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.     

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.     

Forensic identity SNPs: Characterisation of flanking region variation using massively parallel sequencing

Single nucleotide polymorphisms (SNPs) can be analysed for identity or kinship applications in forensic genetics to either provide an adjunct to traditional STR typing or as a stand-alone approach. The advent of massively parallel sequencing technology (MPS) has provided a useful opportunity to more easily deploy SNP typing in a forensic context, given the ability to simultaneously amplify a large number of markers. Furthermore, MPS also provides valuable sequence data for the targeted regions, which enables the detection of any additional variation seen in the flanking regions of amplicons. In this study we genotyped 977 samples across five UK-relevant population groups (White British, East Asian, South Asian, North-East African and West African) for 94 identity-informative SNP markers using the ForenSeq DNA Signature Prep Kit. Examination of flanking region variation allowed for the identification of 158 additional alleles across all populations studied. Here we present allele frequencies for all 94 identity-informative SNPs, both including and excluding the flanking region sequence of these markers. We also present information on the configuration of these SNPs in the ForenSeq DNA Signature Prep Kit, including performance metrics for the markers and investigation of bioinformatic and chemistry-based discordances. Overall, the inclusion of flanking region variation in the analysing workflow for these markers reduced the average combined match probability 2175 times across all populations, with a maximum reduction of 675,000-fold in the West African population. The gain due to flanking region-based discrimination increased the heterozygosity of some loci above that of some of the least useful forensic STR loci; thus demonstrating the benefit of enhanced analysis of currently targeted SNP markers for forensic applications.     

MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.     

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.     

Evaluation of the QIAGEN 140-SNP forensic identification multiplex from latent DNA using massively parallel sequencing

ABSTRACT

Direct PCR can be used to successfully generate full STR profiles from latent DNA. However, some substrates have been shown to be more problematic and in some cases only partial profiles are recovered. As latent DNA is present on the surface of objects in very low quantity, and potentially low quality, the fragment lengths targeted by STR typing may be too large to successfully amplify all markers. As an alternative, QIAGEN have developed a 140-SNP multiplex that targets much shorter amplicons and generates extremely low probabilities of any two unrelated individuals having identical genotypes. Here, we present the first forensic identification SNP data from latent DNA using massively parallel sequencing. We applied the QIAGEN 140-SNP forensic identification multiplex to swabs collected from multiple substrates (including mobile phone, fingerprint, wire, zip-lock bag and SIM card) and multiple donors.     

Evaluation of large-scale highly polymorphic microhaplotypes in complex DNA mixtures analysis using RMNE method

DNA mixture interpretation is one of the most challenging problems in forensics. Complex DNA mixtures are more difficult to analyze when there are more than two contributors or related contributors. Microhaplotypes (MHs) are polymorphic genetic markers recently discovered and employed in DNA mixture analysis. However, the evidentiary interpretation of the MH genotyping data needs more debate. The Random Man Not Excluded (RMNE) method analyzes DNA mixtures without using allelic peak height data or the number of contributors (NoC) assumptions. This study aimed to assess how well RMNE interpreted mixed MH genotyping data. We classified the MH loci from the 1000 Genomes Project database into groups based on their Ae values. Then we performed simulations of DNA mixtures with 2–10 unrelated contributors and DNA mixtures with a pair of sibling contributors. For each simulated DNA mixture, incorrectly included ratios were estimated for three types of non-contributors: random men, parents of contributors, and siblings of contributors. Meanwhile, RMNE probability was calculated for contributors and three types of non-contributors, allowing loci mismatch. The results showed that the MH number, the MH Ae values, and the NoC affected the RMNE probability of the mixture and the incorrectly included ratio of non-contributors. When there were more MHs, MHs with higher Ae values, and a mixture with less NoC, the RMNE probability, and the incorrectly included ratio decreased. The existence of kinship in mixtures complicated the mixture interpretation. Contributors’ relatives as non-contributors and related contributors in the mixture increased the demands on the genetic markers to identify the contributors correctly. When 500 highly polymorphic MHs with Ae values higher than 5 were used, the four individual types could be distinguished according to the RMNE probabilities. This study reveals the promising potential of MH as a genetic marker for mixed DNA interpretation and the broadening of RMNE as a parameter indicating the relationship of a specific individual with a DNA mixture in the DNA database search.     

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.     

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.     

