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Indel markers: Genetic diversity of 38 polymorphisms in Brazilian populations
and application in a paternity investigation with post mortem material
Fernanda Mantaa, Alexandre Caiafaa, Rui Pereirab, Dayse Silvaa, Anto ´nio Amorimb,c,
Elizeu F. Carvalhoa,*, Leonor Gusma ˜ob
aDNA Diagnostic Laboratory – Institute of Biology, State University of Rio de Janeiro, Rio de Janeiro, Brazil
bIPATIMUP – Institute of Pathology and Molecular Immunology of the University of Porto, Porto, Portugal
cFaculty of Sciences of the University of Porto, Porto, Portugal
In human genetic identification, it is important to select highly
polymorphic markers to reach an elevated discrimination power,
even for a restricted number of loci. Nevertheless, very high levels
of polymorphism can be associated with high mutation rates,
which are a limitation in kinship analysis. Due to their high levels
of variation, Short Tandem Repeats (STRs) are the most frequently
used DNA markers in human identification. The universal forensic
use of STRs, for almost two decades, has allowed the establishment
of well structured national databases, simple methodologies, and
to evaluate relevant statistical parameters. Despite the above
advantages, most STRs present relatively large amplicon sizes,
between 150 and 450 base pairs (bp), which can negatively
influence the analysis of challenging forensic samples .
Limitations of sample quantity and/or degradation can also create
problems in forensic casework, where DNA samples are usually far
from pristine. In cases where the DNA is exposed to the
environment, degradation can occur due to bacterial, biochemical,
or oxidative processes . It has been demonstrated that smaller
amplicons are much more likely to be amplified in samples
containing degraded DNA .
In order to overcome the limitations of STRs outlined above,
slow mutating bi-allelic markers analysed in shorter amplicons,
including Single Nucleotide Polymorphisms (SNPs) and insertion/
deletion polymorphisms (indels), have been recently introduced to
the forensic identification field [4–8]. The lower mutation rate
SNPs and indels present when compared to STRs contributes to
their usefulness in paternity cases showing just a few observed
incompatibilities [9–11]. Moreover, they have proved to be
consistently better than STRs with conventional amplicon lengths
for the analysis of degraded DNA samples [4,10,12].
In recent years, indels have received growing attention. After an
aimed to adopt indels as a tool in human genetic studies. In 2006,
Mills et al.  systematically identified indel polymorphisms
using a new computational strategy, resulting in an initial
insertion/deletion map containing more than 415,000 unique
polymorphisms, with an average density of one indel per 7.2 kb.
Recent follow-up studies have reported around 2 million small
insertions/deletionsrangingfrom1 bpto10,000 bpinlengthinthe
genomes of 79 diverse humans . These small indels, involving
Forensic Science International: Genetics 6 (2012) 658–661
A R T I C L EI N F O
Received 12 September 2011
Received in revised form 28 December 2011
Accepted 30 December 2011
Rio de Janeiro
A B S T R A C T
Aiming to evaluate the usefulness of 38 non-coding bi-allelic autosomal indels in genetic identification
and kinship testing, three Brazilian population samples were studied: two from Rio de Janeiro (including
a sample of individuals with self-declared African ancestry) and one Native American population of
Terena from Mato Grosso do Sul. Based on the observed allele frequencies, parameters of forensic
relevance were calculated. The combined power of discrimination of the 38 indels was high in all studied
groups (PD ? 0.9999999999997), although slightly lower in Native Americans. Genetic distance analysis
showed significant differences between the allele frequencies in the Rio de Janeiro population and those
previously reported for Europeans, Africans and Asians explained by its intermediate position between
Europeans and Africans. As expected, the Terena sample was significantly different from all the other
populations: Brazilians from Rio de Janeiro general population and with self-declared African ancestry,
Europeans, Africans and East Asians. Finally, the performance of the 38-indel multiplex assay was tested
in post-mortem material with positive results, supporting the use of short amplicon bi-allelic markers as
an additional tool to STR analysis when DNA molecules are degraded.
? 2012 Published by Elsevier Ireland Ltd.
* Corresponding author at: Laborato ´rio de Diagno ´stico por DNA, Sa ˜o Francisco
Xavier, 524 Maracana ˜ CEP: 21550-013 Rio de Janeiro, RJ, Brazil.
Tel.: +55 21 23342183; fax: +55 21 23340594.
E-mail address: email@example.com (E.F. Carvalho).
Contents lists available at SciVerse ScienceDirect
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1872-4973/$ – see front matter ? 2012 Published by Elsevier Ireland Ltd.
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apparently random DNA sequences, represent ?41% of all indels,
and show a wide range of allele length variation, with the majority
under 100 bp [14,15].
