Mitochondrial DNA heteroplasmy in the emerging field of massively parallel sequencing

Long an important and useful tool in forensic genetic investigations, mitochondrial DNA (mtDNA) typing continues to mature. Research in the last few years has demonstrated both that data from the entire molecule will have practical benefits in forensic DNA casework, and that massively parallel sequencing (MPS) methods will make full mitochondrial genome (mtGenome) sequencing of forensic specimens feasible and cost-effective. A spate of recent studies has employed these new technologies to assess intraindividual mtDNA variation. However, in several instances, contamination and other sources of mixed mtDNA data have been erroneously identified as heteroplasmy. Well vetted mtGenome datasets based on both Sanger and MPS sequences have found authentic point heteroplasmy in approximately 25% of individuals when minor component detection thresholds are in the range of 10–20%, along with positional distribution patterns in the coding region that differ from patterns of point heteroplasmy in the well-studied control region. A few recent studies that examined very low-level heteroplasmy are concordant with these observations when the data are examined at a common level of resolution. In this review we provide an overview of considerations related to the use of MPS technologies to detect mtDNA heteroplasmy. In addition, we examine published reports on point heteroplasmy to characterize features of the data that will assist in the evaluation of future mtGenome data developed by any typing method.     

Application of a custom haplotype caller to analyze sequence-based data of 56 microhaplotypes

Microhaplotypes (microhaps) are recently introduced markers that aim to complement the limitations of conventional forensic markers such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). With the potential of microhaps in forensics becoming clearer through massively parallel sequencing (MPS), MPS-based studies on microhaps are being actively reported. However, simpler workflow schemes for the generation and analysis of MPS data are still required to facilitate the practical application of MPS in forensics. In this study, we developed an in-house MPS panel that simultaneously amplifies 56 microhaps and a custom haplotype caller, Visual Microhap. The developed tool works on a web browser and provides four analysis options to extract SNP-based haplotypes from sequence-based data obtained by STRait Razor 3.0. To demonstrate the utility of the MPS panel and data analysis workflow scheme, we also analyzed 56 microhaps of 286 samples from four populations (African-American, Caucasian, Hispanic, and Korean). The average effective number of alleles (A_e) for the four groups was 3.45, ranging from 1.74 to 6.98. Forensic statistical parameters showed that this microhap panel is more powerful than conventional autosomal STRs for human identification. Meanwhile, the 56-plex panel mostly comprised microhaps with high Ae; however, the four populations were grossly distinguishable from each other by cluster analysis. Consequently, the developed in-house MPS panel for 56 microhaps and the adopted workflow using open-source tools can increase the utility of microhap MPS in forensic research and practice.     

Degradation in forensic trace DNA samples explored by massively parallel sequencing

Routine forensic analysis using STRs will fail if the DNA is too degraded. The DNA degradation process in biological stain material is not well understood. In this study we sequenced old semen and blood stains by massively parallel sequencing. The sequence data coverage was used to measure degradation across the genome. The results supported the contention that degradation is uniform across the genome, showing no evidence of regions with increased or decreased resistance towards degradation. Thus the lack of genetic regions robust to degradation removes the possibility of using such regions to further optimize analysis performance for degraded DNA.     

Development and inter-laboratory validation of the VISAGE enhanced tool for age estimation from semen using quantitative DNA methylation analysis

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool.     

A nomenclature for sequence-based forensic DNA analysis

Forensic DNA analysis of casework samples using massively parallel sequencing (MPS) technology requires a system of nomenclature for uniquely labeling sequence-based alleles and artifacts. The DNA Commission of the ISFG has published considerations concerning a nomenclature format that addresses the requirement for unique labeling of sequences. Nomenclatures based on this format can be used in databasing, or communicating sequence types, but the format is lengthy for software interfaces. The sequence identifier (SID) nomenclature addresses this gap by generating short labels able to uniquely identify all sequences (allelic and artifactual) in single-source or casework profiles. Sequences in casework profiles can be uniquely labeled with only two or three SID characters, making the format compact. SID labels can be used in algorithms for identifying and filtering artifacts, and for expressing associations between artifacts and their likely parent alleles. The nomenclature is suitable for use in downstream mixture analysis by any software able to accept character values rather than numeral values. The SID nomenclature is described, and its ability to discriminate sequence-based alleles and artifacts is demonstrated, and its applicability to forensic mixture analysis is demonstrated.     