Predicting the origin of stains from whole miRNome massively parallel sequencing data

In this study, we have screened the six most relevant forensic body fluids / tissues, namely blood, semen, saliva, vaginal secretion, menstrual blood and skin, for miRNAs using a whole miRNome massively parallel sequencing approach. We applied partial least squares (PLS) and linear discriminant analysis (LDA) to predict body fluids based on the expression of the miRNA markers. We estimated the prediction accuracy for models including different subsets of miRNA markers to identify the minimum number of markers needed for sufficient prediction performance. For one selected model consisting of 9 miRNA markers we calculated their importance for prediction of each of the six different body fluid categories.     

Forensic STR analysis using massive parallel sequencing

We explore the applicability of second generation sequencing (SGS) to sequence multiplexed forensic STR amplicons, both in a single contributor sample as in multiple-person mixtures with different ratios. We compare the results of a commercial STR profiling kit (Applied Biosystems AmpFlSTR^® Profiler Plus^®), analyzed both with capillary electrophoresis and with Roche GS FLX sequencing. An easy to use open-source software pipeline is provided, chaining together the different steps needed to start the analysis from a GS FLX FASTA file, resulting in a FASTA file containing the called and quantified alleles present in the data. Sequencing of multiplexed STR amplicons using Roche GS FLX titanium technology is technically feasible but the technology is not ideal for this purpose. The fraction of full length reads is small and the homopolymer sequencing error rate is high. The pipeline compresses the homopolymers to a single base to avoid false results caused by these homopolymers. The qualitative and quantitative results from the SGS STR analysis pipeline are comparable to the electrophoresis method. Additionally, the SGS method provides extra information and is able to call allele subtypes based on STR sequences in a database. In mixed samples, all alleles were reported from individuals that contributed at least 10% to the mixture.     

Forensic Y-SNP analysis beyond SNaPshot: High-resolution Y-chromosomal haplogrouping from low quality and quantity DNA using Ion AmpliSeq and targeted massively parallel sequencing

Y-chromosomal haplogroups assigned from male-specific Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal bio-geographic ancestry inference, both being relevant in forensic genetics. However, most previously developed forensic Y-SNP tools did not provide Y haplogroup resolution on the high level needed in forensic applications, because the limited multiplex capacity of the DNA technologies used only allowed the inclusion of a relatively small number of Y-SNPs. In a proof-of-principle study, we recently demonstrated that high-resolution Y haplogrouping is feasible via two AmpliSeq PCR analyses and simultaneous massively parallel sequencing (MPS) of 530 Y-SNPs allowing the inference of 432 Y-haplogroups. With the current study, we present a largely improved Y-SNP MPS lab tool that we specifically designed for the analysis of low quality and quantity DNA often confronted with in forensic DNA analysis. Improvements include i) Y-SNP marker selection based on the “minimal reference phylogeny for the human Y chromosome” (PhyloTree Y), ii) strong increase of the number of targeted Y-SNPs allowing many more Y haplogroups to be inferred, iii) focus on short amplicon length enabling successful analysis of degraded DNA, and iv) combination of all amplicons in a single AmpliSeq PCR and simultaneous sequencing allowing single DNA aliquot use. This new MPS tool simultaneously analyses 859 Y-SNPs and allows inferring 640 Y haplogroups. Preliminary forensic developmental validation testing revealed that this tool performs highly accurate, is sensitive and robust. We also provide a revised software tool for analysing the sequencing data produced by the new MPS lab tool including final Y haplogroup assignment. We envision the tools introduced here for high-resolution Y-chromosomal haplogrouping to determine a man’s paternal lineage and/or paternal bio-geographic ancestry to become widely used in forensic Y-chromosome DNA analysis and other applications were Y haplogroup information from low quality / quantity DNA samples is required.     

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.     

SEQ Mapper: A DNA sequence searching tool for massively parallel sequencing data

The development of massively parallel sequencing (MPS) has increased greatly the scale of DNA sequencing. The analysis of massive data-files from single MPS analysis can be a major challenge if examining the data for potential polymorphic loci. To aid in the analysis of both short tandem repeat (STR) and single nucleotide polymorphisms (SNP), we have designed a new program called SEQ Mapper to search for genetic polymorphisms within a large number of reads generated by MPS. This new program has been designed to perform sequence mapping between reference data and generated reads. As a proof-of-concept, sequences derived from the allelic ladders of five STR loci and data from the amelogenin locus were used as reference data sets. Detecting and recording the polymorphic nature of each STR loci was performed using four levels of search criteria: the entire STR locus spanning the two primers; the STR region plus the two primer sequences; the STR region only; and the two primers only. All the genotypes of 5 STR loci and the amelogenin gene were identified correctly using SEQ Mapper when compared to results obtained from capillary electrophoresis based on 10 test samples in this study. SEQ Mapper is a useful tool to detect STR or SNP alleles generated by MPS in both clinical medicine and forensic genetics.     

