Development of an epigenetic age predictor for costal cartilage with a simultaneous somatic tissue differentiation system.

Age prediction from DNA has been a topic of interest in recent years due to the promising results obtained when using epigenetic markers. Since DNA methylation gradually changes across the individual's lifetime, prediction models have been developed accordingly for age estimation. The tissue-dependence for this biomarker usually necessitates the development of tissue-specific age prediction models, in this way, multiple models for age inference have been constructed for the most commonly encountered forensic tissues (blood, oral mucosa, semen). The analysis of skeletal remains has also been attempted and prediction models for bone have now been reported. Recently, the VISAGE Enhanced Tool was developed for the simultaneous DNA methylation analysis of 8 age-correlated loci using targeted high-throughput sequencing. It has been shown that this method is compatible with epigenetic age estimation models for blood, buccal cells, and bone. Since when dealing with decomposed cadavers or postmortem samples, cartilage samples are also an important biological source, an age prediction model for cartilage has been generated in the present study based on methylation data collected using the VISAGE Enhanced Tool. In this way, we have developed a forensic cartilage age prediction model using a training set composed of 109 samples (19-74 age range) based on DNA methylation levels from three CpGs in FHL2, TRIM59 and KLF14, using multivariate quantile regression which provides a mean absolute error (MAE) of ± 4.41 years. An independent testing set composed of 72 samples (19-75 age range) was also analyzed and provided an MAE of ± 4.26 years. In addition, we demonstrate that the 8 VISAGE markers, comprising EDARADD, TRIM59, ELOVL2, MIR29B2CHG, PDE4C, ASPA, FHL2 and KLF14, can be used as tissue prediction markers which provide reliable blood, buccal cells, bone, and cartilage differentiation using a developed multinomial logistic regression model. A training set composed of 392 samples (n = 87 blood, n = 86 buccal cells, n = 110 bone and n = 109 cartilage) was used for building the model (correct classifications: 98.72%, sensitivity: 0.988, specificity: 0.996) and validation was performed using a testing set composed of 192 samples (n = 38 blood, n = 36 buccal cells, n = 46 bone and n = 72 cartilage) showing similar predictive success to the training set (correct classifications: 97.4%, sensitivity: 0.968, specificity: 0.991). By developing both a new cartilage age model and a tissue differentiation model, our study significantly expands the use of the VISAGE Enhanced Tool while increasing the amount of DNA methylation-based information obtained from a single sample and a single forensic laboratory analysis. Both models have been placed in the open-access Snipper forensic classification website.

Development and evaluations of the ancestry informative markers of the VISAGE Enhanced Tool for Appearance and Ancestry.

The VISAGE Enhanced Tool for Appearance and Ancestry (ET) has been designed to combine markers for the prediction of bio-geographical ancestry plus a range of externally visible characteristics into a single massively parallel sequencing (MPS) assay. We describe the development of the ancestry panel markers used in ET, and the enhanced analyses they provide compared to previous MPS-based forensic ancestry assays. As well as established autosomal single nucleotide polymorphisms (SNPs) that differentiate sub-Saharan African, European, East Asian, South Asian, Native American, and Oceanian populations, ET includes autosomal SNPs able to efficiently differentiate populations from Middle East regions. The ability of the ET autosomal ancestry SNPs to distinguish Middle East populations from other continentally defined population groups is such that characteristic patterns for this region can be discerned in genetic cluster analysis using STRUCTURE. Joint cluster membership estimates showing individual co-ancestry that signals North African or East African origins were detected, or cluster patterns were seen that indicate origins from central and Eastern regions of the Middle East. In addition to an augmented panel of autosomal SNPs, ET includes panels of 85 Y-SNPs, 16 X-SNPs and 21 autosomal Microhaplotypes. The Y- and X-SNPs provide a distinct method for obtaining extra detail about co-ancestry patterns identified in males with admixed backgrounds. This study used the 1000 Genomes admixed African and admixed American sample sets to fully explore these enhancements to the analysis of individual co-ancestry. Samples from urban and rural Brazil with contrasting distributions of African, European, and Native American co-ancestry were also studied to gauge the efficiency of combining Y- and X-SNP data for this purpose. The small panel of Microhaplotypes incorporated in ET were selected because they showed the highest levels of haplotype diversity amongst the seven population groups we sought to differentiate. Microhaplotype data was not formally combined with single-site SNP genotypes to analyse ancestry. However, the haplotype sequence reads obtained with ET from these loci creates an effective system for de-convoluting two-contributor mixed DNA. We made simple mixture experiments to demonstrate that when the contributors have different ancestries and the mixture ratios are imbalanced (i.e., not 1:1 mixtures) the ET Microhaplotype panel is an informative system to infer ancestry when this differs between the contributors.