Recent advances in Forensic DNA Phenotyping of appearance,ancestry and age

Forensic DNA Phenotyping (FDP) comprises the prediction of a person’s externally visible characteristics regarding appearance, biogeographic ancestry and age from DNA of crime scene samples, to provide investigative leads to help find unknown perpetrators that cannot be identified with forensic STR-profiling. In recent years, FDP has advanced considerably in all of its three components, which we summarize in this review article. Appearance prediction from DNA has broadened beyond eye, hair and skin color to additionally comprise other traits such as eyebrow color, freckles, hair structure, hair loss in men, and tall stature. Biogeographic ancestry inference from DNA has progressed from continental ancestry to sub-continental ancestry detection and the resolving of co-ancestry patterns in genetically admixed individuals. Age estimation from DNA has widened beyond blood to more somatic tissues such as saliva and bones as well as new markers and tools for semen. Technological progress has allowed forensically suitable DNA technology with largely increased multiplex capacity for the simultaneous analysis of hundreds of DNA predictors with targeted massively parallel sequencing (MPS). Forensically validated MPS-based FDP tools for predicting from crime scene DNA i) several appearance traits, ii) multi-regional ancestry, iii) several appearance traits together with multi-regional ancestry, and iv) age from different tissue types, are already available. Despite recent advances that will likely increase the impact of FDP in criminal casework in the near future, moving reliable appearance, ancestry and age prediction from crime scene DNA to the level of detail and accuracy police investigators may desire, requires further intensified scientific research together with technical developments and forensic validations as well as the necessary funding.     

Targeted DNA methylation analysis and prediction of smoking habits in blood based on massively parallel sequencing

Tobacco smoking is a frequent habit sustained by > 1.3 billion people in 2020 and the leading preventable factor for health risk and premature mortality worldwide. In the forensic context, predicting smoking habits from biological samples may allow broadening DNA phenotyping. In this study, we aimed to implement previously published smoking habit classification models based on blood DNA methylation at 13 CpGs. First, we developed a matching lab tool based on bisulfite conversion and multiplex PCR followed by amplification-free library preparation and targeted paired-end massively parallel sequencing (MPS). Analysis of six technical duplicates revealed high reproducibility of methylation measurements (Pearson correlation of 0.983). Artificially methylated standards uncovered marker-specific amplification bias, which we corrected via bi-exponential models. We then applied our MPS tool to 232 blood samples from Europeans of a wide age range, of which 90 were current, 71 former and 71 never smokers. On average, we obtained 189,000 reads/sample and 15,000 reads/CpG, without marker drop-out. Methylation distributions per smoking category roughly corresponded to previous microarray analysis, showcasing large inter-individual variation but with technology-driven bias. Methylation at 11 out of 13 smoking-CpGs correlated with daily cigarettes in current smokers, while solely one was weakly correlated with time since cessation in former smokers. Interestingly, eight smoking-CpGs correlated with age, and one displayed weak but significant sex-associated methylation differences. Using bias-uncorrected MPS data, smoking habits were relatively accurately predicted using both two- (current/non-current) and three- (never/former/current) category model, but bias correction resulted in worse prediction performance for both models. Finally, to account for technology-driven variation, we built new, joint models with inter-technology corrections, which resulted in improved prediction results for both models, with or without PCR bias correction (e.g. MPS cross-validation F₁-score > 0.8; 2-categories). Overall, our novel assay takes us one step closer towards the forensic application of viable smoking habit prediction from blood traces. However, future research is needed towards forensically validating the assay, especially in terms of sensitivity. We also need to further shed light on the employed biomarkers, particularly on the mechanistics, tissue specificity and putative confounders of smoking epigenetic signatures.     

Assessing the utility of quantitative and qualitative metrics in the DNA quantification process of skeletal remains for autosomal and Y-chromosome STR amplification purposes

