Improved pairwise kinship analysis using massively parallel sequencing

In the present study, 67 individuals from two families were analyzed to explore the efficacy of the ForenSeq^™ DNA Signature Prep Kit for pairwise kinship analysis. Six types of pairwise relationships including 81 parent-offspring, 60 full siblings, 48 grandparent-grandchildren, 147 uncle/aunt-nephew/nieces, 97 first cousins and 190 non-relatives were generated from these two families and the corresponding likelihood ratio (LR) was calculated using either sequence-based or length-based STR genotype data (i.e., LR_sequence and LR_length). In addition, 10,000 pairs of different relationships were simulated to estimate the system powers of the STRs and SNPs in this panel. The results showed that 54, 9 and 5 additional alleles were observed based on sequence for 27 autosomal STRs, 24 Y-STRs and 7 X-STRs, respectively, compared to those based on length information and 11 novel alleles were identified. Five mutations were found for 58 STRs in 81 parent-offspring but no mutations were observed for SNPs. For 27 autosomal STR loci, the LRs were increased from 9.20, 7.87, 2.01, 2.07, 0.42 for log₁₀LR_length to 11.52, 10.12, 2.61, 2.60, 0.52 for log₁₀LR_sequence for paternity index (PI), full siblings index (FSI), grandparent-grandchild index (GI), uncle/aunt-nephew/niece index (UNI) and first cousins index (FCI), respectively. PI values for 94 SNPs separated more than those of 27 STRs if two individuals were non parent-offspring relatives. For the simulation study, the effectiveness was 1 for the parent-offspring relationship at the thresholds of t₁ = − 4 and t₂ = 4 and was 0.9998 for full siblings (t₁ = − 2, t₂ = 2). With an error rate of 0.42%, 93.02% of second degree relatives could be identified at the thresholds of t₁ = − 1 and t₂ = 1. However, the effectiveness was only 0.4300 for first cousins with a relatively high error rate of 2.68% (t₁ = − 1, t₂ = 1). In conclusion, STR typing according to the sequence information is more polymorphic, which increases the discrimination power for kinship testing. Compared to these 27 STR markers, 94 SNP markers in this panel have advantages in paternity testing especially when mutated STRs are involved or when a relative is an alleged parent. This panel is powerful enough to resolve paternity and full sibling testing. Most of the second degree relationships could be identified with low error rate while more markers are still needed for first cousins testing.     

Developmental validation of a 381 Y-chromosome SNP panel for haplogroup analysis in the Chinese populations

Y-chromosome single nucleotide polymorphism (Y-SNP) shows great variation in geographical distribution and population heterogeneity and can be used to map population genetics around the world. Massive parallel sequencing (MPS) methodology enables high-resolution Y-SNP haplogrouping for a certain male and is widely used in forensic genetics and evolutionary studies. In this present study, we used MPS to develop a customized 381 Y-SNP panel (SifaMPS 381 Y-SNP panel) to investigate the basic structure and subbranches of the haplogroup tree of the Chinese populations. The SifaMPS 381 Y-SNP panel covers all the Y-SNPs from our previously designed 183 Y-SNP panel and additional SNPs under the predominant haplogroups in the Chinese populations based on certain criteria. We also evaluated the sequencing matrix, concordance, sensitivity, repeatability of this panel and the ability to analyze mixed and case-type samples based on the Illumina MiSeq System. The results demonstrated that the novel MPS Y-SNP panel possessed good sequencing performance and generated accurate Y-SNP genotyping results. Although the recommended DNA input was greater than 1.25 ng, we observed that a lower DNA amount could still be used to analyze haplogroups correctly. In addition, this panel could handle mixed samples and common case-type samples and had higher resolution among Chinese Han males than previously reported. In conclusion, the SifaMPS 381 Y-SNP panel showed an overall good performance and offers a better choice for Y-SNP haplogrouping of the Chinese population, thereby facilitating paternal lineage classification, familial searching and other forensic applications.     

