples and a series of 31 two-DNA mixtures, indicated that FDSTools
correction module lowers the stutter ratio from > 20% to < 3%.
Recently, Vilsen et al. proposed an alternative approach to predict
the stutter ratio, called block length of the missing motif (BLMM) [163].
This model can be made dependent on both the STR locus and motif.
Understanding the origin of stutter within a specific sequence and how
various repeat motifs influence the formation of stutter peaks may
provide additional useful information on the interpretation of mixed-
source DNA profiles. Consequently, “poly-stuttering” of complex STR
structure and the sensitivity of MPS may introduce an additional level
of complexity and uncertainty to the deconvolution of imbalanced STR
mixtures.
5.2. Initial evaluation of MPS STR mixture profiling
This section provides a general overview of the mixture perfor-
mance of both early and recent panels of STRs on MPS platforms, and
also a comparison to CE analysis; however, it is not meant to be an
exhaustive description of all MPS STR kits. To date, MiSeq™ (Illumina)
and Ion Torrent™ (Thermo Fisher Scientific) are the two most-estab-
lished MPS platforms for forensic applications. Different STR panels,
including the expanded CODIS, the ESS core STR loci, and additional
autosomal and Y chromosome STR loci, are commercially available
[164].
For sequence-based STR analysis on the MiSeq™ platform, Zeng
et al. reported on the use of the prototype PowerSeq™ Auto System of 22
STR loci, one Y-STR and the Amelogenin locus [165]. They compared
the results to mixtures generated using the PowerPlex Fusion System
(Promega) on a CE platform. Mixtures at 19:1, 9:1, 6:1, 4:1, 2:1, 1:1,
1:2, 1:4, 1:6, 1:9 ratios were amplified using 0.5 ng input of DNA along
with mock forensic casework samples (blood/saliva and saliva/semen
mixtures). Overall, the MPS results indicated that the prototype Pow-
erSeq™ STR multiplex system could provide results that are concordant
and comparable in performance with CE fragment analysis for inter-
pretation of two-person DNA mixtures. The MPS PowerSeq™ Auto
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Forensic Science International: Genetics 41 (2019) 107–119
System, however, enabled the detection of more alleles than CE frag-
ment analysis with partial profiles of the minor contributor reported up
to 19:1 ratio.
Van der Gaag investigated a prototype version of the PowerSeq™
(Promega) system on the MiSeq™ sequencer. The kit includes 17 auto-
somal STRs and the Amelogenin locus [166] and was tested on a series
of mixture ratios (1:99, 5:95, 10:90, 20:80, 50:50, 80:20, 90:10, 95:5
and 99:1) using a total DNA input varying from 0.5–6 ng, with a
minimum of 60 pg of the minor donor. The sequence reads representing
the minor contributor down to 1% of the total DNA profile could only
be recovered for a few STR loci. This was due to the presence of extra
PCR amplification or sequence error variants for some of the loci that
could complicate the interpretation of STR profiles, without prior
knowledge of the mixture ratio, and also stutter issues [166]. Stutter
ratios of sequence-based STR alleles generally resemble those of sized-
based STR allele aside from complex STR loci for which CE alleles can
be separated into their sequenced alleles.
Moreover, Guo et al. tested the performance of ForenSeq™ DNA
Signature Prep Kit from MiSeq FGx™ Forensic Genomics (Illumina)
System in a mixture study using 2800 M and 9947 A Control DNA
samples. Mixtures at various ratios (1:49, 1:19, 1:9, 1:1, 9:1, 19:1 and
49:1) were prepared at 1 ng of input DNA [167]. Robust performance in
typing the minor alleles was reported at 1:9 and 9:1 ratios, and also at
19:1 ratio, but it was not confirmed at 1:19 ratio. However, a decrease
in the number of minor alleles was reported significantly at 1:49.
Though the ForenSeq Kit™ showed better performance than conven-
tional CE-STR kits to generate the full profile of the minor contributor,
mixture deconvolution was challenging without using reference DNA
profiles.