Elevated mRNA expression of CHAC1 splicing variants is associated with poor outcome for breast and ovarian cancer patients.

BACKGROUND: The role of CHAC1 (cation transport regulator-like protein 1), a recently identified component of the unfolded protein response (UPR) pathway, in gynaecological cancers has not yet been characterised. Now, this work illustrates CHAC1 mRNA expression and associated clinical outcome in breast and ovarian cancer. METHODS: The prognostic value of CHAC1 and its two transcript variants was investigated in 116 breast and 133 ovarian tissues using quantitative real-time reverse-transcriptase PCR. Subsequently, we conducted functional studies using short-interfering RNA-mediated knockdown and plasmid-mediated overexpression of CHAC1 in breast and ovarian cancer cells. RESULTS: Poorly differentiated tumours exhibited higher CHAC1 mRNA expression (breast cancer: P=0.004; ovarian cancer: P=0.024). Hormone receptor-negative breast tumours and advanced-staged ovarian cancers demonstrated elevated CHAC1 mRNA expression levels (P<0.001 and P=0.026, respectively). The multivariate survival analysis showed a prognostic value of both transcript variants in breast cancer (transcript variant 1: RR(death) 6.7 (2.4-18.9); P<0.001), RR(relapse) 6.7 (2.1-21.3); P=0.001); (transcript variant 2: RR(death) 4.9 (2.0-12.4); P<0.001), RR(relapse) 8.0 (2.4-26.8); P<0.001). Ovarian cancer patients aged younger than 62.6 years with high CHAC1 mRNA expression showed poorer relapse-free- and overall-survival (P=0.030 and P=0.012, respectively). In functional studies CHAC1 knockdown suppressed cell migration, whereas ectopic overexpression opposed these effects. CONCLUSION: High CHAC1 mRNA expression could be an independent indicator for elevated risk of cancer recurrence in breast and ovarian cancer.

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 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.

Building a custom large-scale panel of novel microhaplotypes for forensic identification using MiSeq and Ion S5 massively parallel sequencing systems.

A large number of new microhaplotype loci were identified in the human genome by applying a directed search with selection criteria emphasizing short haplotype length (<120 nucleotides) and maximum levels of polymorphism in the composite SNPs. From these searches, 107 autosomal microhaplotypes and 11 X chromosome microhaplotypes were selected, with well-spaced autosomal positions to ensure their independence in relationship tests. The 118 microhaplotypes were assembled into a single multiplex assay for the analysis of forensic DNA with massively parallel sequencing (MPS). A single AmpliSeq-adapted primer set was made for Illumina MiSeq and Thermo Fisher Ion S5 MPS platforms and the performance of the assay was comprehensively evaluated in both systems. Five microhaplotypes showed critical sequencing failures in both MPS platforms and were removed, while a further 13 required manual checks and the application of sequence quality thresholds in one or both systems to ensure the successful analysis of low-level DNA in these loci. The targeting of short microhaplotype spans during marker selection, with an average length of 51 nucleotides in the 118 loci, led to a high level of sensitivity for the panel when sequencing the very degraded DNA typically encountered in forensic casework and the identification of missing persons.

Development and optimization of the VISAGE basic prototype tool for forensic age estimation.

The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay's reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20 ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicates.

Forensic evaluation of the Asia Pacific ancestry-informative MAPlex assay.

DNA intelligence, and particularly the inference of biogeographical ancestry (BGA) is increasing in interest, and relevance within the forensic genetics community. The majority of current MPS-based forensic ancestry-informative assays focus on the differentiation of major global populations. The recently published MAPlex (Multiplex for the Asia Pacific) panel contains 144 SNPs and 20 microhaplotypes and aims to improve the differentiation of populations in the Asia Pacific region. This study reports the first forensic evaluation of the MAPlex panel using AmpliSeq technology and Ion S5 sequencing. This study reports on the overall performance of MAPlex including the assay's sequence coverage distribution and stability, baseline noise and description of problematic SNPs. Dilution series, artificially degraded and mixed DNA samples were also analysed to evaluate the sensitivity of the panel with challenging or compromised forensic samples. As the first panel to combine biallelic SNPs, multiple-allele SNPs and microhaplotypes, the MAPlex assay demonstrated an enhanced capacity for mixture detection, not easily performed with common binary SNPs. This performance evaluation indicates that MAPlex is a robust, stable and highly sensitive assay that is applicable to forensic casework for the prediction of BGA.

Body fluid identification and assignment to donors using a targeted mRNA massively parallel sequencing approach - results of a second EUROFORGEN / EDNAP collaborative exercise.