Pairwise kinship analysis of 17 pedigrees using massively parallel sequencing

With the tremendous development of massively parallel sequencing (MPS) in the last decade, it has been widely applied in basic science, clinical diagnostics, microbial genomics, as well as forensic genetics. MPS has lots of advantages that may facilitate the kinship analysis. In this study, 243 Chinese Han individuals from 17 families were involved and sequenced using the ForenSeq™ DNA Signature Prep Kit (Verogen, Inc., San Diego, USA), which provided the sequence information of 27 autosomal STRs (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative SNPs (iSNPs). A total of 275 pairs of parent-child, 123 pairs of full siblings, 1 pair of twins, 1 pair of half siblings, 158 pairs of grandparent-grandchild, 222 pairs of uncle/aunt-nephew/niece and 121 pairs of first cousins, as well as 701 pairs of unrelated individuals were identified. Using both likelihood ratio (LR) and identical by state (IBS) methods, the kinship analysis was conducted among these relative and non-relative pairs based on the A-STRs and SNPs. As a result, the ForenSeq Signature Kit could solve the analysis of parent-child (t1 = −4, t2 = 4), full siblings (t1 = −2, t2 = 2) and most second-degree kinships (t1 = −1, t2 = 1) using the LR method. When the IBS method was applied, 123 full sibling pairs had a higher average IBS value than other kinship groups in this study. And the IBS method could play a role in the testing of parent-child and full siblings.     

An evaluation of the PowerSeq™ Auto System: A multiplex short tandem repeat marker kit compatible with massively parallel sequencing

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq™ Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62 pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles.     

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48 × 10⁻²¹, higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA.In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.     

Genotyping polymorphic microhaplotype markers through the Illumina® MiSeq platform for forensics

Microhaplotype markers are emerging forensic genetic markers that have received broad attention in forensics and may supplement existing genetic marker panels. Short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs) are the general genetic markers at present. Stutter and the high mutation rate of STR markers and the low polymorphism of SNP markers obstruct the solving of certain cases. Kidd proposed microhaplotype markers that encompass 2–4 SNPs. In this study, we screened microhaplotype loci through three criteria, and chose the Illumina^® MiSeq platform to sequence the new markers. A new nomenclature was proposed and Perl-based tool FLfinder was designed to genotype the microhaplotype marker. After counting the number of haplotypes in samples that were sequenced and calculating common forensic parameters, 13 loci with high polymorphism were reported. Twelve of the 13 loci had an average allele coverage ratio (ACR) of 0.72 to 0.92. Structure analysis showed that 2504 samples (1000 genome project) could be divided into 5 groupings of populations, and each one representing a continental origin. The finding indicates that microhaplotype markers could be used for individual identification and ancestry inference, and a new choice is provided for forensic practice in the future.     

A new approach for forensic analysis of saliva-containing body fluid mixtures based on SNPs and methylation patterns of nearby CpGs

Saliva samples obtained from crime scenes often contain body fluids from other people, which makes it difficult to not only interpret the obtained DNA profiles, but also interpret saliva identification test results. α-amylase activity, an indicator of most saliva identification methods, can be slightly detected in other body fluids. This study aimed to overcome these difficulties. Here, we identified 13 saliva-specific methylated regions and five saliva-specific unmethylated regions neighboring common single nucleotide polymorphisms (SNPs) by array-based genome-wide methylation analysis of pooled saliva, blood, semen, or vaginal swab samples. Bisulfite sequencing by massively parallel sequencing (MPS) technology was then performed using individual body fluid samples to evaluate the saliva-specificity of each CpG of the three regions selected from the identified candidates. Although no single CpG demonstrated complete saliva-specificity, we found that the reads that were simultaneously (un)methylated at the selected neighboring two to three CpGs of each region were highly specific for saliva DNA. Based on these findings, we then designed MPS-based bisulfite sequencing assays for each region to analyze the selected CpGs and SNP(s) on the same read. These assays could identify the saliva of a target person from body fluid mixtures of known contributors (individual-specific saliva identification) by calculating the ratios of simultaneous (un)methylation at the selected CpGs within the reads containing SNP alleles unique to the target person. Moreover, these assays could indicate the SNP types of saliva DNA (saliva-specific genotyping) from body fluid mixtures by analyzing the alleles of the reads simultaneously (un)methylated at the selected CpGs, while careful attention should be paid to interpret the results of heterologous genotypes. Although further regions should be identified, especially for saliva-specific individual identification, the CpG-SNP approach may be an effective method to interpret the complicated results obtained from saliva-containing body fluid mixtures.     

