Development and validation of the VISAGE AmpliSeq basic tool to predict appearance and ancestry from DNA

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 laboratoriesThe 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 3UM1HG008901-03S1S