In 2009, Pereira et al.  described a new multiplex for human
identification combining 38 small non-coding biallelic autosomal
indels into a single multiplex. This multiplex benefits from using a
single PCR where the products are then detected directly by
standard Capillary Electrophoresis (CE) analysers. Furthermore,
amplicon length was designed to be shorter than a maximum of
160 bp. The same study also demonstrated the feasibility of
obtaining complete profiles using 0.3 ng of DNA, while successful
genotyping of degraded samples was possible in cases where
standard STR typing had partially failed .
The aim of the present work was to evaluate the usefulness of
the 38-indel multiplex  applied to Brazilian forensic casework.
We have studied three populations from Brazil, a sample of Native
Americans from a Terena tribe living in Mato Grosso do Sul, plus
two samples from individuals living in Rio de Janeiro; one selected
from the general population and the other including self-declared
African admixed individuals. These samples were typed for the 38
autosomal indels, and forensic informativeness parameters were
calculated. We also tested the 38-indel multiplex with samples
from human remains in order to evaluate the use of indels as a
complementary tool in kinship analysis.
2. Materials and methods
2.1. DNA samples
A random sample of 137 unrelated individuals was selected
from paternity cases, in Rio de Janeiro, South East Brazil.
Additionally, two samples were selected to represent Brazilian
groups withdifferent ancestrycontributions:45unrelatedTerenas
(Native Americans living in the state of Mato Grosso do Sul, Central
West Brazil) and 115 unrelated individuals living in Rio de Janeiro
and self-declared as having African ancestry.
Samples were collected with informed voluntary donation for
research purposes approved by the Ethics Committee of the
Federal University of Mato Grosso do Sul (CI n8 015/98) and the
State University of Rio de Janeiro (CAAE: 0067.0.228.000-09).
Genomic DNA was extracted from peripheral blood samples using
a salting out method.
Although Rio de Janeiro is an admixed population where three
different ancestries of European, African and Native American
origins contribute to high population variability, no population
substructure has been described. For this reason, we did not take
into account ancestry information when select a sample to
represent a forensic reference population of the people currently
living in Rio de Janeiro. As it is known that a diverse pattern of
admixture exists in Brazil, we included a sample from Rio de
Janeiro with a high African ancestry to infer the expected diversity
of Afro-descendant groups for the indel loci typed and to evaluate
the impact of substructure on the results of identification when
using a single Brazilian database.
In order to extend the worldwide coverage of population data
for the 38-indel multiplex and to evaluate the discrimination
power of this set of markers in Native American populations, the
Terena sample was studied and compared to the European, African
and East Asian population groups originally reported .
Finally, to evaluate the performance of indels in paternity cases
involving post-mortem samples, we selected a mother-child-
alleged father trio where the father had deceased and no
amplification was achieved with standard STRs for his sample
(AmpFlSTR1Identifiler kit used; Applied Biosystems, CA, USA).
Blood samples were obtained from the mother and child and DNA
was extracted using a standard Chelex method. DNA was extracted
from teeth and bones of the alleged father using the DNA IQTM
Casework Sample Kit for Maxwell116 (Promega Corporation, WI,
USA), following manufacturer’s instructions. AmpFlSTR1MiniFiler
(Applied Biosystems) typing was then used to compare to indel
2.2. Indel genotyping
The 38 indels were genotyped using the PCR multiplex protocol
originally described . Amplification products were separated by
electrophoresis in a 3130 Genetic Analyzer (Applied Biosystems)
and fragment sizes and allele calls were determined automatically
using GeneMapper v3.2 ID software (Applied Biosystems).
2.3. Data analysis
Allele frequencies, Hardy–Weinberg equilibrium analysis and
estimation of population pairwise genetic distances (FST) were
computed using the Arlequin v3.0 software . Gene diversity
was calculated as the expected heterozygosity for diploid data,
using the Nei’s formula, as implemented in Arlequin. Statistical
parameters of forensic interest: power of discrimination (PD),
power of exclusion (PE) and matching probability (MP) were
calculated using PowerStats v1.2 (Promega Corporation). Genetic
distances were visualized in two-dimensional plots using the
multidimensional scaling (MDS) method included in data analysis
software STATISTICA v8.0 (StatSoft, Inc. 2007; www.statsoft.com).
3. Results and discussion
3.1. Forensic utility of the 38 indels
The allele frequencies of the 38 indels in the three Brazilian
populations are outlined in Table 1. As suggested by previous allele
a high genetic diversity was observed in the sample of Rio de
Janeiro and in individuals with self-declared African ancestry, with
all 38 indel markers exhibiting high heterozygosities in both cases
(0.283 < GD < 0.499; Table 1).
In the Terena population sample, a lower mean diversity value
was found compared to the Rio de Janeiro samples as well as to
those previously reported in Africans, Europeans and East Asians
. Although this result can be expected for Amerindian
populations, more subject to genetic drift, the reduced genetic
diversity in such populations may also reflect the selection criteria
originally used, focusing on highly polymorphic indels in those
three major population groups .