Similarly, the EU funded project DNASeqEx (DNA-STR Massive
Sequencing & International Information Exchange) investigated the
Forenseq™ DNA Signature Prep Kit on the MiSeq FGx™platform. A series
of male:female (1:1, 1:5, 1:10, 1:15, 1:20, 20:1, 15:1, 10:1, 5:1 ratios) as
well as male:male (1:1, 1:5, 1:10, 1:15, 1:20 ratios) and male:female
(1:100, 1:500 and 1:1000 ratios) mixtures was prepared using 1–2 ng of
DNA. Overall, the minor DNA’s profile (shared and unshared, obligate
or unique) was detected down to a ratio of 1:20 in all female-male and
male-male while no allele could be detected at more extreme ratios
(1:1000) using either MPS or CE platforms, as previously reported by
Jäger et al. [168]. When comparing the results of extreme mixtures
using the Forenseq™ DNA Signature Prep Kit to CE (NGM Select™ PCR
Amplification Kit and PowerPlex® Y23 System) more alleles of the
minor donor could be reported using the sequence-based STR kit. When
comparing the performance of the comprehensive MPS multiplex Kit
(autosomal STRs, Y-STRs and X-STRs, and individual identification
SNPs) to CE methods, the separate PowerPlex® Y23 kit could detect
most of the male minor alleles at severely underrepresented male
mixture contributions. However, this could not be reported by se-
quenced-STRs because of the potential primer competition (preferential
amplification) between female and male donors.
For sequence-based STR analysis on the Ion Torrent platform,
Fordyce et al. initially evaluated the human identification STR 10-plex
on the Ion PGM [169] platform. The panel was tested on a series of
mixtures that covered balanced and imbalanced ratios (1000:1, 100:1,
50:1, 20:1, 10:1, 5:1, 2:1, and 1:1) and using specific input DNA amount
variable from 1 ng to 10 ng. The authors reported on the ability to de-
convolute mixtures up to 20:1 ratio akin to CE-STR analysis. However,
the reads from the minor contributor proved difficult to distinguish
from sequences related to stutter and baseline noise for more extreme
mixture ratios
Wang et al. evaluated the mixture performance of the Precision ID
GlobalFiler™ NGS 32-plex STR Panel (Thermo Fisher Scientific) on the
Ion PGM platform. The kit enables the multiplex amplification of 29
autosomal STRs, one Y-STR locus and Y-indel, and Amelogenin. A series
of mixtures (i.e., 29:1, 19:1, 9:1, 4:1, 1:1, 1:4, 1:9, 1:19 and 1: 29) at a
total amount of 1 ng was created [170]. All non-overlapping minor
F. Oldoni and D. Podini
alleles (i.e., control DNA samples 007 and 9947 A) were detected at 1:4
and 1:9 ratio while partial minor STR profiles (8–10 of the 25 non-
overlapping alleles) were detected at approximately 1:19 ratio. No
minor alleles could be detected at most loci at higher order mixture
(1:29 or 29:1).
More recently, Wang et al. [171] reported on the first mixture in-
vestigation of an early access 25-plex STR panel (Thermo Fisher Sci-
entific) on the Ion S5 XL™ system. A series of balanced, moderately and
highly imbalanced two-person mixtures (1:49, 1:19, 1:9, 1:6, 1:3, 1:1,
3:1, 6:1, 9:1, 19:1, and 49:1) at 1 ng of total DNA was prepared. Similar
to the aforementioned studies, alleles from the minor donor could be
reliably genotyped within a 1:9 to 9:1 ratio range and with most minor
alleles reported at 19:1 when no overlapping alleles between the two
DNA contributors were analysed.
Overall, these preliminary MPS investigations of STR mixtures in-
dicated that, if properly applied and interpreted, MPS could be con-
sistently more sensitive, powerful, informative, and perform better in
mixture detection and deconvolution than conventional CE fragment
analysis. Indeed, MPS displays the potential of improving further mix-
ture deconvolution by enabling the detection of specific intra-repeat
variations within STR allelic variants of the minor contributor(s) in
mixed samples. However, these initial studies also highlighted the
current limitations in detecting the minor alleles due to the undesirable
preferential amplification of the major’s allele prior to library pre-
paration [25–27,166]. The benefit of mega multiplex MPS kits, in-
cluding multiple classes of DNA markers, is, however, counterbalanced
by the lack of flexibility shown by conventional CE-STR typing kits.
