Towards Genetic Identification with Male-specific Mutations

The identification and application of new genetic variants for differentiation patrilineally related male relatives using Y chromosomal short tandem repeats (Y-STRs)

A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs

A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518, DYS526ab, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. This study reflects the proof of concept to analyse all currently known RM Y-STRs simultaneously and describes the optimization of the multiplex assay. The RM-Yplex assay generated complete RM Y-STR profiles down to 62.5 pg of male template DNA, and from male–female DNA mixtures at all ratios tested. We herewith introduce and make available for widespread use in forensic and anthropological studies, an effective and sensitive single multiplex assay for simultaneous genotyping of 13 RM Y-STRs

The HIrisPlex system for simultaneous prediction of hair and eye colour from DNA

AbstractRecently, the field of predicting phenotypes of externally visible characteristics (EVCs) from DNA genotypes with the final aim of concentrating police investigations to find persons completely unknown to investigating authorities, also referred to as Forensic DNA Phenotyping (FDP), has started to become established in forensic biology. We previously developed and forensically validated the IrisPlex system for accurate prediction of blue and brown eye colour from DNA, and recently showed that all major hair colour categories are predictable from carefully selected DNA markers. Here, we introduce the newly developed HIrisPlex system, which is capable of simultaneously predicting both hair and eye colour from DNA. HIrisPlex consists of a single multiplex assay targeting 24 eye and hair colour predictive DNA variants including all 6 IrisPlex SNPs, as well as two prediction models, a newly developed model for hair colour categories and shade, and the previously developed IrisPlex model for eye colour. The HIrisPlex assay was designed to cope with low amounts of template DNA, as well as degraded DNA, and preliminary sensitivity testing revealed full DNA profiles down to 63pg input DNA. The power of the HIrisPlex system to predict hair colour was assessed in 1551 individuals from three different parts of Europe showing different hair colour frequencies. Using a 20% subset of individuals, while 80% were used for model building, the individual-based prediction accuracies employing a prediction-guided approach were 69.5% for blond, 78.5% for brown, 80% for red and 87.5% for black hair colour on average. Results from HIrisPlex analysis on worldwide DNA samples imply that HIrisPlex hair colour prediction is reliable independent of bio-geographic ancestry (similar to previous IrisPlex findings for eye colour). We furthermore demonstrate that it is possible to infer with a prediction accuracy of >86% if a brown-eyed, black-haired individual is of non-European (excluding regions nearby Europe) versus European (including nearby regions) bio-geographic origin solely from the strength of HIrisPlex eye and hair colour probabilities, which can provide extra intelligence for future forensic applications. The HIrisPlex system introduced here, including a single multiplex test assay, an interactive tool and prediction guide, and recommendations for reporting final outcomes, represents the first tool for simultaneously establishing categorical eye and hair colour of a person from DNA. The practical forensic application of the HIrisPlex system is expected to benefit cases where other avenues of investigation, including STR profiling, provide no leads on who the unknown crime scene sample donor or the unknown missing person might be

Simultaneous Whole Mitochondrial Genome Sequencing with Short Overlapping Amplicons Suitable for Degraded DNA Using the Ion Torrent Personal Genome Machine

Whole mitochondrial (mt) genome analysis enables a considerable increase in analysis throughput, and improves the discriminatory power to the maximum possible phylo

An efficient multiplex genotyping approach for detecting the major worldwide human Y-chromosome haplogroups

The Y chromosome is paternally inherited and therefore serves as an evolutionary marker of patrilineal descent. Worldwide DNA variation within the non-recombining portion of the Y chromosome can be represented as a monophyletic phylogenetic tree in which the branches (haplogroups) are defined by at least one SNP. Previous human population genetics research has produced a wealth of knowledge about the worldwide distribution of Y-SNP haplogroups. Here, we apply previous and very recent knowledge on the Y-SNP phylogeny and Y-haplogroup distribution by introducing two multiplex genotyping assays that allow for the hierarchical detection of 28 Y-SNPs defining the major worldwide Y haplogroups. PCR amplicons were kept small to make the method sensitive and thereby applicable to DNA of limited amount and/or quality such as in forensic settings. These Y-SNP assays thus form a valuable tool for researchers in the fields of forensic genetics and genetic anthropology to infer a man's patrilineal bio-geographic ancestry from DNA

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM

Abstract DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine® (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1 ng of genomic DNA and mRNA analysis from 10 ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material

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 study was supported by 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. M.d.l.P. 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 (ED481D-2021–008). J.R. is supported by the “Programa de axudas á etapa predoutoral” funded by the Consellería de Cultura, Educación e Ordenación Universitaria e da Consellería de Economía, Emprego e Industria from Xunta de Galicia, Spain (ED481A-2020/039). C.P., A.F.A., A.M.M., M.d.l.P., M.V.L. and the work to compile ancestry informative tri-allelic SNPs and microhaplotypes 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. The population studies by S.O. at University of Santiago de Compostela, were financed by the Fundação de Apoio a Pesquisa do Distrito Federal (FAPDF), BrazilS

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers

Short tandem repeat polymorphisms on the male‐specific part of the human Y‐chromosome (Y‐STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (~10−3 mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y‐STR markers with untypically high mutation rates (>10−2 ), termed rapidly mutating (RM) Y‐STRs, and showed that they improved male relative differentiation over standard Y‐STRs. By applying a newly developed in silico search approach to the Y‐chromosome reference sequence, we identified 27 novel RM Y‐STR candidates. Genotyping them in 1,616 DNA‐confirmed father–son pairs for mutation rate estimation empirically highlighted 12 novel RM Y‐STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y‐STRs, it was 44%, 69%, and 83%. Of the 647 Y‐STR mutations o