Analysis of nondegraded and degraded DNA mixtures of close relatives using massively parallel sequencing

Identification of the minor contributor in DNA mixture of close relatives remains a dilemma in forensic genetics. Massively parallel sequencing (MPS) can analyze multiple short tandem repeats (STRs) and single nucleotide polymorphism (SNPs) concurrently and detect non-overlapping alleles of the minor contributors in DNA mixtures. A commercial kit for MPS of 59 identity informative STRs (iiSTRs) and 94 autosomal identity-informative SNPs (iiSNPs) was used to analyzed 34 nondegraded and 33 highly degraded two-person artificial DNA mixtures of close relatives with various minor to major ratios (1:9, 1:19, 1:29, 1:39, 1:79, 1:99). EuroForMix software was used to determine the minor contributors in the mixtures based on the likelihood ratios calculated from the MPS data, and relMix software was used to perform kinship analysis of the contributors. The STRs and SNPs of the 34 nondegraded and 33 degraded DNA mixtures were genotyped using MPS. Using EuroForMix based on the genotypes of autosomal iiSTRs and autosomal iiSNPs, 82.4% (28/34) and 54.5% (18/33) of minor donors could be accurately assigned for the nondegraded and degraded DNA mixtures, respectively. The relMix software correctly inferred the relationship between contributors in 97.1% (33/34) of nondegraded mixtures and in 97.0% (32/33) of degraded mixtures. In conclusion, combined EuroForMix and MPS data of STRs and SNPs can assist in the assignment of minor donors in nondegraded DNA mixtures of close relatives, and relMix can be used to infer relationship among contributors.     

Mitigating the effects of reference sequence bias in single-multiplex massively parallel sequencing of the mitochondrial DNA control region

Sequence analysis of the mitochondrial DNA (mtDNA) control region can provide forensically useful information, particularly in challenging samples where autosomal DNA profiling fails. Sub-division of the 1122-bp region into shorter PCR fragments improves data recovery, and such fragments can be analysed together via massively parallel sequencing (MPS). Here, we generate mtDNA data using the prototype PowerSeq™ Auto/Mito/Y System (Promega) MPS assay, in which a single PCR reaction amplifies ten overlapping amplicons of the control region, in a set of 101 highly diverse samples representing most major clades of the mtDNA phylogeny. The overlapping multiplex design leads to non-uniform coverage in the regions of overlap, where it is further increased by short amplicons generated alongside the intended products. Primer sequences in targeted amplification libraries are a potential source of reference sequence bias and thus should be removed, but the proprietary nature of the primers in commercial kits necessitates an alternative approach that minimises data loss: here, we introduce the bioinformatic selection of sequencing reads spanning putative primer sites (Overarching Read Enrichment Option, OREO). While OREO performs well in mitigating the effects of primer sequences at the ends of sequence reads, we still find evidence of the internalisation of primer-derived sequences by overlap extension, which may compromise the ability to call variants or to measure heteroplasmy in primer-binding regions. The commercially available PowerSeq™ CRM Nested System design prevents primer internalisation, as shown in a reanalysis of a subset of 57 samples that contain possible heteroplasmies. In combination with OREO, the CRM Nested kit mitigates reference sequence bias, allowing heteroplasmic variants to be estimated down to a 5% threshold. Provided appropriate steps are taken in data processing, single-reaction multiplex assays represent robust tools to analyse mtDNA control region variation. The OREO approach will allow users to bypass the effects of unknown primer sequences in any single-reaction tiled multiplex and eliminate primer-derived bias in overlapping amplicon sequencing studies, in both forensic and non-forensic settings.     

