Development of a SNP-based panel for human identification for Indian populations

The widely employed short tandem repeat (STR)-based panels for forensic human identification (HID) have limitations while dealing with challenging forensic samples involving DNA degradation, resulting in dropping-out of higher molecular weight alleles/loci. To address this issue, bialleic markers like single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), which can be scored even when the template DNA is heavily degraded (<100 bp), have been suggested as alternative markers for HID testing. Recent studies have highlighted their utility in forensic HID and several panels based on biallelic markers have been described for worldwide populations. However, there has been very little information about the behavior of such DNA markers in Indian populations, which is known to possess great genetic diversity. This study describes a two-step approach for designing a SNP-based panel consisting of 70 SNPs for HID testing in Indian populations. In the first step, candidate SNPs were shortlisted from public databases by screening them for several criteria including allelic distribution, genomic location, potential phenotypic expression or functionality and species specificity. The second step involved genotyping the shortlisted SNPs in various Indian populations followed by shortlisting of the best performers for identity-testing. Starting with 592,652 SNPs listed in Human660W-Quad Beadchip (Illumina Inc.), we shortlisted 275 candidate SNPs for identity-testing and genotyped them in 462 unrelated individuals from different population groups in India. Post genotyping and statistical analyses based on biogeographic regions, 206 SNPs demonstrated desired allelic distribution (Heterozygosity ≥ 0.4 and F_ST ≤ 0.02), from which 2–4 widely separated (>20 Mb apart) SNPs from each chromosome were finally selected to construct a panel of 70 SNPs. This panel on average possessed match probability 10e-29 and probability of paternity of 0.99999997, which was orders of magnitude higher than most of the currently employed STR-based chemistries and SNP-based panels that were proposed previously for HID testing. For comparison purpose, genotyping previously reported SNPs for HID in our samples led us to conclude that the panel developed in this study is much more efficient and robust and better suited for the Indian populations.     

Completion of a worldwide reference panel of samples for an ancestry informative Indel assay

The use of ancestry informative markers (AIMs) in forensic analysis is of considerable utility since ancestry inference can progress an investigation when no identification has been made of DNA from the crime-scene. Short-amplicon markers, including insertion deletion polymorphisms, are particularly useful in forensic analysis due to their mutational stability, capacity to amplify degraded samples and straightforward amplification technique. In this study we report the completion of H952 HGDP–CEPH panel genotyping with a set of 46 AIM-Indels. The study adds Central South Asian and Middle Eastern population data, allowing a comparison of patterns of variation in Eurasia for these markers, in order to enhance their use in forensic analyses, particularly when combined with sets of ancestry informative SNPs. Ancestry analysis using principal component analysis and Bayesian methods indicates that a proportion of classification error occurs with European–Middle East population comparisons, but the 46 AIM-Indels have the capability to differentiate six major population groups when European–Central South Asian comparisons are made. These findings have relevance for forensic ancestry analyses in countries where South Asians form much of the demographic profile, including the UK, USA and South Africa. A novel third allele detected in MID-548 was characterized – despite a low frequency in the HGDP–CEPH panel samples, it appears confined to Central South Asian populations, increasing the ability to differentiate this population group. The H952 data set was implemented in a new open access SPSmart frequency browser – forInDel: Forensic Indel browser.     

A single nucleotide polymorphism panel for individual identification and ancestry assignment in Caucasians and four East and Southeast Asian populations using a machine learning classifier

Single nucleotide polymorphism (SNP) profiling is an effective means of individual identification and ancestry inferences in forensic genetics. This study established a SNP panel for the simultaneous individual identification and ancestry assignment of Caucasian and four East and Southeast Asian populations. We analyzed 220 SNPs (125 autosomal, 17 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs) of the DNA samples from 563 unrelated individuals of five populations (89 Caucasian, 234 Taiwanese Han, 90 Filipino, 79 Indonesian and 71 Vietnamese) and 18 degraded DNA samples. Informativeness for assignment (In) was used to select ancestry informative SNPs (AISNPs). A machine learning classifier, support vector machine (SVM), was used for ancestry assignment. Of the 220 SNPs, 62 were individual identification SNPs (IISNPs) (51 autosomal and 11 X-chromosomal SNPs) and 191 were AISNPs (100 autosomal, 13 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs). The 51 autosomal IISNPs offered cumulative random match probabilities (cRMPs) ranging from 1.56 × 10−21 to 3.16 × 10−22 among these five populations. Using AISNPs with the SVM, the overall accuracy rate of ancestry inference achieved in the testing dataset between Caucasian, Taiwanese Han, and Filipino populations was 88.9%, whereas it was 70.0% between Caucasians and each of the four East and Southeast Asian populations. For the 18 degraded DNA samples with incomplete profiling, the accuracy rate of ancestry assignment was 94.4%. We have developed a 220-SNP panel for simultaneous individual identification and ethnic origin differentiation between Caucasian and the four East and Southeast Asian populations. This SNP panel may assist with DNA analysis of forensic casework.     

