There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

Department of Forensic and Analytical Science Faculty of Life Science King's College London UK

Department of Forensic Biology Norwegian Institute of Public Health Oslo Norway

Department of Forensic Genetics Institute of Criminalistics Prague Czech Republic

Department of Forensic Molecular Biology Erasmus MC University Medical Centre Rotterdam Rotterdam The Netherlands

Department of Human Biological Traces Netherlands Forensic Institute The Hague The Netherlands

ESR Private Bag 92021 Auckland New Zealand

Federal Criminal Police Office Wiesbaden Germany

Forensic Genetic and Biology Service Centre Branch National Institute of Legal Medicine and Forensic Sciences Coimbra Portugal

Forensic Genetics Laboratory Institute of Legal Medicine Università Cattolica del Sacro Cuore Rome Italy

Forensic Genetics Unit Institute of Forensic Sciences University of Santiago de Compostela Santiago de Compostela Spain

Forensic Science Laboratory Dublin Ireland

Institute of Forensic Science Ministry of the Interior Department of Biology and DNA Analysis Slovenská Lupca Slovakia

Institute of Legal Medicine Faculty of Medicine University of Cologne Cologne Germany

Institute of Legal Medicine Medical University of Innsbruck Innsbruck Austria

Institute of Legal Medicine Medical University of Innsbruck Innsbruck Austria; Forensic Science Program The Pennsylvania State University University Park PA USA

Material Measurement Laboratory National Institute of Standards and Technology Gaithersburg MD USA

National Institute of Criminalistics and Criminology Chaussée de Vilvoorde 100 Brussels Belgium

Office of the Chief Forensic Scientist Forensic Services Department Victoria Police Australia

School of Biological Sciences Flinders University Adelaide South Australia 5042 Australia

Section of Forensic Genetics Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen Frederik V's Vej 11 Copenhagen Denmark

Section of Forensic Genetics Department of Forensic Medicine Faculty of Health and Medical Sciences University of Copenhagen Frederik V's Vej 11 Copenhagen Denmark; National Institute of Criminalistics and Criminology Chaussée de Vilvoorde 100 Brussels Belgium

Section of Forensic Genetics Institute of Forensic Research Kraków Poland

Zurich Institute of Forensic Medicine University of Zurich Zurich Switzerland

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