In a previous EUROFORGEN/EDNAP collaborative exercise, we tested two assays for targeted mRNA massively parallel sequencing for the identification of body fluids/tissues, optimized for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms, respectively. The task of the second EUROFORGEN/EDNAP collaborative exercise was to analyze dried body fluid stains with two different multiplexes, the former Illumina 33plex mRNA panel for body fluid/tissue identification and a 35plex cSNP panel for assignment of body fluids/tissues to donors that was introduced in a proof-of-concept study recently. The coding region SNPs (cSNPs) are located within the body fluid specific mRNA transcripts and represent a direct link between the body fluid and the donor. We predicted the origin of the stains using a partial least squares discriminant analysis (PLS-DA) model, where most of the single source samples were correctly predicted. The mixed body fluid stains showed poorer results, however, at least one component was predicted correctly in most stains. The cSNP data demonstrated that coding region SNPs can give valuable information on linking body fluids/tissues with donors in mixed body fluid stains. However, due to the unfavorable performance of some cSNPs, the interpretation remains challenging. As a consequence, additional markers are needed to increase the discrimination power in each body fluid/tissue category.

The V108M mutation decreases the structural stability of catechol O-methyltransferase.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

Dispersed polaron simulations of electron transfer in photosynthetic reaction centers.

A microscopic method for simulating quantum mechanical, nuclear tunneling effects in biological electron transfer reactions is presented and applied to several electron transfer steps in photosynthetic bacterial reaction centers. In this "dispersed polaron" method the fluctuations of the protein and the electron carriers are projected as effective normal modes onto an appropriate reaction coordinate and used to evaluate the quantum mechanical rate constant. The simulations, based on the crystallographic structure of the reaction center from Rhodopseudomonas viridis, focus on electron transfer from a bacteriopheophytin to a quinone and the subsequent back-reaction. The rates of both of these reactions are almost independent of temperature or even increase with decreasing temperature. The simulations reproduce this unusual temperature dependence in a qualitative way, without the use of adjustable parameters for the protein's Franck-Condon factors. The observed dependence of the back-reaction on the free energy of the reaction also is reproduced, including the special behavior in the "inverted region."

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.

Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: II. AFLP analysis reflects human-aided local adaptation of a global pest species.

Originally resident in southeastern Europe, the codling moth (Cydia pomonella L.) (Tortricidae) has achieved a nearly global distribution, being one of the most successful pest insect species known today. As shown in our accompanying study, mitochondrial genetic markers suggest a Pleistocenic splitting of Cydia pomonella into two refugial clades which came into secondary contact after de-glaciation. The actual distribution pattern shows, however, that Central European codling moths have experienced a geographic splitting into many strains and locally adapted populations, which is not reflected by their mitochondrial haplotype distribution. We therefore have applied, in addition to mitochondrial markers, an approach with a higher resolution potential at the population level, based on the analysis of amplification fragment length polymorphisms (AFLPs). As shown in the present study, AFLP markers elucidate the genetic structure of codling moth strains and populations from different Central European apple orchard sites. While individual genetic diversity within codling moth strains and populations was small, a high degree of genetic differentiation was observed between the analyzed strains and populations, even at a small geographic scale. One of the main factors contributing to local differentiation may be limited gene flow among adjacent codling moth populations. In addition, microclimatic, ecological, and geographic constraints also may favour the splitting of Cydia pomonella into many local populations. Lastly, codling moths in Central European fruit orchards may experience considerable selective pressure due to pest control activities. As a consequence of all these selective forces, today in Central Europe we see a patchy distribution of many locally adapted codling moth populations, each of them having its own genetic fingerprint. Because of the complete absence of any correlation between insecticide resistance and geographic or genetic distances among populations, AFLP markers do not have a prognostic value for predicting an outbreak of pesticide resistance in the field. By combining mitochondrial genetic data and AFLP analysis it was possible, however, to track the recent evolutionary history of Cydia pomonella on three different time scales: from population splitting in Pleistocene, to interbreeding of mitochondrial haplotypes in Holocene, to human-aided complete intermixing and splitting into many locally adapted populations in very recent times. The case of Cydia pomonella is reminiscent of examples of sympatric speciation and another example of a human-induced globally successful pest species.

Molecular phylogeny and population structure of the codling moth (Cydia pomonella) in Central Europe: I. Ancient clade splitting revealed by mitochondrial haplotype markers.

