Building a forensic ancestry panel from the ground up: The EUROFORGEN Global AIM-SNP set

Emerging next-generation sequencing technologies will enable DNA analyses to add pigmentation predictive and ancestry informative (AIM) SNPs to the range of markers detectable from a single PCR test. This prompted us to re-appraise current forensic and genomics AIM-SNPs and from the best sets, to identify the most divergent markers for a five population group differentiation of Africans, Europeans, East Asians, Native Americans and Oceanians by using our own online genome variation browsers. We prioritized careful balancing of population differentiation across the five group comparisons in order to minimize bias when estimating co-ancestry proportions in individuals with admixed ancestries. The differentiation of European from Middle East or South Asian ancestries was not chosen as a characteristic in order to concentrate on introducing Oceanian differentiation for the first time in a forensic AIM set. We describe a complete set of 128 AIM-SNPs that have near identical population-specific divergence across five continentally defined population groups. The full set can be systematically reduced in size, while preserving the most informative markers and the balance of population-specific divergence in at least four groups. We describe subsets of 88, 55, 28, 20 and 12 AIMs, enabling both new and existing SNP genotyping technologies to exploit the best markers identified for forensic ancestry analysis.     

A SNaPshot of next generation sequencing for forensic SNP analysis

Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot^®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30–40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies’ Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.     

Analysis of the South Australian Aboriginal population using the Global AIMs Nano ancestry test

We investigate the ability of the 31 SNP loci in the Global AIMs Nano set to distinguish self-declared Australian Aboriginal individuals from European, Oceanic, African, Native American and East Asian populations. Human evolution suggests that Australian Aboriginal individuals came to Australia approximately 50 000 years ago, during the time it made up part of Sahul. Since then the colonisation of Australia by Europeans has meant significant admixture within the Australian Aboriginal population. These two events present themselves in our study with the Aboriginal population creating a continuous genetic cline between the Oceanic and European groups. We also assigned the Aboriginal individuals into their traditional regional groups to determine whether there was any ability to distinguish these from each other. We found similar results to studies using other markers, namely that the more remote regions (that have been less affected by admixture) diverged from the rest. Overall, we found the ability of the GNano system to differentiate self-declared Australian Aboriginal individuals was reasonable but had limitations that need to be recognised if these assignments are applied to unknown individuals.     

Eurasiaplex-2: Shifting the focus to SNPs with high population specificity increases the power of forensic ancestry marker sets

To compile a new South Asian-informative panel of forensic ancestry SNPs, we changed the strategy for selecting the most powerful markers for this purpose by targeting polymorphisms with near absolute specificity – when the South Asian-informative allele identified is absent from all other populations or present at frequencies below 0.001 (one in a thousand). More than 120 candidate SNPs were identified from 1000 Genomes datasets satisfying an allele frequency screen of ≥ 0.1 (10 % or more) allele frequency in South Asians, and ≤ 0.001 (0.1 % or less) in African, East Asian, and European populations. From the candidate pool of markers, a final panel of 36 SNPs, widely distributed across most autosomes, were selected that had allele frequencies in the five 1000 Genomes South Asian populations ranging from 0.4 to 0.15. Slightly lower average allele frequencies, but consistent patterns of informativeness were observed in gnomAD South Asian datasets used to validate the 1000 Genomes variant annotations. We named the panel of 36 South Asian-specific SNPs Eurasiaplex-2, and the informativeness of the panel was evaluated by compiling worldwide population data from 4097 samples in four genome variation databases that largely complement the global sampling of 1000 Genomes. Consistent patterns of allele frequency distribution, which were specific to South Asia, were observed in all populations in, or closely sited to, the Indian sub-continent. Pakistani populations from the HGDP-CEPH panel had markedly lower allele frequencies, highlighting the need to develop a statistical system to evaluate the ancestry inference value of counting the number of population-specific alleles present in an individual.     

First successful assay of Y-SNP typing by SNaPshot minisequencing on ancient DNA

In the present study, a set of 13 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) selected for the identification of the most frequent Asian Y-haplogroups was included in an allele-specific primer extension assay. Single nucleotide polymorphism (SNP) genotyping was accomplished by co-amplification of these 13 DNA fragments within 2 multiplex PCRs followed by detection with 1 minisequencing reaction using the SNaPshot™ Multiplex kit and analysis of extension products by capillary electrophoresis. First developed on modern samples, the assay was optimized for the analysis of 11 ancient DNA (aDNA) samples from the Krasnoyarsk region (southern Siberia) that were dated from 5,500–1,800 years before present (YBP). SNP typing was successful for most of them, which were all assigned to Y-haplogroup R1a1 except one. These results show that SNPs are well-suited for the analysis of aged and degraded DNA samples. Moreover, we found that the SNaPshot minisequencing methodology is a convenient, robust, and efficient method for SNP typing. To our knowledge, this study reports the first successful investigation of Y-SNPs on aDNA samples. The potential use of Y-SNPs in both evolutionary and forensic fields is also discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A rapid mtDNA assay of 22 SNPs in one multiplex reaction increases the power of forensic testing in European Caucasians