In historical cases, ancient DNA investigations and missing persons identification, teeth or bone samples are often the only and almost always the best biological material available for DNA typing. On the other hand, DNA obtained from bone material may be characterized by a high degradation index (DI) or its low content, or DNA tests cannot be repeated due to bone piece size limitation. That is often the effect of the environment in which the material was placed and the time during which exposure to unfavorable environmental factors took place. Therefore, it is very important to use appropriate procedures related to STR analysis. For our study, we selected 80 challenging bone samples. The amount of DNA was compared in qPCR using Quantifiler™ Trio DNA Quantification Kit and Investigator® Quantiplex® Pro RGQ. All qPCR results were confirmed by PCR-CE. The results of DNA concentrations and the assigned degradation index (DI) differed significantly within analyzed samples (~10%). Additionally, the Y-chromosome DI also differed from the autosomal DI in the samples. The difference in degradation indexes could explain the lower Y-chromosome amplification success rate compared to autosomal e.g. during human identification process. The results indicate that performing two DNA quantifications with the use of two different kits (primers sets) allows for a much more precise evaluation of the DNA quality and quantity in the isolate. We suggest that at least one of two suggested DNA concentration measurements should be based on an additional determination of the Y chromosome degradation index. Altogether, it allows for rational isolate management, especially when the volume is limited and the sample is unique.     

Evaluating 130 microhaplotypes across a global set of 83 populations

Today the primary DNA markers used in forensics are short tandem repeat (STR) polymorphisms (STRPs), initially selected because they are highly polymorphic. However, the increasingly common need to deal with samples with a mixture of DNA from two or more individuals sometimes is complicated by the inherent stutter involved with PCR amplification, especially in strongly unbalanced mixtures when the minor component coincides with the stutter range of the major component. Also, the STRPs in use provide little evidence of ancestry of a single source sample beyond broad “continental” resolution. Methodologies for analyzing DNA have become much more powerful in recent years. Massively parallel sequencing (MPS) is a new method being considered for routine use in forensics. Primarily to aid in mixture deconvolution and avoid the issue of stutter, we have begun to investigate a new type of forensic marker, microhaplotype loci, that will provide useful information on mixtures of DNA and on ancestry when typed using massively parallel sequencing (MPS). We have identified 130 loci and estimated their haplotype (allele) frequencies in 83 different population samples. Many of these loci are shown to be highly informative for individual identification and for mixture identification and deconvolution.     

Considering DNA damage when interpreting mtDNA heteroplasmy in deep sequencing data

Resolution of mitochondrial (mt) DNA heteroplasmy is now possible when applying a massively parallel sequencing (MPS) approach, including minor components down to 1%. However, reporting thresholds and interpretation criteria will need to be established for calling heteroplasmic variants that address a number of important topics, one of which is DNA damage. We assessed the impact of increasing amounts of DNA damage on the interpretation of minor component sequence variants in the mtDNA control region, including low-level mixed sites. A passive approach was used to evaluate the impact of storage conditions, and an active approach was employed to accelerate the process of hydrolytic damage (for example, replication errors associated with depurination events). The patterns of damage were compared and assessed in relation to damage typically encountered in poor quality samples. As expected, the number of miscoding lesions increased as conditions worsened. Single nucleotide polymorphisms (SNPs) associated with miscoding lesions were indistinguishable from innate heteroplasmy and were most often observed as 1–2% of the total sequencing reads. Numerous examples of miscoding lesions above 2% were identified, including two complete changes in the nucleotide sequence, presenting a challenge when assessing the placement of reporting thresholds for heteroplasmy. To mitigate the impact, replication of miscoding lesions was not observed in stored samples, and was rarely seen in data associated with accelerated hydrolysis. In addition, a significant decrease in the expected transition:transversion ratio was observed, providing a useful tool for predicting the presence of damage-induced lesions. The results of this study directly impact MPS analysis of minor sequence variants from poorly preserved DNA extracts, and when biological samples have been exposed to agents that induce DNA damage. These findings are particularly relevant to clinical and forensic investigations.     

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.     

Development and inter-laboratory evaluation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA

Responding to the growing scientific and practical interest in forensic DNA phenotyping, the VISible Attributes through GEnomics (VISAGE) Consortium was founded in 2017 with the main goal of developing and validating new and reliable molecular and statistical tools to predict appearance, ancestry and age from DNA. Here, we describe the development and inter-laboratory evaluation and validation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA. The VISAGE Enhanced Tool for Appearance and Ancestry is the first forensic-driven genetic laboratory tool that comprises well-established markers for eye, hair and skin color with more recently discovered DNA markers for eyebrow color, freckling, hair shape and male pattern baldness and bio-geographic ancestry informative DNA markers. The bio-geographic ancestry markers include autosomal SNPs (bi- and tri-allelic SNPs), X-SNPs, Y-SNPs and autosomal Microhaplotypes. In total, primers targeting 524 SNPs (representing a 97.6% assay conversion rate) were successfully designed using AmpliSeq into a single primer pool (i.e., one multiplex assay) and sequenced with the Ion S5. In a collaborative framework, five VISAGE laboratories tested the VISAGE Enhanced Tool for Appearance and Ancestry on reproducibility, sensitivity, genotyping concordance, mixtures, species specificity and performance in relevant forensic conditions, including inhibitor-spiked, mock casework and artificially degraded samples. Based on our results, the VISAGE Enhanced Tool for Appearance and Ancestry is a robust, reproducible, and – for the large SNP number - fairly sensitive MPS assay with high concordance rates. With the VISAGE Enhanced Tool for Appearance and Ancestry introduced here, the VISAGE Consortium delivers the first single DNA-test for combined appearance prediction based on seven traits together with bio-geographic ancestry inference based on major continental regions for separated bi-parental and paternal ancestry, which represents the most comprehensive validated laboratory tool currently available for Forensic DNA Phenotyping.     