Set up of cutoff thresholds for kinship determination using SNP loci

The usefulness of single nucleotide polymorphism (SNP) loci for kinship testing has been demonstrated in many case works, and suggested as a promising marker for relationship identification. For interpreting results based on the calculation of the likelihood ratio (LR) in kinship testing, it is important to prepare cutoffs for respective relatives which are dependent on genetic relatedness. For this, analysis using true pedigree data is significant and reliable as it reflects the actual frequencies of markers in the population. In this study, the kinship index was explored through 1209 parent-child pairs, 1373 full sibling pairs, and 247 uncle-nephew pairs using 136 SNP loci. The cutoffs for LR were set up using different numbers of SNP loci with accuracy, sensitivity, and specificity. It is expected that this study can support the application of SNP loci-based kinship testing for various relationships.     

Multiplex Y-STRs analysis using the ion torrent personal genome machine (PGM)

Massively parallel sequencing (MPS) technologies allow parallel sequencing analyses of many targeted regions of multiple samples at desirable depth of coverage. Routine use of MPS for forensic genetics is on the horizon. In this study, we explore the application of MPS technology in forensic Y-STR analysis. We designed a multiplex assay with 13 Y-STR loci (DYS19, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS437, DYS438, DYS439, DYS448, DYS456, DYS635, GATA-H4) for the purpose of MPS. The multiplex Y-STR assay was amplified in 42 unrelated male individuals and amplicons were sequenced simultaneously using the ion torrent personal genome machine (PGM) system. All loci were detected successfully, except for DYS389 II that exhibited a failure rate of 1.8% due to the relatively long amplicon sizes. We observed 7, 3, 2, 6 and 5 new alleles, respectively in DYS389 II, DYS390, DYS437, DYS448 and DYS635 due to the presence of sub-repeat composition differences, and a new allele in DYS438 because of nucleotide substitution. One allele of DYS390 was inconsistent with allele call from conventional capillary electrophoresis (CE) because of 4 bp deletions upstream of the core repeat unit. This study demonstrates that Y-STR typing by MPS can provide more genetic information, holding the promise for high discriminatory power.     

Screening for single nucleotide polymorphisms in highly degraded DNA by using the amplified fragment length polymorphism technique

Short tandem repeat (STR) analysis is generally used for human identification of forensic samples; however, standard STR analysis sometimes fails to generate full profiles since DNA is frequently degraded by various environmental factors. Recently, single nucleotide polymorphism (SNP) analysis has attracted attention for human identification since the shorter amplicons are better suited for degraded samples. Though various SNP loci are used for analysis of degraded samples, it is unclear which ones are more appropriate. To identify SNPs that were resistant to degradation, we artificially degraded DNA obtained from the buccal swabs of six volunteers and the K562 cell line by heat treatment. Subsequently, the amplified fragment length polymorphism (AFLP) technique was used for SNP screening. We focused on the AFLP bands detected in both the heat-treated and untreated samples, and DNA extracted from these bands was directly sequenced. DNA degradation increased as the duration of heat treatment increased, and no STR profiles could be generated after 6 h of heat treatment. When the AFLP band patterns were compared between 6 h heat-treated and untreated samples, eight common bands were detected. The sequences of the DNA fragments of these common bands had higher adenine-thymine (A-T) content and included 17 SNPs. The SNPs detected in the heat-treated and untreated samples were considered to be resistant to degradation. Although there was a little information available in databases regarding the nine SNPs identified in this study, this study shows that some of these SNPs might be useful for human identification of extremely degraded DNA.     

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.     

