Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q.

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.

Population data for 21 STRs for the Salvadoran population using GlobalFiler Express and GlobalFiler STR Amplification Kits.

With the advent of expanded STR (short tandem repeats) typing kits, it was necessary to determine allele frequencies and other appropriate population data parameters for El Salvador. Samples were collected from the central, east, and west regions of the country and typed for 21 forensically relevant STR loci. The data indicate that all loci are highly polymorphic, the three regions are genetically similar, and the population data are similar to those of US Hispanics. The results of this study support that the allele frequency data described herein can be used for statistical calculations for human identity testing in El Salvador.

The Ancestry of Eastern Paraguay: A Typical South American Profile with a Unique Pattern of Admixture.

Immigrants from diverse origins have arrived in Paraguay and produced important demographic changes in a territory initially inhabited by indigenous Guarani. Few studies have been performed to estimate the proportion of Native ancestry that is still preserved in Paraguay and the role of females and males in admixture processes. Therefore, 548 individuals from eastern Paraguay were genotyped for three marker sets: mtDNA, Y-SNPs and autosomal AIM-InDels. A genetic homogeneity was found between departments for each set of markers, supported by the demographic data collected, which showed that only 43% of the individuals have the same birthplace as their parents. The results show a sex-biased intermarriage, with higher maternal than paternal Native American ancestry. Within the native mtDNA lineages in Paraguay (87.2% of the total), most haplogroups have a broad distribution across the subcontinent, and only few are concentrated around the Paraná River basin. The frequency distribution of the European paternal lineages in Paraguay (92.2% of the total) showed a major contribution from the Iberian region. In addition to the remaining legacy of the colonial period, the joint analysis of the different types of markers included in this study revealed the impact of post-war migrations on the current genetic background of Paraguay.

Second GHEP-ISFG exercise for DVI: "DNA-led" victims' identification in a simulated air crash.

The Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) has organized a second collaborative exercise on a simulated case of Disaster Victim Identification (DVI), with the participation of eighteen laboratories. The exercise focused on the analysis of a simulated plane crash case of medium-size resulting in 66 victims with varying degrees of fragmentation of the bodies (with commingled remains). As an additional difficulty, this second exercise included 21 related victims belonging to 6 families among the 66 missings to be identified. A total number of 228 post-mortem samples were represented with aSTR and mtDNA profiles, with a proportion of partial aSTR profiles simulating charred remains. To perform the exercise, participants were provided with aSTR and mtDNA data of 51 reference pedigrees -some of which deficient-including 128 donors for identification purposes. The exercise consisted firstly in the comparison of the post-mortem genetic profiles in order to re-associate fragmented remains to the same individual and secondly in the identification of the re-associated remains by comparing aSTR and mtDNA profiles with reference pedigrees using pre-established thresholds to report a positive identification. Regarding the results of the post-mortem samples re-associations, only a small number of discrepancies among participants were detected, all of which were from just a few labs. However, in the identification process by kinship analysis with family references, there were more discrepancies in comparison to the correct results. The identification results of single victims yielded fewer problems than the identification of multiple related victims within the same family groups. Several reasons for the discrepant results were detected: a) the identity/non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, b) some laboratories failed to use all family references to report the DNA match, c) In families with several related victims, some laboratories firstly identified some victims and then unnecessarily used their genetic information to identify the remaining victims within the family, d) some laboratories did not correctly use "prior odds" values for the Bayesian treatment of the episode for both post-mortem/post-mortem re-associations as well as the ante-mortem/post-mortem comparisons to evaluate the probability of identity. For some of the above reasons, certain laboratories failed to identify some victims. This simulated "DNA-led" identification exercise may help forensic genetic laboratories to gain experience and expertize for DVI or MPI in using genetic data and comparing their own results with the ones in this collaborative exercise.

Allele frequency data for 23 aSTR for different ethnic groups from Republic of Zimbabwe.