6. Microhaplotype profiling
6.1. Marker features
Microhaplotypes (i.e., microhaps or MHs) are novel genetic markers
recently introduced to complement conventional STR analysis used in
mixture profiling [172]. They are short loci with an expanse of DNA
that is generally < 300 nucleotides in size (i.e., “micro”) consisting of
two or more closely linked SNPs associated in three or more allelic
combinations (i.e., “haplotypes”). Since the SNPs are clustered, re-
combination rates are extremely low [173–175] and because microhaps
are single-copy multi-SNP loci, they can provide, on a per locus basis,
more information than a single SNP (Fig. 2). Sanger sequencing has
historically been the mainstay methodology for DNA sequencing;
however, it does not allow the determination of the cis/trans relation-
ship among individual SNP alleles (i.e., process referred to as phase)
from genomic DNA when two or more SNP sites are heterozygous. On
the contrary, massively parallel sequencing overcomes such limitation
by clonal sequencing of each DNA strand, and thereby distinguishing
the parental haplotypes at one specific locus and unambiguously re-
vealing the SNP phase [176,177].
Microhaps present some advantages over standard STRs that include
by definition their multi-allelic nature, absence of an STR sequence,
which prevents Taq polymerase slippage and stutter peak generation.
The preferential PCR amplification of shorter alleles, which commonly
occurs with STRs, is absent as microhap amplicons are the same size
within each locus. In addition, lower mutation rates than STRs render
microhaps a potentially effective tool for relationship testing and
missing-persons identification. However, microhaps also display some
disadvantages, including the presence of fewer alleles than most STRs
and consequently, the requirement for a larger panel of microhaps to
reach comparable values to STRs. The generation of artificial haplo-
types not observed among the DNA sample donors can occur due to
technical sequencing issues. Moreover, since allele frequencies vary
across different populations [178], population-specific allele fre-
quencies are needed to calculate the random match probability or the
likelihood ratio in forensic caseworks. The implementation of this novel
DNA-typing approach within the global forensic DNA community could
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Forensic Science International: Genetics 41 (2019) 107–119
be delayed by the laborious and time-consuming workflow of MPS,
which requires appropriate pipelines for data assembly and analysis
[179]. Microhaps are selected on the basis of two main statistics: high
effective number of alleles (Ae) within a population and informative-
ness across different populations (In). For mixture deconvolution, larger
Ae values correlate with a greater ability of microhap loci to detect and
deconvolute two or more than two-person DNA mixtures, and de-
termine the number of donors.
6.2. MPS panels of microhaps for mixture analysis
An initial assessment of microhaps for mixture deconvolution was
performed on the Kidd proof-of-concept set of 31 loci [180], which
showed an overall average Ae range of 1.9−2.8. The additional Kidd
panel of 130 microhaps includes 21 loci with Ae values from 3.0–3.9,
five loci from 4.0–4.9, and only two loci of Ae > 5.0 and with a prob-
ability of mixture detection of 99.99956%, 99.52% and 94.62%, re-
spectively. The Kidd subset of 65 microhaps also includes 22 promising
loci with Ae > 3.0.
A proof-of-concept Kidd set of 36 microhap loci, selected from a
larger panel of 130 microhaps [181], potentially useful for forensic
applications, has been recently developed and its effectiveness on
mixture deconvolution evaluated on both CE size analysis and a beta
version of MPS AmpliSeq™ kit (Thermo Fisher Scientific) [182]. The
interpretation of STR mixtures using CE was questionable because
minor DNA donor(s) were under the analytical/stochastic threshold and
in stutter range position. Conversely, MPS-microhap typing of the same
mixed samples enabled the detection of several contributors in two,
three, and four mixed source-samples at different contributor ratios,
respectively, as well as in some representative forensic-type traces using
low input of DNA [182]. Additionally, preliminary results on a novel
extended Kidd 74MHplex MPS panel sensitive down to approximately
50 pg of input DNA confirmed the ability to deconvolute highly im-
balanced mixtures of sister alleles from two contributors at > 19:1 ratio
and using both 1 and 10 ng of input DNA, though uneven performance
among microhap loci was observed [183,184]. The same study also
displayed the potential of using the same panel to simultaneously detect
and infer the biogeographic ancestry of the minor contributor [172].