Allele frequencies and other forensic parameters of the HID-Ion AmpliSeq™ Identity Panel markers in Basques using the Ion Torrent PGM™ platform

The HID-Ion AmpliSeq™ Identity Panel amplifies 90 autosomal SNPs and 34 Y- SNPs with massively parallel sequencing (MPS) using the Ion Torrent PGM™ platform. In the present study, 105 Basques were analyzed to assess this panel. All loci were in Hardy-Weinberg equilibrium and no association between them was detected. Forensic parameters were calculated as 5.74 × 10⁻³⁶ for combined match probability and 99.99998% for combined power of exclusion. In conclusion, the HID Identity panel and the use of this new MPS technology are very promising tools for paternity testing and human identification in routine casework in the forensic field.     

A custom hybridisation enrichment forensic intelligence panel to infer biogeographic ancestry,hair and eye colour,and Y chromosome lineage

Massively parallel sequencing can provide genetic data for hundreds to thousands of loci in a single assay for various types of forensic testing. However, available commercial kits require an initial PCR amplification of short-to-medium sized targets which limits their application for highly degraded DNA. Development and optimisation of large PCR multiplexes also prevents creation of custom panels that target different suites of markers for identity, biogeographic ancestry, phenotype, and lineage markers (Y-chromosome and mtDNA). Hybridisation enrichment, an alternative approach for target enrichment prior to sequencing, uses biotinylated probes to bind to target DNA and has proven successful on degraded and ancient DNA. We developed a customisable hybridisation capture method, that uses individually mixed baits to allow tailored and targeted enrichment to specific forensic questions of interest. To allow collection of forensic intelligence data, we assembled and tested a custom panel of hybridisation baits to infer biogeographic ancestry, hair and eye colour, and paternal lineage (and sex) on modern male and female samples with a range of self-declared ancestries and hair/eye colour combinations. The panel correctly estimated biogeographic ancestry in 9/12 samples (75%) but detected European admixture in three individuals from regions with admixed demographic history. Hair and eye colour were predicted correctly in 83% and 92% of samples respectively, where intermediate eye colour and blond hair were problematic to predict. Analysis of Y-chromosome SNPs correctly assigned sex and paternal haplogroups, the latter complementing and supporting biogeographic ancestry predictions. Overall, we demonstrate the utility of this hybridisation enrichment approach to forensic intelligence testing using a combined suite of biogeographic ancestry, phenotype, and Y-chromosome SNPs for comprehensive biological profiling.     

Forensic and population genetic characteristics of 62 X chromosome SNPs revealed by multiplex PCR and MALDI-TOF mass spectrometry genotyping in 4 North Eurasian populations

X chromosome genetic markers are widely used in basic population genetic research as well as in forensic genetics. In this paper we analyze the genetic diversity of 62 X chromosome SNPs in 4 populations using multiplex genotyping based on multi-locus PCR and MALDI-TOF mass spectrometry, and report forensic and population genetic features of the panel of X-linked SNPs (XSNPid). Studied populations represent Siberian (Buryat and Khakas), North Asian (Khanty) and Central Asian (Kazakh) native people. Khanty, Khakas and Kazakh population demonstrate average gene diversity over 0.45. Only East Siberian Buryat population is characterized by lower average heterozygosity (0.436). AMOVA analysis of genetic structure reveals a relatively low but significant level of genetic differentiation in a group of 4 population studied (F_ST = 0.023, p = 0.0000). The XSNPid panel provides a very high discriminating power in each population. The combined probability of discrimination in females (PDf) for XSNPid panel ranged between populations from 0.99999999999999999999999982 in Khakas to 0.9999999999999999999999963 in Buryats. The combined discriminating power in males (PDm) varies from 0.999999999999999792 to 0.9999999999999999819. The developed multiplex set of X chromosome SNPs can be a useful tool for population genetic studies and for forensic identity and kinship testing.     

