Search

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 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). CP, AFA, AMM, MdlP, MVL are supported by MAPA, Multiple Allele Polymorphism Analysis (BIO2016-78525-R), a research project funded by the Spanish Research State Agency (AEI), and co-financed with ERDF funds. 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 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

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.

This study investigated the mitochondrial DNA (mtDNA) control region variation in Middle Eastern populations (610 individuals from Lebanon, Jordan and the Kingdom of Bahrain) for which population data are scarce. FST comparison among populations revealed that there are significant differences in mtDNA distributions between Bahrain and the two other populations, while Lebanon and Jordan showed no significant differences. This was also reflected by the distribution of the observed lineages that differed prominently between Bahrain and the other two investigated populations. Jordan and Lebanon fit the hitherto known genetic results of the Levant population. Data are available via EMPOP (https://empop.online) and GenBank.

Measuring the quantity of DNA present in a forensic sample is relevant in a number of ways. First, it informs the analyst about the general DNA content to adjust the volume of DNA extract used for the genotyping assay to the optimal conditions (when possible). Second, quantification values can serve as plausibility checks for the performance of the DNA extraction method used as extraction positive and negative controls demand expected values. Third and relevant to highly compromised specimens, DNA quantification can inform about the degradation state of the DNA extracted from the unknown biological sample and aid the choice of downstream genotyping assays. While there are different, commercial products for the quantification of nuclear DNA available, commercial mitochondrial DNA (mtDNA) quantification systems are rare. Even more so, the simultaneous quantification of nuclear and mtDNA that is of relevance in highly degraded forensic specimens has rarely been described. We present here a novel real-time qPCR based tetraplex system termed SD quants that targets two different-sized mtDNA and a nuclear DNA region and includes an internal positive control to monitor potential inhibition. SD quants was compared to other existing quantification systems and subjected to analysis of severely degraded DNA present in ancient DNA and aged forensic specimens. This study complies with the MIQE (Bustin et al., 2009) guidelines (when applicable).

In 1976 human remains of seven individuals were discovered in a storage pit located within the Late Bronze Age (9th century B.C.) settlement Stillfried an der March, Austria. In contrast to the common funeral rite of cremation typical for the Urnfield culture (1300-800 B.C.) the individuals' skeletal remains were found outstandingly preserved (Figure S1). As a result, the burial was subject to various investigations, including two conflicting genealogical pedigree reconstructions, one of which was favoured by later geological fingerprinting. We performed mitochondrial (mt)DNA testing in order to genetically characterize the remains and shed light into the matrilineal relationship of the seven individuals that were earlier anthropologically identified as three adults (two women and a man) and four subadults (one female and three males). MtDNA was analysed using Primer Extension Capture and Massively Parallel Sequencing. The results were by and large in conflict with both pedigree models but confirmed some of the details that were elaborated in previous studies. Whereas both pedigree models suggested that all children were related to one or both females, mtDNA analyses revealed that only one subadult male resulted in the same mitotype as one adult female. All other children yielded different mitotypes indicating that they were maternally unrelated to the two females and between each other.

Massively Parallel (Next Generation) Sequencing (MPS) technologies have recently been proven useful and successful in typing various markers relevant in forensic genetics, such as STRs, SNPs and mitochondrial genomes. Early studies investigated self-developed DNA libraries, commercially supplied kits are currently being made available to allow a smoother and gradual implementation of such technologies in forensic laboratories. The ForenSeq™ DNA Signature Prep Kit (Illumina, CA) is the first commercially available STR kit that can be used on the MiSeq FGx™ (Illumina, CA) benchtop high-throughput sequencer. This kit allows the simultaneous typing of 59 STRs and up to 172 SNPs in a single reaction and presents a short library preparation protocol adapted to contemporary forensic requirements. In this study, we evaluated the beta version of the ForenSeq DNA Signature Prep Kit MiSeq FGx system by investigating reproducibility, sensitivity, mixtures, concordance, casework-type and aDNA samples and found it to perform successfully, proving to be a robust method for future forensic applications. MPS brings the possibility of complex multiplexing, high sensitivity and sequencing resolution to forensics; however, the need for consensual directions on databasing, data storage and nomenclature must be taken into consideration.

Though shed hairs are one of the most commonly encountered evidence types, they are among the most limited in terms of DNA quantity and quality. As a result, DNA testing has historically focused on the recovery of just about 600 base pairs of the mitochondrial DNA control region. Here, we describe our success in recovering complete mitochondrial genome (mtGenome) data (∼16,569bp) from single shed hairs. By employing massively parallel sequencing (MPS), we demonstrate that particular hair samples yield DNA sufficient in quantity and quality to produce 2-3kb mtGenome amplicons and that entire mtGenome data can be recovered from hair extracts even without PCR enrichment. Most importantly, we describe a small amplicon multiplex assay comprised of sixty-two primer sets that can be routinely applied to the compromised hair samples typically encountered in forensic casework. In all samples tested here, the MPS data recovered using any one of the three methods were consistent with the control Sanger sequence data developed from high quality known specimens. Given the recently demonstrated value of complete mtGenome data in terms of discrimination power among randomly sampled individuals, the possibility of recovering mtGenome data from the most compromised and limited evidentiary material is likely to vastly increase the utility of mtDNA testing for hair evidence.