We have developed a multiplex mitochondrial (mtDNA) assay of 21 coding region single nucleotide polymorphisms (SNPs) and one control region SNP outside hypervariable region 1 (HVR1) and hypervariable region 2 (HVR2) that can be amplified in a single reverse touchdown polymerase chain reaction. Single base extension using the SNaPshot technique is also carried out as one multiplex. Besides the nine major European haplogroups (i.e. H, I, J, K, T, U, V, W, and X), 16 additional subclades (i.e. N1, X2, X2b, U2′-4/7′-9′, J/T, J1, J1c, HV, H1, H1a1, H1c, H3, H4, H6a, H7a H10) can be detected and classified into a phylogenetic mtDNA tree. By analyzing 130 Caucasoid samples from Germany, 36 different haplotypes were found resulting in a power of discrimination of 93.2%. Although 49% of all samples belonged to superhaplogroup H, the most common haplotype, i.e., haplogroup-specific SNPs plus haplogroup unspecific SNPs, had a frequency of only 18%. This assay is applicable for high-throughput mtDNA analysis and forensic mass screening. It will give additional information to the common control region sequencing of HVR1 and HVR2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of the Precision ID Ancestry Panel for crime case work: A SNP typing assay developed for typing of 165 ancestral informative markers

The application of massive parallel sequencing (MPS) methodologies in forensic genetics is promising and it is gradually being implemented in forensic genetic case work. One of the major advantages of these technologies is that several traditional electrophoresis assays can be combined into one single MPS assay. This reduces both the amount of sample used and the time of the investigations.This study assessed the utility of the Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) in forensic genetics. This assay was developed for the Ion Torrent PGM™ System and genotypes 165 ancestry informative SNPs. The performance of the assay and the accompanying software solution for ancestry inference was assessed by typing 142 Danes and 98 Somalis. Locus balance, heterozygote balance, and noise levels were calculated and future analysis criteria for crime case work were estimated. Overall, the Precision ID Ancestry Panel performed well, and only minor changes to the recommended protocol were implemented. Three out of the 165 loci (rs459920, rs7251928, and rs7722456) had consistently poor performance, mainly due to misalignment of homopolymeric stretches. We suggest that these loci should be excluded from the analyses.The different statistical methods for reporting ancestry in forensic genetic case work are discussed.     

The art of traditional native PAGE: The APLP 48-ID assay for human identification

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing. Forty-seven SNPs were selected from all 22 autosomes, showing high diversity in European, Nigerian, Han Chinese, and Japanese population in the HapMap data. PCR primers were designed to generate amplicons 40–100 bp in length to increase the robustness of the PCR.The APLP 48-ID assay consisted of four independent PCR reactions followed by electrophoretic run on four lanes in a polyacrylamide gel. Complete profiles were obtained when more than 1.2 ng of DNA was used. We applied this assay for genotyping of 236 Japanese individuals. The random matching probability was 3.3E-20, and the power of exclusion was greater than 0.9999999. This method is a rapid, robust, and cost-effective approach for human identification and paternity testing.     

The role of Na+/K+ ATPase activity during low flow ischemia in preventing myocardial injury: A 31P, 23Na and 87Rb NMR spectroscopic study

An increase in intracellular Na⁺ during ischaemia has been associated with myocardial injury. In this study, we determined whether inhibition of Na⁺/K⁺ ATPase activity contributes to this increase and whether Na⁺/K⁺ ATPase activity can be maintained by provision of glucose to perfused rat hearts during low flow, 0.5 ml/min, ischemia. We used ³¹P NMR spectroscopy to determine changes in myocardial energetics and intracellular and extracellular volumes. ²³Na NMR spectroscopy, with DyTTHA^3- present as a shift reagent, was used to measure changes in intracellular Na⁺ and ⁸⁷Rb NMR spectroscopy was used to estimate Na⁺/K⁺ ATPase activity from Rb⁺ influx rates, Rb⁺ being an NMR-sensitive congener of K⁺. In hearts provided with 11 mM glucose throughout ischemia, glycolysis continued and ATP was twofold higher than in hearts without glucose. In the glucose-hearts, Rb⁺ influx rate was threefold higher, intracellular Na⁺ was fivefold lower at the end of ischemia and functional recovery during reperfusion was twofold higher. We propose that continuation of glycolysis throughout low flow ischemia allowed maintenance of sufficient Na⁺/K⁺ ATPase activity to prevent the increase in intracellular Na⁺ that would otherwise have led to myocardial injury.     

The use of the STRs HUMTH01, HUMVWA31/A,HUMF13A1, HUMFES/FPS,HUMLPL in forensic application: Validation studies and population data for Galicia (NW Spain)

The 5 tetranucleotide short tandem repeats, HUMTHOI, HUMVWA31/A, HUMF13A1, HUMFES/FPS and HUMLPL were studied using different electrophoretic methods and PCR amplification conditions in order to optimize the typing conditions. A genetic population study in the population of Galicia was carried out and the allele and genotype frequencies are given. Compliance with the Hardy-Weinberg equilibrium was tested using different statistical parameters, with clear advantages resulting in favor of using the exact test (Guo-Thompson method) instead of conventional chi-square methods. Some statistical parameters of forensic interest (PD, CE, h) were also calculated. There were no mutations found in a total of 73 paternal meioses and 101 maternal meioses. Abnormal electrophoretic mobility was found in the AT-rich STR HUMF13AI under non-denaturing conditions and, therefore, the use of denaturing conditions is absolutely necessary. No "stutter" bands were found, although double peaks in the HUMFES/FPS system were observed in some samples. The advantage of using automated sequencers with fluorescent technology is also reported.