A 21 marker insertion deletion polymorphism panel to study biogeographic ancestry

Insertion/deletion polymorphisms have recently received increased interest in the forensic genetics community. This class of markers combines the advantageous genetic properties of single nucleotide polymorphisms (i.e., low mutation rate, genetic stability, and short amplicon size) with the technical advantage of short tandem repeat markers (simple detection by fluorescence-labelled PCR and capillary electrophoresis). For a large number of indel markers significant differences in allele frequencies between the major populations have been reported, making this class of markers suitable for the analysis of biogeographic ancestry. We have developed a multiplex PCR assay designed to establish the biogeographic ancestry of forensic DNA samples based on insertion/deletion polymorphisms. A panel of 21 short indels with allele frequency differences between three major population groups (European, African and Asian) was selected to be incorporated into a single-tube multiplex PCR assay. The assay is highly sensitive, requiring less than 0.5 ng of genomic DNA for successful typing. Due to the short fragment lengths below 200 bp, the assay is ideally suited for the typing of challenging forensic genetic case work samples. A population genetic study has been performed proving the performance of the assay in inferring the ancestral population of individuals. The chosen 21 markers are sufficient to distinguish between three major global population groups. Furthermore, the assay design leaves room for an extension in order to cover additional population groups.     

EurEAs_Gplex—A new SNaPshot assay for continental population discrimination and gender identification

Assays that allow analysis of the biogeographic origin of biological samples in a standard forensic laboratory have to target a small number of highly differentiating markers. Such markers should be easy to multiplex and the assay must perform well in the degraded and scarce biological material. SNPs localized in the genome regions, which in the past were subjected to differential selective pressure in various populations, are the most widely used markers in the studies of biogeographic affiliation. SNPs reflecting biogeographic differences not related to any phenotypic traits are not sufficiently explored.The goal of our study was to identify a small set of SNPs not related to any known pigmentation/phenotype-specific genes, which would allow efficient discrimination between populations of Europe and East Asia. The selection of SNPs was based on the comparative analysis of representative European and Chinese/Japanese samples (B-lymphocyte cell lines), genotyped using the Infinium HumanOmniExpressExome microarray (Illumina). The classifier, consisting of 24 unlinked SNPs (24-SNP classifier), was selected. The performance of a 14-SNP subset of this classifier (14-SNP subclassifier) was tested using genotype data from several populations. The 14-SNP subclassifier differentiated East Asians, Europeans and Africans with ∼100% accuracy; Palestinians, representative of the Middle East, clustered with Europeans, while Amerindians and Pakistani were placed between East Asian and European populations.Based on these results, we have developed a SNaPshot assay (EurEAs_Gplex) for genotyping SNPs from the 14-SNP subclassifier, combined with an additional marker for gender identification. Forensic utility of the EurEAs_Gplex was verified using degraded and low quantity DNA samples. The performance of the EurEAs_Gplex was satisfactory when using degraded DNA; tests using low quantity DNA samples revealed a previously not described source of genotyping errors, potentially important for any SNaPshot-based assays.     

Next generation sequencing of SNPs using the HID-Ion AmpliSeq™ Identity Panel on the Ion Torrent PGM™ platform