Forensic parameter estimates comprised a combined matching
probabilityvalueof1.4 ? 10?15(PD = 0.9999999999999990)forRio
de Janeiro population; 3.2 ? 10?15for Rio de Janeiro Afro-descen-
dant population (PD = 0.999999999999997); and 2.7 ? 10?13for
the Terena population (PD = 0.9999999999997). The combined
In summary, the 38-indel multiplex was observed to be highly
informative in the Rio de Janeiro population, showing an increased
efficiency in comparison to values for Europeans and Africans .
de Janeiro are in line with values that can be expected for
populations with African and European admixture. Moreover,
these values did not differ significantly between the two Rio de
Janeiro samples, although they present differing average admix-
ture component proportions. This result can be explained by the
criteria used in the original indel selection with markers chosen to
show high diversities both in Africans and Europeans. Therefore,
similarly high discrimination power can be expected for other
F. Manta et al./Forensic Science International: Genetics 6 (2012) 658–661
Author's personal copy
Brazilian populations, evenwith contrastingproportions of African
and European ancestries.
In Native Americans, the results of this study show reduced
diversity but still present a high discrimination power and a fair
power of exclusion, providing a suitable short amplicon marker set
to complement STRs applied to a fourth population group,
characterised for the first time for these 38 indels.
3.2. Population analysis
All analysed loci met the Hardy–Weinberg equilibrium in both
Rio de Janeiro and Terena populations, after applying the
Bonferroni correction for multiple tests (exact p values ? 0.066
and 0.004, respectively). Therefore, we do not expect significant
levels of substructure in these two Brazilian populations. On the
other hand, it is not expected a large frequency of null alleles in
Native Americans, similarly to previous findings in other
continental groups .
Pairwise genetic distance analysis was performed between the
studied samples and those representatives of three continental
population groups: Europe, Africa and East Asia (Portugal; Angola
and Mozambique; Macau and Taiwan, respectively). In the
comparison of both Rio de Janeiro samples the observed genetic
distance was low, but showing a significant p value between them
(FST= 0.0054; p ? 10?5). For the remaining pairwise comparisons,
high values and significant genetic distances were observed in all
cases (Fig. 1). The MDS plot of Fig. 1, indicates the two samples
from Rio de Janeiro placed between the European and African
reference samples, with the Afro-descendants plotted closer to the
Africans. In comparison the Native Americans are more distantly
positioned from the other three groups.
3.3. Application of indels to a challenging paternity investigation
Conventional STR analysis of the degraded DNA from the
alleged father showed most loci failing amplification (Supplemen-
tary data Fig. S1) and no analysis were possible from the data. In
comparison, the AmpFlSTR1Minifiler (Applied Biosystems) geno-
typed 6 of 8 STR loci (Supplementary data Fig. S2) giving a
paternity index (PI) of 2307 (Table 2) and a near-complete profile
was obtained for the 38-indel multiplex. Some evidence of allele
imbalance and allelic dropout was observed (Supplementary data
Figs. S3 and S4) so considering just the concordant genotypes
between replicated analyses, 26 out of 38 indel loci provided a PI of
Allele frequencies, gene diversity, power of discrimination and power of exclusion in Rio de Janeiro general population (RJ); and self-declared Afro-descendants (AFD); and in
Terena from Mato Grosso do Sul, Brazil.
Loci coders number Allele frequencies (Short al-
Gene diversityPower of discrimination Power of exclusion
RJAFD Terena RJAFD TerenaRJ AFD TerenaRJ AFDTerena
Paternity indices (PI) and probabilities based on the results obtained with STRs,
indels, and with both type of markers.
STRs (Minifiler)INDELs (HID Indel-plex)STRs+INDELs
F. Manta et al./Forensic Science International: Genetics 6 (2012) 658–661
Author's personal copy Download full-text
2147. Joining the information from indels and miniaturised
STRs provided a considerably enhanced PI of almost 5 millions
allowing a sufficiently informative PI to be achieved when
individual short-amplicon markers set analysis would have given
less definitive likelihoods. These results further indicate the
suitability of indels for typing degraded forensic samples, while
commercial STR kits.
The 38 autosomal indels further characterised in this study
were observed to be suitable for general forensic purposes in
admixed populations, such as the Brazilian samples of Rio de
Janeiro we analysed. The allele frequency estimates for Terena
Native Americans provide reference data to compliment those of
the original study of the 38 indels. Indels provide an efficient way
to amplify degraded DNA, especially in situations where conven-
tional STRs can be inefficient, or may give uninformative paternity
This paper follows the guidelines for publication of population
data requested by the journal .
Financial support was granted by Coordenac ¸a ˜o de Aper-
feic ¸oamento de Pessoal de Nı ´vel Superior (CAPES), Conselho
Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico (CNPq) and
DNA Program – State University and Justice Court of Rio de Janeiro,
University of Porto (IPATIMUP) is an Associate Laboratory of the
Portuguese Ministry of Education and Science and is partially
supported by FCT.
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F. Manta et al./Forensic Science International: Genetics 6 (2012) 658–661