In order to determine the population allele frequencies of autosomal STR markers of forensic interest in the Zimbabwean population, we analyzed a sample of 478 individuals from 19 different ethnic groups using the PowerPlex® Fusion 6C Kit (Promega Corp, Madison, Wisconsin). The data obtained were compared among the different Zimbabwean ethnic groups as well as with several African populations to establish whether significant differences exist among them. No significant differences were found among the ethnic groups in Zimbabwe. Statistically significant differences were observed between allele frequencies in Zimbabwe and some other African populations, although FST with neighboring Bantu populations from South and Southeast regions were low (below 0.005 in most single locus comparisons).

Evaluation of mitogenome sequence concordance, heteroplasmy detection, and haplogrouping in a worldwide lineage study using the Precision ID mtDNA Whole Genome Panel.

The emergence of Massively Parallel Sequencing technologies enabled the analysis of full mitochondrial (mt)DNA sequences from forensically relevant samples that have, so far, only been typed in the control region or its hypervariable segments. In this study, we evaluated the performance of a commercially available multiplex-PCR-based assay, the Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific), for the amplification and sequencing of the entire mitochondrial genome (mitogenome) from even degraded forensic specimens. For this purpose, more than 500 samples from 24 different populations were selected to cover the vast majority of established superhaplogroups. These are known to harbor different signature sequence motifs corresponding to their phylogenetic background that could have an effect on primer binding and, thus, could limit a broad application of this molecular genetic tool. The selected samples derived from various forensically relevant tissue sources and were DNA extracted using different methods. We evaluated sequence concordance and heteroplasmy detection and compared the findings to conventional Sanger sequencing as well as an orthogonal MPS platform. We discuss advantages and limitations of this approach with respect to forensic genetic workflow and analytical requirements.

The maternal inheritance of Alto Paraná revealed by full mitogenome sequences.

Most studies on maternal lineages of South America populations are restricted to control region (CR) markers and, for some geographical regions, the number of studied samples does not adequately represent the existing diversity. This is the case of mitochondrial DNA (mtDNA) studies on Paraguay that are limited to two Native ethnic groups. To overcome this deficiency, we analysed the mitogenomes from 105 individuals living in Alto Paraná, the second most populated department of the country. Using the Precision ID mtDNA Whole Genome Panel, the molecule was sequenced on Ion S5. The majority of the haplotypes belong to the Native American lineages A, B, C and D. Analyses of maximum parsimony using mitogenome data retrieved from publications and in The 1000 Genomes Project showed a high number of new native American subclades in Paraguay. Also, none of the haplotypes found in Alto Paraná match the remaining South American samples, which include admixed populations from Colombia, Peru and Ecuador, and natives from Colombia and Ecuador. FST genetic distance analysis showed that the native genetic background of Alto Paraná has an intermediate position between the Amazonian groups and the admixed populations from Peru and Ecuador, supporting the theory about the Amazonian origin of the Tupi-Guarani and, at the same time, showing the influence of other linguistic groups.

Early human dispersals within the Americas.

Studies of the peopling of the Americas have focused on the timing and number of initial migrations. Less attention has been paid to the subsequent spread of people within the Americas. We sequenced 15 ancient human genomes spanning from Alaska to Patagonia; six are ≥10,000 years old (up to ~18× coverage). All are most closely related to Native Americans, including those from an Ancient Beringian individual and two morphologically distinct "Paleoamericans." We found evidence of rapid dispersal and early diversification that included previously unknown groups as people moved south. This resulted in multiple independent, geographically uneven migrations, including one that provides clues of a Late Pleistocene Australasian genetic signal, as well as a later Mesoamerican-related expansion. These led to complex and dynamic population histories from North to South America.

Investigator® HDplex (Qiagen) reference population database for forensic use in Argentina.