The HID-Ion AmpliSeq™ Identity Panel (the HID Identity Panel) is designed to detect 124-plex single nucleotide polymorphisms (SNPs) with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 90 individual identification SNPs (IISNPs) on autosomal chromosomes and 34 lineage informative SNPs (LISNPs) on Y chromosome. In this study, we evaluated performance for the HID Identity Panel to provide a reference for NGS-SNP application, focusing on locus strand balance, locus coverage balance, heterozygote balance, and background signals. Besides, several experiments were carried out to find out improvements and limitations of this panel, including studies of species specificity, repeatability and concordance, sensitivity, mixtures, case-type samples and degraded samples, population genetics and pedigrees following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. In addition, Southern and Northern Chinese Han were investigated to assess applicability of this panel. Results showed this panel led to cross-reactivity with primates to some extent but rarely with non-primate animals. Repeatable and concordant genotypes could be obtained in triplicate with one exception at rs7520386. Full profiles could be obtained from 100 pg input DNA, but the optimal input DNA would be 1 ng–200 pg with 21 initial PCR cycles. A sample with ≥20% minor contributor could be considered as a mixture by the number of homozygotes, and full profiles belonging to minor contributors could be detected between 9:1 and 1:9 mixtures with known reference profiles. Also, this assay could be used for case-type samples and degraded samples. For autosomal SNPs (A-SNPs), F_ST across all 90 loci was not significantly different between Southern and Northern Chinese Han or between male and female samples. All A-SNP loci were independent in Chinese Han population. Except for 18 loci with H_e <0.4, most of the A-SNPs in the HID Identity Panel presented high polymorphisms. Forensic parameters were calculated as >99.999% for combined discrimination power (CDP), 0.999999724 for combined power of exclusion (CPE), 1.390 × 10¹¹ for combined likelihood ratio (CLR) of trios, and 2.361 × 10⁶ for CLR of motherless duos. For Y-SNPs, a total of 8 haplotypes were observed with the value of 0.684 for haplotype diversity. As a whole, the HID Identity Panel is a well-performed, robust, reliable and high informative NGS-SNP assay and it can fully meet requirements for individual identification and paternity testing in forensic science.     

Pacifiplex: an ancestry-informative SNP panel centred on Australia and the Pacific region

The analysis of human population variation is an area of considerable interest in the forensic, medical genetics and anthropological fields. Several forensic single nucleotide polymorphism (SNP) assays provide ancestry-informative genotypes in sensitive tests designed to work with limited DNA samples, including a 34-SNP multiplex differentiating African, European and East Asian ancestries. Although assays capable of differentiating Oceanian ancestry at a global scale have become available, this study describes markers compiled specifically for differentiation of Oceanian populations. A sensitive multiplex assay, termed Pacifiplex, was developed and optimized in a small-scale test applicable to forensic analyses. The Pacifiplex assay comprises 29 ancestry-informative marker SNPs (AIM-SNPs) selected to complement the 34-plex test, that in a combined set distinguish Africans, Europeans, East Asians and Oceanians. Nine Pacific region study populations were genotyped with both SNP assays, then compared to four reference population groups from the HGDP-CEPH human diversity panel. STRUCTURE analyses estimated population cluster membership proportions that aligned with the patterns of variation suggested for each study population’s currently inferred demographic histories. Aboriginal Taiwanese and Philippine samples indicated high East Asian ancestry components, Papua New Guinean and Aboriginal Australians samples were predominantly Oceanian, while other populations displayed cluster patterns explained by the distribution of divergence amongst Melanesians, Polynesians and Micronesians. Genotype data from Pacifiplex and 34-plex tests is particularly well suited to analysis of Australian Aboriginal populations and when combined with Y and mitochondrial DNA variation will provide a powerful set of markers for ancestry inference applied to modern Australian demographic profiles. On a broader geographic scale, Pacifiplex adds highly informative data for inferring the ancestry of individuals from Oceanian populations. The sensitivity of Pacifiplex enabled successful genotyping of population samples from 50-year-old serum samples obtained from several Oceanian regions that would otherwise be unlikely to produce useful population data. This indicates tests primarily developed for forensic ancestry analysis also provide an important contribution to studies of populations where useful samples are in limited supply.     

EASplex: A panel of 308 AISNPs for East Asian ancestry inference using next generation sequencing