Characterizing the amplification of STR markers in multiplex polymerase chain displacement reaction using massively parallel sequencing

Polymerase chain displacement reaction (PCDR) showed advantages in forensic low-template DNA analysis with improved amplification efficiency, higher allele detection capacity, and lower stutter artifact than PCR. However, characteristics of STR markers after PCDR amplification remain unclarified for the limited resolving power of capillary electrophoresis (CE). This issue can be addressed by massively parallel sequencing (MPS) technology with higher throughput and discriminability. Here, we developed a multiplex PCDR system including 24 STRs and amelogenin. In addition, a PCR reference was established for comparison. After amplification, products were subjected to PCR-free library construction and sequenced on the Illumina NovaSeq system. We implemented a sequence-matching pipeline to separate different amplicon types of PCDR products from the combination of primers. In the sensitivity test, the PCDR multiplex obtained full STR profiles with as low as 125 pg 2800M control DNA. Based on that, single-source DNA samples were tested. First, highly concordant genotypes were observed among the PCDR multiplex, the PCR reference, and CE-based STR kits. Next, read counts of different PCDR amplicon types were investigated, showing a relative abundance of 78:12:12:1 for the shortest amplicon S, the two medium amplicons M1 and M2, and the longest amplicon L. We also analyzed the stutter artifacts for distinct amplicon types, and the results revealed the reduction of N − 1 and N − 2 contraction stutters, and the increase of N + 1 and N + 2 elongation stutters in PCDR samples. Moreover, we confirmed the feasibility of PCDR for amplifying degraded DNA samples and unbalanced DNA mixtures. Compared to the previous proof of principle study, our work took a further step to characterize the complete profile of STR markers in the PCDR context. Our results suggested that the PCDR-MPS workflow is an effective approach for forensic STR analysis. Corresponding findings in this study may help the development of PCDR-based assays and probabilistic methods in future studies.     

Comparison of whole mitochondrial genome variants between hair shafts and reference samples using massively parallel sequencing

International Journal of Legal Medicine - Hair shafts are one of the most common types of evidence at crime scenes, and mitochondrial DNA (mtDNA) has been analyzed as a valuable genetic marker for...     

Development of a multiplex forensic identity panel for massively parallel sequencing and its systematic optimization using design of experiments

The application of massively parallel sequencing (MPS) in forensic sciences enables high-resolution short tandem repeat (STR) genotyping for the characterization of biological evidence. While MPS supports multiplexing of a large number of forensic markers, the performance of an MPS-STR panel depends on good primer design and optimal PCR conditions. However, conventional strategies for multifactorial assay optimization are labor-intensive and do not necessarily allow the experimenter to identify optimum factor settings.Here we describe our new multiplex PCR assay, monSTR, which supports the simultaneous amplification of 21 forensic markers followed by targeted sequencing on the Illumina MiSeq. The selection of STR markers adapts on the expanded European Standard Set (ESS), including the highly polymorphic locus SE33, for compatibility with existing forensic DNA databases. Primer engineering involved bioinformatics tools to create a multiplex-compatible primer set. Primer quality was evaluated in silico and in vitro. We demonstrate the systematic optimization of multiplex PCR thermocycling conditions using Design of Experiments (DOE) methodology. The objective was to yield a specific, balanced, low-noise amplification of forensic targets. A central composite face design of experiments enabled an efficient simultaneous investigation of multiple critical process parameters and their interactions. Optimal multiplex PCR conditions were predicted using software-aided modelling based on DOE data. Verification experiments suggested a balanced, reproducible amplification of all markers with reduced formation of artefacts. Fully concordant STR profiles were obtained for the investigated reference samples even with challenging input DNA concentrations. We found that application of DOE principles enabled an experimentally practical and economically justifiable assay development and optimization, even beyond the field of forensic genetics.     

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.     

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.