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SummaryEpidemiological models of notifiable livestock disease are typically framed at a national level and targeted for specific diseases. There are inherent difficulties in extending models beyond national borders as details of the livestock population, production systems and marketing systems of neighbouring countries are not always readily available. It can also be a challenge to capture heterogeneities in production systems, control policies, and response resourcing across multiple countries, in a single transboundary model.In this paper we describe EuFMDiS, a continental-scale modelling framework for transboundary animal disease, specifically designed to support emergency animal disease planning in Europe. EuFMDiS simulates the spread of livestock disease within and between countries and allows control policies to be enacted and resourced on per-country basis. It provides a sophisticated decision support tool that can be used to look at the risk of disease introduction, establishment and spread; control approaches in terms of effectiveness and costs; resource management; and post-outbreak management issues.

In the last 10 years, forensic genetic analysis has been extended beyond identification tests that link a suspect to crime scene evidence using standard DNA profiling, to new supplementary tests that can provide information to investigators about a suspect in the absence of a database hit or eyewitness testimony. These tests now encompass the prediction of physical appearance, ancestry and age. In this review, we give a comprehensive overview of the full range of DNA-based ancestry inference tests designed to work with forensic contact traces, when the level of DNA is often very low or highly degraded. We outline recent developments in the design of ancestry-informative marker sets, forensic assays that use capillary electrophoresis or massively parallel sequencing, and the statistical analysis frameworks that examine the test profile and compares it to reference population variation. Three casework ancestry analysis examples are described which were successfully accomplished in the authors’ laboratory, where the ancestry information obtained was critical to the outcome of the DNA analyses made.

DNA methylation is known as a biomarker for age with applications in forensics. Here we describe the VISAGE (VISible Attributes through GEnomics) Consortium’s enhanced tool for epigenetic age estimation in somatic tissues. The tool is based on eight DNA methylation markers (44 CpGs), bisulfite multiplex PCR followed by sequencing on the MiSeq FGx platform, and three statistical prediction models for blood, buccal cells and bones. The model for blood is based on six CpGs from ELOVL2, MIR29B2CHG, KLF14, FHL2, TRIM59 and PDE4C, and predicts age with a mean absolute error (MAE) of 3.2 years, while the model for buccal cells includes five CpGs from PDE4C, MIR29B2CHG, ELOVL2, KLF14 and EDARADD and predicts age with MAE of 3.7 years, and the model for bones has six CpGs from ELOVL2, KLF14, PDE4C and ASPA and predicts age with MAE of 3.4 years. The VISAGE enhanced tool for age estimation in somatic tissues enables reliable collection of DNA methylation data from small amounts of DNA using a sensitive multiplex MPS assay that provides accurate estimation of age in blood, buccal swabs, and bones using the statistical model tailored to each tissue.

Recently, extensive catalogs of human variation derived from whole-genome sequencing have been released as openly accessible archives of sequence variants—a resource that is highly suitable for selecting markers for new forensic tests using massively parallel sequencing. In particular, the comparison of population patterns in these databases of variants can help identify markers suitable for the inference of ancestry that can then form informative 242forensic assays for this purpose. This chapter outlines in detail the ancestry or co-ancestry composition, genotype data access details, and geographic distributions of the samples in the most extensive variant catalogs of: 1000 Genomes; the HGDP–CEPH panel; Simons Foundation Human Genome Diversity Project; and Estonian Biocentre Genome Diversity Panel. While 1000 Genomes systematically characterizes a large number of samples from a limted number of carefully selected populations and restricts itself to five to six populations per continent, both SGDP and EGDP have analyzed only 2–4 samples per geographic location and these are much more widely dispersed geographically. The pros and cons of each approach are discussed, and details are provided of more recently published variant compilation projects with much larger sample sizes, notably the genome aggregation database gnomAD.