Ancestry inference is useful in many scientific fields, such as forensic genetics, medical genetics, and molecular archaeology. Various ancestry inferring methods have been released for major continental populations. However, few reports refer to sub-populations within the East Asian population using hundreds of ancestry informative SNPs (AISNPs). In this study, we developed a 308-AISNP panel (EASplex NGS DNA panel) using multiplex PCR and next generation sequencing (NGS). This panel included 56 SNPs relevant for the continent-level ancestry inference and 252 Japanese-, Korean-, and/or Han Chinese-specific AISNPs to address the ancestry inference of global populations and regional populations among Japanese (JPT), Korean minority (CHK), and Han Chinese (CHH). A total of 87 CHK and 59 CHH samples were used to check the performance of the EASplex NGS DNA panel. By analyzing 146 profiles of samples with JPT and CHH data from Beijing and South China in 1000 genomes project, the following results were obtained: (1) the 146 tested samples were correctly assigned to the East Asian group; (2) the paired population assignment rate was 99.73% for JPT and CHH, 95% for JPT and CHK, and 90.11% for CHK and CHH; and (3) the whole population assignment was 92.14% for the JPT, CHK, and CHH data. Overall, the EASplex NGS DNA panel displayed informativeness for continental ancestry inference and regional ancestry inference among JPT, CHH, and CHK and has the potential for use in forensic and genetic population studies.     

Identification and characterization of novel DIP-STRs from whole-genome sequencing data

In an attempt to enhance forensic DNA mixture deconvolution several alternative DNA typing approaches have been developed. Among these, DIP-STR compound markers can resolve extremely unbalanced two-source DNA mixtures of same-or-opposite sex donors, up to a 1:1000 minor:major DNA ratio. A forensic set of 10 markers was validated for casework and a larger set of 23 DIP-STRs has proven suitable to biogeographic ancestry inference and for prenatal paternity testing. Yet, to promote the widespread use of this original approach, more markers and multiplex panels need to be developed.To this end, here we describe an extended set of forensic DIP-STRs identified using currently available whole-genome sequencing datasets. Complete lists of Indels and STRs were obtained from reported frequencies of genetic variants of 76,156 genomes. About 3000 identified DIP-STRs candidates were shorter than 200 bp and 500 showed high haplotype variability estimated using the genotypes of individuals homozygous for the DIP or the STR. Here, we present 23 additional DIP-STRs validated for sensitivity, specificity and Swiss population variability. Finally, a set of 30 markers comprising seven previously validated ones is proposed for the prospective development of a forensic DIP-STR multiplex panel.     

Frequencies of the precision ID ancestry panel markers in Basques using the Ion Torrent PGMTM platform

The Precision ID Ancestry Panel for the Ion Torrent PGM™ platform is able to genotype 165 autosomal SNPs by massively parallel sequencing (MPS). In the present study, we evaluated the depth of coverage, the locus strand balance and the heterozygote balance of the above panel. The SNPs rs1569175, rs2306040, rs9845457, rs1407434, and rs10007810 showed a low performance, due either to a low coverage, locus strand imbalance or heterozygote imbalance. To further to assess this panel, we analyzed 108 Basques. All loci proved to be in Hardy-Weinberg equilibrium and we did not detect any association between them with the exception of the pair rs3916235-rs4891825 in chromosome 18. The forensic parameters combined match probability and combined power of exclusion were estimated to be 3.13 × 10⁻³⁵ and 99.9972%, respectively. In conclusion, the Precision ID Ancestry panel along with this new MPS technology constitute a very promising tool for human identification and biogeographical ancestry inference in routine casework in the forensic field.     

Current sequencing technology makes microhaplotypes a powerful new type of genetic marker for forensics

SNPs that are molecularly very close (<10 kb) will generally have extremely low recombination rates, much less than 10⁻⁴. Multiple haplotypes will often exist because of the history of the origins of the variants at the different sites, rare recombinants, and the vagaries of random genetic drift and/or selection. Such multiallelic haplotype loci are potentially important in forensic work for individual identification, for defining ancestry, and for identifying familial relationships. The new DNA sequencing capabilities currently available make possible continuous runs of a few hundred base pairs so that we can now determine the allelic combination of multiple SNPs on each chromosome of an individual, i.e., the phase, for multiple SNPs within a small segment of DNA. Therefore, we have begun to identify regions, encompassing two to four SNPs with an extent of <200 bp that define multiallelic haplotype loci. We have identified candidate regions and have collected pilot data on many candidate microhaplotype loci. Here we present 31 microhaplotype loci that have at least three alleles, have high heterozygosity, are globally informative, and are statistically independent at the population level. This study of microhaplotype loci (microhaps) provides proof of principle that such markers exist and validates their usefulness for ancestry inference, lineage–clan–family inference, and individual identification. The true value of microhaplotypes will come with sequencing methods that can establish alleles unambiguously, including disentangling of mixtures, because a single sequencing run on a single strand of DNA will encompass all of the SNPs.     