These findings were confirmed in an independent study on two-person
DNA mixtures at an approximate 1:10 ratio, which demonstrated the
possibility to recover the full set of alleles from 11 loci of both DNA
donors [185].
Moreover, Chen et al. developed a panel of 26 tiny microhaps
(< 50bp) on the Hiseq X® Sequencer® (Illumina) platform [186]. These
markers presented a high effective number of alleles (Ae) and high
heterozygosity. However, only 14 out of 26 were successfully multi-
plexed and tested for mixture analysis. The performance of the assay
was evaluated on two simulated two-donor mixtures at a 1:1 ratio and
600 ng input of DNA. The preliminary results indicated that one-third of
the loci displayed three or more haplotypes in the artificially prepared
mixtures, thereby suggesting their promise for mixture deconvolution.
Van der Gaar et al. [187] developed a set of 16 short loci that showed
that the power to detect a third allele in a two-person mixture for at
least one of the 16 microhaps ranged from 0.995 to 0.9989 in Asian and
Dutch populations, respectively and up to 0.99992 in African popula-
tion.
Finally, Voskoboinik et al. also suggested the use of additional
polymorphic haplotypes (Ae > 15) for mixture analysis [188]. They
selected 10 loci that included > 10 SNPs and performed a simulation
analysis using sequence reads from mixtures simulated to replicate the
reads from the MinION sequencing platform (Oxford Nanopore). Mix-
tures were prepared by mixing a fixed number of contributors at dif-
ferent ratios and with each contributor resulting from randomly sam-
pled haplotypes at each locus. Although marker development and
validation is required, preliminary results suggested the potential of
using these hypervariable loci to include one contributor to a four or
F. Oldoni and D. Podini
five mixed-source sample.
These initial MPS findings raise promising expectations for the
possibility of using microhaps as a supplemental DNA marker to detect
and deconvolute two- and more than two-person DNA mixtures. In
addition, microhaps are useful for biogeographic ancestry inference
[187,189,190], analysis of degraded samples, human and missing-per-
sons identification, and relationship testing [172,191,192].
7. MPS application of SNP/indel and candidate compound
markers
MPS platforms may provide great expectations for the analysis of
mixtures when also typed using SNP/indel assays [193]. As shown in a
recent study, a custom-designed 1204-SNP and indel panel was tested
on a set of degraded and non-degraded mixed samples and detection of
the minor contributor down to a 1:99 ratio was reported using a total
amount of 80–156.25 ng input DNA [194]. By taking advantage of
parallel sequencing, the combination of prospective panels of com-
pound markers on MPS platform(s) may prove useful to complement
MPS-STR typing of challenging genomic mixtures. The MPS im-
plementation of STR, SNP/indel, microhaplotype, and compound
markers represents a comprehensive molecular typing approach that
may enormously benefit analysis of evidence samples in the near future.
8. Concluding remarks
In a nutshell, MPS implementation of robust consensus panels of
compound markers along with new/optimized assays of existing/ad-
ditional STR, SNP/indel and microhaplotype assays may remarkably
aid increase the discriminating power of DNA evidence while simulta-
neously providing additional forensically useful information on the
minor donor(s) detected.
Disclosure statement
The authors declare no conflict of interest.
Acknowledgements
This study was partially supported by Swiss National Science
Foundation through grant No. 2017-P2LAP3_174742 awarded to Fabio
Oldoni and National Institute of Justice through grant No. 2017-DN-BX-
0164 awarded to Daniele Podini.
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