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.     

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.     

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.     

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.     

Estimating number of contributors in massively parallel sequencing data of STR loci

In recent years a number of computer-based algorithms have been developed for the deconvolution of complex DNA mixtures in forensic science. These procedures utilize likelihood ratios that quantify the evidence for a hypothesis for the presence of a person of interest in a DNA profile compared to an alternative hypothesis. Proper operation of these software systems requires an assumption regarding the total number of contributors present in the mixture. Unfortunately, estimates based on counting the number of alleles at a locus can be inaccurate due to the sharing and masking of alleles at individual loci. The effects of allele masking become increasingly severe as the number of contributors increases, rendering estimates about high-order mixtures uncertain. The accuracy of these estimates can be improved by increasing the number of STR markers in panels, and by using highly polymorphic markers. Increasing the number of STR markers from 13 to 20 (expanded CODIS panel) improves the accuracy of allele count-based estimation methods for low-order mixtures, but accuracy for high-order mixtures (> 3 contributors) remains poor due to allele masking. An alternative technique, massively parallel sequencing, holds great potential to improve the accuracy of the estimate of number of contributors due to its ability to detect sequence polymorphisms within alleles. This process results in an expansion of the number of alleles when compared to that obtained using capillary electrophoresis. Here, we show that the detection of these additional sequence-defined alleles in 22-marker panels improves number of contributor estimates in conceptual mixtures of 4 and 5 contributors.     

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.     

Integrating the human microbiome in the forensic toolkit: Current bottlenecks and future solutions

Over the last few years, advances in massively parallel sequencing technologies (also referred to next generation sequencing) and bioinformatics analysis tools have boosted our knowledge on the human microbiome. Such insights have brought new perspectives and possibilities to apply human microbiome analysis in many areas, particularly in medicine. In the forensic field, the use of microbial DNA obtained from human materials is still in its infancy but has been suggested as a potential alternative in situations when other human (non-microbial) approaches present limitations. More specifically, DNA analysis of a wide variety of microorganisms that live in and on the human body offers promises to answer various forensically relevant questions, such as post-mortem interval estimation, individual identification, and tissue/body fluid identification, among others. However, human microbiome analysis currently faces significant challenges that need to be considered and overcome via future forensically oriented human microbiome research to provide the necessary solutions. In this perspective article, we discuss the most relevant biological, technical and data-related issues and propose future solutions that will pave the way towards the integration of human microbiome analysis in the forensic toolkit.     

A phylogenetic approach for haplotype analysis of sequence data from complex mitochondrial mixtures

Massively parallel (next-generation) sequencing provides a powerful method to analyze DNA from many different sources, including degraded and trace samples. A common challenge, however, is that many forensic samples are often known or suspected mixtures of DNA from multiple individuals. Haploid lineage markers, such as mitochondrial (mt) DNA, are useful for analysis of mixtures because, unlike nuclear genetic markers, each individual contributes a single sequence to the mixture. Deconvolution of these mixtures into the constituent mitochondrial haplotypes is challenging as typical sequence read lengths are too short to reconstruct the distinct haplotypes completely. We present a powerful computational approach for determining the constituent haplotypes in massively parallel sequencing data from potentially mixed samples. At the heart of our approach is an expectation maximization based algorithm that co-estimates the overall mixture proportions and the source haplogroup for each read individually. This approach, implemented in the software package mixemt, correctly identifies haplogroups from mixed samples across a range of mixture proportions. Furthermore, our method can separate fragments in a mixed sample by the most likely originating contributor and generate reconstructions of the constituent haplotypes based on known patterns of mtDNA diversity.