Development and validation of a custom panel including 256 Y-SNPs for Chinese Y-chromosomal haplogroups dissection

Y-chromosomal haplogroups determined by Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal biogeographic ancestry inference, which has attracted a lot of interest in the forensic community. Recently, a comprehensive Y-SNP tool with dominant markers targeting haplogroups in R, E and I branches has been reported, which allows the inference of 640 Y haplogroups. It had a very good performance and could provide a high level of Y haplogroup resolution in most populations. However, the predominant haplogroups in the Chinese populations are O, C and N, suggesting that more Y-SNPs under these clades are needed to achieve the population-specific high resolution. Herein, aiming at the Chinese population, we presented a largely improved custom Y-SNP MPS panel that contains 256 carefully ascertained Y-SNPs based on our previous studies, and evaluated this panel via a series of tests, including the tests for concordance, repeatability, sensitivity, specificity, and stability, as well as the mixture, degraded and case-type sample analysis. The preliminary developmental validation demonstrated that this panel was highly reliable, sensitive, specific, and robust. In the sensitivity test, even when the DNA input was reduced to as low as 0.5 ng, the sample could still be assigned to the correct Y haplogroup. For mixture analysis, even the 1:99 (Male: Female) mixtures had no effects on the assignation of the Y haplogroup of the male contributor. In summary, this assay has provided a high-resolution Y-chromosomal haplogrouping workflow to determine a male’s paternal lineage and/or paternal biogeographic ancestry and could be widely used for Chinese Y-chromosomal haplogroups dissection.     

First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with the Identitas v1 Forensic Chip

When a forensic DNA sample cannot be associated directly with a previously genotyped reference sample by standard short tandem repeat profiling, the investigation required for identifying perpetrators, victims, or missing persons can be both costly and time consuming. Here, we describe the outcome of a collaborative study using the Identitas Version 1 (v1) Forensic Chip, the first commercially available all-in-one tool dedicated to the concept of developing intelligence leads based on DNA. The chip allows parallel interrogation of 201,173 genome-wide autosomal, X-chromosomal, Y-chromosomal, and mitochondrial single nucleotide polymorphisms for inference of biogeographic ancestry, appearance, relatedness, and sex. The first assessment of the chip’s performance was carried out on 3,196 blinded DNA samples of varying quantities and qualities, covering a wide range of biogeographic origin and eye/hair coloration as well as variation in relatedness and sex. Overall, 95 % of the samples (N = 3,034) passed quality checks with an overall genotype call rate >90 % on variable numbers of available recorded trait information. Predictions of sex, direct match, and first to third degree relatedness were highly accurate. Chip-based predictions of biparental continental ancestry were on average ~94 % correct (further support provided by separately inferred patrilineal and matrilineal ancestry). Predictions of eye color were 85 % correct for brown and 70 % correct for blue eyes, and predictions of hair color were 72 % for brown, 63 % for blond, 58 % for black, and 48 % for red hair. From the 5 % of samples (N = 162) with <90 % call rate, 56 % yielded correct continental ancestry predictions while 7 % yielded sufficient genotypes to allow hair and eye color prediction. Our results demonstrate that the Identitas v1 Forensic Chip holds great promise for a wide range of applications including criminal investigations, missing person investigations, and for national security purposes.     

Performance of ancestry-informative SNP and microhaplotype markers

The use of microhaplotypes (MHs) for ancestry inference has added to an increasing number of ancestry-informative markers (AIMs) for forensic application that includes autosomal single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). This study compares bi-allelic and tri-allelic SNPs as well as MH markers for their ability to differentiate African, European, South Asian, East Asian, and American population groups from the 1000 Genomes Phase 3 database. A range of well-established metrics were applied to rank each marker according to the population differentiation potential they measured. These comprised: absolute allele frequency differences (δ); Rosenberg’s informativeness for (ancestry) assignment (I_n); the fixation index (F_ST); and the effective number of alleles (A_e). A panel consisting of all three marker types resulted in the lowest mean divergence per population per individual (MDPI = 2.16%) when selected by I_n. However, when marker types were not mixed, MHs were the highest performing markers by most metrics (MDPI < 4%) for differentiation between the five continental populations.     

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.     

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.