Currently, autosomal Short Tandem Repeat (STR) markers represent the method of election in forensic human identification. Commercial kits of most common use nowadays -e.g. PowerPlex®Fusion, Promega Corp.; AmpFlSTR GlobalFiler, Thermofisher scientific; Investigator 24Plex QS,Qiagen-, allow the co-amplification of 23 highly polymorphic STR loci providing a high discrimination power in human identity testing. However, in complex kinship analysis and familial database searches involving distant relationships, additional DNA typing is often required in order to achieve well-founded conclusions. The recently developed kit Investigator® HDplex (Qiagen) co-amplify twelve autosomal STRs markers (D7S1517, D3S1744, D12S391, D2S1360, D6S474, D4S2366, D8S1132, D5S2500, D18S51, D21S2055, D10S2325, SE33), nine of which are not present in the above mentioned kits, providing a set of efficient supplementary markers for human identification purposes. In this study we genotyped a sample of 980 individuals from urban areas of ten Argentinean provinces using the Investigator® HDplex kit, aiming to provide forensic estimates for use in forensic casework and parentage testing in Argentina. We report reference allelic frequency databases for each of the provinces studied as well as for the combined samples. No deviation of Hardy-Weinberg equilibrium was observed. A reasonable discrimination capacity and power of exclusion was estimated which allowed predicting an acceptable forensic behavior of this kit, either to be used as the main STR panel for simple cases or as an auxiliary tool in complex cases. Additionally, population comparison tests showed that the studied samples are relatively homogeneous across the country for these STR set.

A comprehensive Y-STR portrait of Argentinean populations.

A study of 23 Y-STRs was conducted in 257 individuals living in urban areas from eight Argentinean provinces. The data were meta-analyzed together with 364 profiles obtained from the literature that represent other five provinces. A total of 255 different haplotypes were observed (253 singletons). Genetic structure estimated from analysis of molecular variance (AMOVA) and exploring different grouping scenarios, yielded high within population variance. Not surprisingly, analyses of genetic distances with respect to main ancestral continental populations indicated Argentinean haplotypes to be closely related to European ones. Overall, these results provide a quite complete picture of the patterns of Y chromosome variation in Argentina, notably contributing to increase the previous national database, and consequently allowing a better estimation of parameters of interest in forensic casework and parentage testing.

Characterization of 5' promoter and exon 1-3 polymorphism of the RAET1E gene.

NKG2D is an activating receptor utilized by natural killer (NK) cells that recognizes upregulated ligands on infected, tumorigenic and damaged cells, leading to their cytolysis. However, the NKG2D ligand (NKG2DL) system is very complex with eight known gene loci encoding slightly different molecules. Furthermore, most NKG2DL gene loci such as MICA and MICB are highly polymorphic with potential for functional differences. NKG2DL expression on tumors varies depending on the malignancy and tumors can also release soluble NKG2DL that exert anergic effects on NK cells when engagement with NKG2D occurs, allowing escape from NK cell immunosurveillance. We carried out RAET1E typing of IHW cell line DNA, including a 580 bp proximal promoter fragment and exons 1-3 identifying 13 of 15 known RAET1E alleles. We determined 7 polymorphisms within the promoter region, including 2 already known that contributed to 9 promoter types. RAET1E alleles with variability in the extracellular region also differed with respect to promoter type and one allele, RAET1E(∗)003, associated with 5 promoter types. We then identified putative transcription factor binding sites for RAET1E, and found 5 of the 7 promoter polymorphisms may disrupt these sites, abrogating binding of transcription factors and varying the potential level of expression.

GHEP-ISFG collaborative simulated exercise for DVI/MPI: Lessons learned about large-scale profile database comparisons.

The GHEP-ISFG Working Group has recognized the importance of assisting DNA laboratories to gain expertise in handling DVI or missing persons identification (MPI) projects which involve the need for large-scale genetic profile comparisons. Eleven laboratories participated in a DNA matching exercise to identify victims from a hypothetical conflict with 193 missing persons. The post mortem database was comprised of 87 skeletal remain profiles from a secondary mass grave displaying a minimal number of 58 individuals with evidence of commingling. The reference database was represented by 286 family reference profiles with diverse pedigrees. The goal of the exercise was to correctly discover re-associations and family matches. The results of direct matching for commingled remains re-associations were correct and fully concordant among all laboratories. However, the kinship analysis for missing persons identifications showed variable results among the participants. There was a group of laboratories with correct, concordant results but nearly half of the others showed discrepant results exhibiting likelihood ratio differences of several degrees of magnitude in some cases. Three main errors were detected: (a) some laboratories did not use the complete reference family genetic data to report the match with the remains, (b) the identity and/or non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, and (c) many laboratories did not properly evaluate the prior odds for the event. The results suggest that large-scale profile comparisons for DVI or MPI is a challenge for forensic genetics laboratories and the statistical treatment of DNA matching and the Bayesian framework should be better standardized among laboratories.