Improving the system power of complex kinship analysis by combining multiple systems

Complex kinship analysis is a critical issue in forensic genetics. To address this issue, 55 STRs and 94 SNPs collected from four commercial forensic typing kits [three kits were based on a capillary electrophoresis (CE) platform and one was based on a next-generation sequencing (NGS) platform] were employed to test the system power for 2nd-degree and 3rd-degree kinship analysis. To measure the kinship index in related individuals, likelihood ratios (LRs) were calculated based on length and sequence polymorphism information (LR_length and LR_sequence, respectively) from simulation as well as true pedigree samples. LRs calculated based on sequence information are generally higher than those based on length information. The sensitivity, specificity, and effectiveness to distinguish the 2nd- and 3rd-degree kinship were estimated from four marker sets with different numbers of markers. As expected, system power for kinship analysis improved by increasing the number of markers and using LR_sequence, instead of LR_length. Furthermore, the system power based on 55 STRs from the CE platform is equal to the 40 STRs and 94 SNPs from one CE kit and the kit based on NGS platform for both 2nd-degree and 3rd-degree kinship analysis. For discrimination of 2nd-degree kinship, the system effectiveness is 86.63% with an error ratio < 0.01 using the 55 STRs from the CE platform. Using sequence information from the 55 STRs and 94 SNPs, the system effectiveness is 94.43%, with an error ratio < 0.001 for 2nd-degree kinship analysis and 64.34% with an error ratio < 0.05 for 3rd-degree kinship analysis, indicating that these markers are powerful for 2nd-degree kinship analysis and can be used for 3rd-degree kinship analysis.     

Assessing non-LUS stutter in DNA sequence data

Forensic DNA analysis is among the most well-recognized and well-developed forensic disciplines. The field’s use of DNA markers known as short tandem repeats (STRs) offer a robust means of discriminating individuals while also introducing challenges to the analysis. One of these challenges, stutter, is the result of a non-biological artifact introduced during PCR. The formation and amplification of these stutter products can occur at rates as high as 15–20% of the parent allele. The challenge inherent in this process is differentiating stutter artifacts from true alleles, particularly in the presence of a minor contributor. Traditionally, DNA profiles are obtained using capillary electrophoresis (CE), where amplified DNA fragments are separated by size, not sequence, and the identification of stutter is performed on a locus-specific level. The use of CE-based fragment data rather than sequence-based data, has limited the community’s understanding of the precise behavior of stutter. Massively parallel sequencing (MPS) data provides an opportunity to better characterize stutter, permitting a more accurate means of detecting both size- or longest uninterrupted stretch (LUS)-based stutter but also allele and motif-specific stutter characteristics. This study sheds light on the value of characterizing motif- and allele-specific stutter, including non-LUS stutter, when using MPS methods. Analysis and characterization of stutter sequences was performed using data generated from 539 samples amplified with the ForenSeq and PowerSeq 46GY library preparation kit and sequenced on the Illumina MiSeq FGx. Assessment of non-LUS stutter begins with calculating stutter rates for all potential stutter products at a given locus (and allele), additionally, the occurrence of these discrete stutter products were quantified. Results show that although the LUS sequence stutters at a higher rate than non-LUS motifs, the non-LUS stutter products do occur at detectable levels and potentially influence sequence-based mixture analysis. The data indicate that the stutter from one motif or allele can be distinguished from another motif or allele based on their unique stutter rates; however, the number of stutter products from each motif or allele may similarly make up the overall pool of stutter products. Motif- and allele-specific stutter models provide the most comprehensive analysis of sequence stutter rates and provide the ability to differentiate stutter sequences more accurately from true allele stutter. This information provides a foundation for including the characterization of non-LUS stutter products when analyzing DNA profiles, specifically mixtures with potential low-level contributors.     

Separation of SNP profiles from DNA mixtures with two contributors via massively parallel sequencing technology

ABSTRACT

Massively parallel sequencing technology offers the opportunity to analyse forensically challenging samples, such as degraded samples and mixtures. In the current study, we developed a perl-based pipeline to separate the DNA mixture into its components and to predict the most probable single nucleotide polymorphism (SNP) genotypes of each contributor to the mixed profile. We examined the usefulness of this method by detecting both artificially constructed DNA mixtures and mixtures from crime cases using the Precision ID Identity Panel on the Ion PGM platform. The separated genotypes of mixtures were validated by genotypes of each of the donors detected independently. The results indicate that the method performed well in identifications of both the artificially constructed mixtures and case-type mixtures, even when the two contributors are immediate relatives (mother and son), which demonstrated the practical usefulness of this method in forensic casework. Our research presents an efficient and different strategy for identification and paternity testing of DNA mixtures in forensic genetics.     