Forensic DNA phenotyping is gaining interest as the number of applications increases within the forensic genetics community. The possibility of providing investigative leads in addition to conventional DNA profiling for human identification provides new insights into otherwise “cold” police investigations. The ability of reporting on the bio-geographical ancestry (BGA), appearance characteristics and age based on DNA obtained from a crime scene sample of an unknown donor makes the exploration of such markers and the development of new methods meaningful for criminal investigations. The VISible Attributes through GEnomics (VISAGE) Consortium aims to disseminate and broaden the use of predictive markers and develop fully optimized and validated prototypes for forensic casework implementation. Here, the first VISAGE appearance and ancestry tool development, performance and validation is reported. A total of 153 SNPs (96.84 % assay conversion rate) were successfully incorporated into a single multiplex reaction using the AmpliSeq™ design pipeline, and applied for massively parallel sequencing with the Ion S5 platform. A collaborative effort involving six VISAGE laboratory partners was devised to perform all validation tests. An extensive validation plan was carefully organized to explore the assay’s overall performance with optimum and low-input samples, as well as with challenging and casework mock samples. In addition, forensic validation studies such as concordance and mixture tests recurring to the Coriell sample set with known genotypes were performed. Finally, inhibitor tolerance and specificity were also evaluated. Results showed a robust, highly sensitive assay with good overall concordance between laboratories The study received support from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 740580 within the framework of the VISible Attributes through GEnomics (VISAGE) Project and Consortium. MdlP is supported by a postdoctoral fellowship awarded by the Consellería de Cultura, Educación e Ordenación Universitaria and the Consellería de Economía, Emprego e Industria from Xunta de Galicia (Modalidade A, ED481B 2017/088). AFA is supported by a post-doctorate grant funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (Modalidade B, ED481B 2018/010). The 1000 Genomes high coverage sequence data were generated at the New York Genome Center with funds provided by NHGRI Grant 3UM1HG008901-03S1 SI

Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place.

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a &ldquo, skull and crossbones&rdquo, orientation. The lid of the 19th century coffin had brass tacks that spelled &ldquo, JB55&rdquo, the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be &ldquo, killed&rdquo, by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname &ldquo, Barber&rdquo, A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of &ldquo, NB13,&rdquo, found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.

The 944 individuals of the CEPH human genome diversity panel (HGDP-CEPH), a standard sample set of 51 globally distributed populations, were sequenced using the Illumina ForenSeq™ DNA Signature Prep Kit. The ForenSeq™ system is a single multiplex for the MiSeq/FGx™ massively parallel sequencing instrument, comprising: amelogenin, 27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 SNPforID+Kiddlab autosomal ID-SNPs (plus optionally detected ancestry and phenotyping SNP sets). We report in detail the patterns of sequence variation observed in the repeat regions of the 58 forensic STR loci typed by the ForenSeq™ system. Sequence alleles were characterized and repeat region structures annotated by aligning the ForenSeq™ sequence output to the latest GRCh38 human reference sequence, necessitating the reversal and re-alignment of STR allele sequences reported by the Forenseq™ system in 20 of 58 STRs (plus the reverse alleles in two Y-STRs with duplicated-inverted repeat regions). Individual population sample sizes of the HGDP-CEPH panel do not allow reliable inferences to be made about levels of genetic variability in low frequency STR alleles-where particular sequence variants are found in only a few individuals; but we assessed the occurrence of both population-specific sequence variants and singleton observations; finding each of these in a sizeable proportion of HGDP-CEPH samples, with consequences for planning the co-ordinated compilation of sequence variation on a much larger scale than was required before by forensic laboratories now adopting massively parallel sequencing.

The possibility of providing investigative leads when conventional DNA identification methods fail to solve a case can be of extreme relevance to law enforcement. Therefore, the forensic genetics community has focused research towards the broadened use of DNA, particularly for prediction of appearance traits, bio-geographical ancestry and age. The VISible Attributes through GEnomics (VISAGE) Consortium expanded the use of DNA phenotyping by developing new molecular and statistical tools for appearance, age and ancestry prediction. The VISAGE basic tool for appearance (EVC) and ancestry (BGA) prediction was initially developed using Ampliseq chemistry, but here is being evaluated using ForenSeq chemistry. The VISAGE basic tool offers a total of 41 EVC and 115 BGA SNPs and thus provides more predictions, i.e., skin color, than achieved with the ForenSeq DNA Signature Prep kit that is based on 24 EVC and 56 BGA SNPs. Five VISAGE laboratories participated in collaborative experiments to provide foreground for developmental validation of the assay. Assessment of assay performance and quality metrics, reproducibility, sensitivity, inhibitor tolerance and species specificity are described. Furthermore, the assay was tested using challenging samples such as mock casework samples and artificially degraded DNA. Two different analysis strategies were applied for this study and output on genotype calls and read depth was compared. Overall, inter-laboratory, inter-method and concordance with publicly available data were analysed and compared. Finally, the results showed a reliable and robust tool, which can be easily applied for laboratories already using a MiSeq FGx with ForenSeq reagents.