Developmental validation of mitochondrial DNA genotyping assays for adept matrilineal inference of biogeographic ancestry at a continental level Lakshmi Chaitanya a , Mannis van Oven a , Natalie Weiler b , Joyce Harteveld b , Laura Wirken c , Titia Sijen b , Peter de Knijff c , Manfred Kayser a, * a Department of Forensic Molecular Biology, Erasmus MC University Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, Zuid-Holland, The Netherlands b Department of Human Biological Traces, Netherlands Forensic Institute, P.O. Box 24044, 2490 AA The Hague, The Netherlands c Forensic Laboratory for DNA Research, Department of Human Genetics, Leiden, University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands 1. Introduction The successful forensic application of DNA analysis methods as tools to exonerate innocents and to convict criminals has been expanding progressively, inculcating the recent technological advancements into practice to decipher the conundrum of human identity. Currently, autosomal short tandem repeats (STRs) are extensively used for human identification and thus aid in solving crimes with the available biological evidence. However, there is a necessity to employ additional DNA markers when conventional STR typing practically fails or does not yield a match to a known person including those whose profile is present in a criminal DNA database. The attractive features of mitochondrial DNA (mtDNA), i.e. its molecular stability, small size (16,570 bp), high cellular copy number, maternal inheritance and haploidity, have made it an indispensable tool in forensic practice. Initially applied as identification tool for maternal lineages by use of DNA sequence information from the first hypervariable segment (HVS-I) (some- times in combination with HVS-II) [1], particular mtDNA single nucleotide polymorphisms (mt-SNPs) are starting to be used for inference of matrilineal bio-geographic origin. Substantiating the matrilineal biogeographic origin of an unknown individual provides an impetus for investigations where an STR profile is unattainable from limited or degraded samples or in the absence of Forensic Science International: Genetics 11 (2014) 39–51 A R T I C L E I N F O Article history: Received 10 September 2013 Received in revised form 12 February 2014 Accepted 15 February 2014 Keywords: Biogeographic origin Genetic ancestry Matrilineal ancestry mtDNA Developmental validation Haplogroups A B S T R A C T Mitochondrial DNA (mtDNA) can be used for matrilineal biogeographic ancestry prediction and can thus provide investigative leads towards identifying unknown suspects, when conventional autosomal short tandem repeat (STR) profiling fails to provide a match. Recently, six multiplex genotyping assays targeting 62 ancestry-informative mitochondrial single nucleotide polymorphisms (mt-SNPs) were developed. This hierarchical system of assays allows detection of the major haplogroups present in Africa, America, Western Eurasia, Eastern Eurasia, Australia and Oceania, thus revealing the broad geographic region of matrilineal origin of a DNA donor. Here, we provide a forensic developmental validation study of five multiplex assays targeting all the 62 ancestry-informative mt-SNPs following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. We demonstrate that the assays are highly sensitive; being able to produce full profiles at input DNA amounts of as little as 1 pg. The assays were shown to be highly robust and efficient in providing information from degraded samples and from simulated casework samples of different substrates such as blood, semen, hair, saliva and trace DNA samples. Reproducible results were successfully achieved from concordance testing across three independent laboratories depicting the ease and reliability of these assays. Overall, our results demonstrate the suitability of these five mt-SNP assays for application to forensic casework and other purposes aiming to establish an individual’s matrilineal genetic ancestry. With this validated tool, it is now possible to determine the matrilineal biogeographic origin of unknown individuals on the level of continental resolution from forensic DNA samples to provide investigative leads in criminal and missing person cases where autosomal STR profiling is uninformative. ß 2014 Elsevier Ireland Ltd. All rights reserved. * Corresponding author. Tel.: +31 10 7038073; fax: +31 10 7044575. E-mail address: m.kayser@erasmusmc.nl (M. Kayser). Contents lists available at ScienceDirect Forensic Science International: Genetics jou r nal h o mep ag e: w ww .elsevier .co m /loc ate/fs ig http://dx.doi.org/10.1016/j.fsigen.2014.02.010 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.