Massively parallel sequencing of 74 microhaplotypes and forensic characteristics in three Chinese Sino-Tibetan populations

Microhaplotype (MH), as an emerging type of forensic genetic marker in recent years, has the potential to support multiple forensic applications, especially for mixture deconvolution and biogeographic ancestry inference. Herein, we investigated the genotype data of 74 MHs included in a novel MH panel, the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel, in three Chinese Sino-Tibetan populations (Han, Tibetan, and Yi) using the Ion Torrent semiconductor sequencing. The sequencing performance, allele frequencies, effective number of alleles (A_e), informativeness (I_n), and forensic parameters were subsequently estimated and calculated. In addition, principal component analysis (PCA) and structure analysis were performed to explore the population relationships among the three populations and the ancestry component distribution. Overall, this novel MH panel is robust and reliable, and has an excellent sequencing performance. The A_e values ranged from 1.0126 to 7.0855 across all samples, and 75.68 % of MHs had A_e values >2.0000. Allele frequencies at some loci varied considerably among the three studied populations, and the mean I_n value was 0.0195. Moreover, the genetic affinity between Tibetans and Yis was closer than that between Tibetans and Hans. The aforementioned results suggest that the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel is highly polymorphic in three investigated populations and could be used as an effective tool for human forensics. Although these 74 MHs have demonstrated the competency in continental population stratification, a higher resolution for distinguishing intracontinental subpopulations and a more comprehensive database with sufficient reference population data still remain to be accomplished.     

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.     

Developmental validation of a custom panel including 165 Y-SNPs for Chinese Y-chromosomal haplogroups dissection using the ion S5 XL system

Massively parallel sequencing (MPS) technology has the capability to genotype hundreds to thousands of SNPs from multiple samples, with high coverage in a single experimental run. MPS, which enables high-resolution Y haplogrouping from limited DNA resources, has attracted much interest in Y-chromosomal applications, particularly human forensics and anthropological studies. Previously, we selected and designed a 72 Y-SNPs MPS panel aiming at the Chinese population for providing the basic structure of the haplogroup tree, and then additional Y-SNPs were screened out for further subdivision into different populations by using pyrosequencing technology. In the present study, we designed a customized MPS panel that includes 165 Y-SNPs based on our previous studies and evaluated the sequencing performance, sensitivity and the ability of the MPS-SNP panel to analyze case-type samples using the Ion S5 XL System. In the concordant study, only four Y-SNPs presented missing genotypes due to low coverage reads, and five SNPs were prone to being designated heterozygotes, whereas the designation of the other SNPs were fully consistent with the results obtained from other Y-SNP typing tools. The analyst-interpreted Y-SNP profiles demonstrated that the targeted Y-SNPs, except for the nine poorly performing Y-SNPs, performed well and could acquire achievable high-resolution. Moreover, this MPS panel could be adapted to case-type samples, and single source complete profiles could be stably obtained using 1 ng of input DNA. In summary, this MPS Y-SNP panel offers a straightforward sample-to-haplogroup workflow that would be beneficial for paternal lineage classification and forensic pedigree searches, and the present results provide support for the application of MPS technology in SNP analysis.     

Robust detection of individual forensic profiles in DNA mixtures

For a forensic identification method to be admissible in international courts, the probability of false match must be quantified. For comparison of individuals against complex mixtures using a panel of single nucleotide polymorphisms (SNPs), the probability of a random man not excluded, P(RMNE) is one admissible standard. While the P(RMNE) of SNP alleles has been previously studied, it remains to be rigorously defined and calculated for experimentally genotyped mixtures. In this report, exact P(RMNE) values were calculated for a range of complex mixtures, verified with Monte Carlo simulations, and compared alongside experimentally determined detection probabilities.