The codling moth (Cydia pomonella L., Tortricidae, Lepidoptera) is an important pest of pome fruit with global distribution. It has adapted successfully to different habitats by forming various ecotypes and populations, often termed strains, which differ among each other in several morphological, developmental, and physiological features. Many strains of Cydia pomonella have developed resistance against a broad range of chemically different pesticides. Obviously, pesticide-resistant strains must have a genetic basis inherent to the gene pool of codling moth populations, and this deserves our particular attention. The primary intention of the present study was to contribute novel information regarding the evolutionary phylogeny and phylogeography of codling moth populations in Central Europe. In addition, we aimed at testing the hypothesis that differential biological traits and response patterns towards pesticides in codling moth populations may be reflected at a mitochondrial DNA level. In particular, we wanted to test if pesticide resistance in codling moths is associated repeatedly and independently with more than one mitochondrial haplotype. To this end, we analyzed mitochondrial DNA and constructed phylogenetic trees based on three mitochondrial genes: cytochrome oxidase I (COI), the A+T-rich region of the control region (CR), and the nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5). The results indicate that Central European populations of Cydia pomonella are clearly divided in two ancient clades. As shown by means of a molecular clock approach, the splitting of the two clades can be dated to a time period between the lower and middle Pleistocene, about 1.29-0.20 million years ago. It is assumed that the cyclic changes of warm and cold periods during Pleistocene may have lead to the geographic separation of codling moth populations due to glaciation, giving rise to the formation of the two separate refugial clades, as already shown for many other European animal species. Due to their inclination towards developing novel detoxification gene variants, codling moth individuals from both clades independently and multifariously may have developed pesticide resistance, and this process may be ongoing. During their more recent evolutionary history, natural events such as the gradual disappearance of climate-specific geographic barriers, as well as human-aided dispersal in recent historic times, may have allowed codling moth haplotypes from the original clades to interbreed and completely merge again, creating a globally successful insect species with a gene pool capable of responding to novel selective challenges by rapid adaptation.

Patterns in genetic diversity of Trifolium pallescens populations do not reflect chronosequence on alpine glacier forelands.

How does genetic diversity within populations of plants develop during primary succession on alpine glacier forelands? Theory predicts that pioneer populations are characterized by low genetic diversity due to founder effects and that genetic diversity increases within populations as they mature and recurrent gene flow occurs. However, few genetic studies have so far been carried out on plants on glacier forelands. In this study, we analysed the development of genetic diversity with time for populations of Trifolium pallescens along successional series (chronosequences) on three parallel glacier forelands in the European Alps, using neutral amplified fragment length polymorphism. No general trend in the development of genetic diversity was observed with increasing population age: even pioneer populations harboured substantial genetic diversity. Assignment tests showed that the latter consist of a genetic sub-sample from several source areas, and not just from other populations on the glacier forelands. We also detected some long distances-that is, inter-valley gene flow events. However, gene flow was not spatially unrestricted, as shown by a weak isolation by distance pattern within glacier valleys. The actual patterns of genetic diversity along the chronosequences are a result of the combination of factors, such as gene flow and growth rate, influenced by site- and species-specific attributes.

"The devil's in the detail": Release of an expanded, enhanced and dynamically revised forensic STR Sequence Guide.

The STR sequence template file published in 2016 as part of the considerations from the DNA Commission of the International Society for Forensic Genetics on minimal STR sequence nomenclature requirements, has been comprehensively revised and audited using the latest GRCh38 genome assembly. The list of forensic STRs characterized was expanded by including supplementary autosomal, X- and Y-chromosome microsatellites in less common use for routine DNA profiling, but some likely to be adopted in future massively parallel sequencing (MPS) STR panels. We outline several aspects of sequence alignment and annotation that required care and attention to detail when comparing sequences to GRCh37 and GRCh38 assemblies, as well as the necessary matching of MPS-based allele descriptions to previously established repeat region structures described in initial sequencing studies of the less well known forensic STRs. The revised sequence guide is now available in a dynamically updated FTP format from the STRidER website with a date-stamped change log to allow users to explore their own MPS data with the most up-to-date forensic STR sequence information compiled in a simple guide.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing.

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.

More evidence for non-maternal inheritance of mitochondrial DNA?

BACKGROUND: A single case of paternal co-transmission of mitochondrial DNA (mtDNA) in humans has been reported so far. OBJECTIVE: To find potential instances of non-maternal inheritance of mtDNA. METHODS: Published medical case studies (of single patients) were searched for irregular mtDNA patterns by comparing the given haplotype information for different clones or tissues with the worldwide mtDNA database as known to date-a method that has proved robust and reliable for the detection of flawed mtDNA sequence data. RESULTS: More than 20 studies were found reporting clear cut instances with mtDNAs of different ancestries in single individuals. As examples, cases are reviewed from recent published reports which, at face value, may be taken as evidence for paternal inheritance of mtDNA or recombination. CONCLUSIONS: Multiple types (or recombinant types) of quite dissimilar mitochondrial DNA from different parts of the known mtDNA phylogeny are often reported in single individuals. From re-analyses and corrigenda of forensic mtDNA data, it is apparent that the phenomenon of mixed or mosaic mtDNA can be ascribed solely to contamination and sample mix up.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.