The complete mitogenome of a 500-year-old Inca child mummy.

In 1985, a frozen mummy was found in Cerro Aconcagua (Argentina). Archaeological studies identified the mummy as a seven-year-old Inca sacrifice victim who lived >500 years ago, at the time of the expansion of the Inca Empire towards the southern cone. The sequence of its entire mitogenome was obtained. After querying a large worldwide database of mitogenomes (>28,000) we found that the Inca haplotype belonged to a branch of haplogroup C1b (C1bi) that has not yet been identified in modern Native Americans. The expansion of C1b into the Americas, as estimated using 203 C1b mitogenomes, dates to the initial Paleoindian settlements (~18.3 thousand years ago [kya]); however, its internal variation differs between Mesoamerica and South America. By querying large databases of control region haplotypes (>150,000), we found only a few C1bi members in Peru and Bolivia (e.g. Aymaras), including one haplotype retrieved from ancient DNA of an individual belonging to the Wari Empire (Peruvian Andes). Overall, the results suggest that the profile of the mummy represents a very rare sub-clade that arose 14.3 (5-23.6) kya and could have been more frequent in the past. A Peruvian Inca origin for present-day C1bi haplotypes would satisfy both the genetic and paleo-anthropological findings.

Association between Y haplogroups and autosomal AIMs reveals intra-population substructure in Bolivian populations.

For the correct evaluation of the weight of genetic evidence in a forensic context, databases must reflect the structure of the population, with all possible groups being represented. Countries with a recent history of admixture between strongly differentiated populations are usually highly heterogeneous and sub-structured. Bolivia is one of these countries, with a high diversity of ethnic groups and different levels of admixture (among Native Americans, Europeans and Africans) across the territory. For a better characterization of the male lineages in Bolivia, 17 Y-STR and 42 Y-SNP loci were genotyped in samples from La Paz and Chuquisaca. Only European and Native American Y-haplogroups were detected, and no sub-Saharan African chromosomes were found. Significant differences were observed between the two samples, with a higher frequency of European lineages in Chuquisaca than in La Paz. A sample belonging to haplogroup Q1a3a1a1-M19 was detected in La Paz, in a haplotype background different from those previously found in Argentina. This result supports an old M19 North-south dispersion in South America, possibly via two routes. When comparing the ancestry of each individual assessed through his Y chromosome with the one estimated using autosomal AIMs, (a) increased European ancestry in individuals with European Y chromosomes and (b) higher Native American ancestry in the carriers of Native American Y-haplogroups were observed, revealing an association between autosomal and Y-chromosomal markers. The results of this study demonstrate that a sub-structure does exist in Bolivia at both inter- and intrapopulation levels, a fact which must be taken into account in the evaluation of forensic genetic evidence.

Ancestry informative markers: inference of ancestry in aged bone samples using an autosomal AIM-Indel multiplex.

Ancestry informative markers (AIMs) can be useful to infer ancestry proportions of the donors of forensic evidence. The probability of success typing degraded samples, such as human skeletal remains, is strongly influenced by the DNA fragment lengths that can be amplified and the presence of PCR inhibitors. Several AIM panels are available amongst the many forensic marker sets developed for genotyping degraded DNA. Using a 46 AIM Insertion Deletion (Indel) multiplex, we analyzed human skeletal remains of post mortem time ranging from 35 to 60 years from four different continents (Sub-Saharan Africa, South and Central America, East Asia and Europe) to ascertain the genetic ancestry components. Samples belonging to non-admixed individuals could be assigned to their corresponding continental group. For the remaining samples with admixed ancestry, it was possible to estimate the proportion of co-ancestry components from the four reference population groups. The 46 AIM Indel set was informative enough to efficiently estimate the proportion of ancestry even in samples yielding partial profiles, a frequent occurrence when analyzing inhibited and/or degraded DNA extracts.