Evaluation of the 124-plex SNP typing microarray for forensic testing Kaarel Krjuts ˇkov a,b,c, *, Triin Viltrop a , Priit Palta b,d , Ene Metspalu g , Erika Tamm g , Siim Suvi c , Katrin Sak c , Alo Merilo k , Helena Sork e , Rita Teek f,g , Tiit Nikopensius a , Toomas Kivisild h,i , Andres Metspalu a,b,j a Department of Biotechnology, IMCB, University of Tartu, 23 Riia St, 51010 Tartu, Estonia b Estonian Biocentre, 23 Riia St, 51010 Tartu, Estonia c Asper Biotech, 17A Vaksali St, 50410 Tartu, Estonia d Department of Bioinformatics, IMCB, University of Tartu, 23 Riia St, 51010 Tartu, Estonia e Institute of Technology, University of Tartu, 1 Nooruse St, 50411, Tartu, Estonia f Department of Oto-Rhino-Laryngology, University of Tartu, 1 Kuperjanovi St, 51003 Tartu, Estonia g Department of Genetics, United Laboratories, Tartu University Hospital, 3 Oru St, 51005 Tartu, Estonia h Leverhulme Centre for Human Evolutionary Studies, University of Cambridge, CB2 1QH Cambridge, UK i Department of Evolutionary Biology, IMCB, University of Tartu, 23 Riia St, 51010 Tartu, Estonia j Estonian Genome Project of University of Tartu, 61b Tiigi St, 50410 Tartu, Estonia k Harcourt Management Consulting, 7 Sonajala St, 61709 Tartu, Estonia 1. Introduction Compared to conventional STR-based methods, single nucleo- tide polymorphisms (SNPs) show increasing potential for forensic case-work studies because a number of features make them attractive: (i) there are several millions of validated SNPs in the human genome; (ii) SNPs can be typed by automated, cost- effective and standardized methods; and (iii) PCR amplicons shorter than 100 bp allow for amplification and unambiguous allele calling even from highly degraded DNA samples [1]. (iv) The mutation rate of SNPs is lower ((2 À 4) Â 10 À8 /site/genera- tion [2]) than those of STRs or VNTRs (variable number tandem repeats, e.g. 0.23%/STR/generation [3]); and (v) as biallelic polymorphisms, SNPs are comparatively easy to validate [4]. Besides many advantages over STR-based systems, significant disadvantages using SNPs, should also be keep in mind. (i) Mixture analysis is still an obstacle. A major advantage with STRs in a forensic setting is that many possible alleles exist, providing the possibility that the multiple contributors to a mixture will have distinguishable (non-overlapping) alleles [5]. (ii) Another reason why SNP technology would not dominate forensic DNA analysis in the near future is that national DNA databases store STR data and SNP profiles cannot be searched against an STR Forensic Science International: Genetics xxx (2009) xxx–xxx ARTICLE INFO Article history: Received 22 December 2008 Received in revised form 16 April 2009 Accepted 17 April 2009 Available online xxx Keywords: Multiplex PCR Forensic test APEX-2 genotyping ABSTRACT Human identification systems such as criminal databases, forensic DNA testing and genetic genealogy require reliable and cost-effective genotyping of autosomal, mitochondrial and Y chromosome markers from different biological materials, including venous blood and saliva. Although many such assays are available, few systems are capable of simultaneously detecting all three targets in a single reaction. Employing the APEX-2 principle, we have characterized a novel 124-plex assay, using specific primer extension, universal primer amplification and single base extension on an oligonucleotide array. The assay has been designed for simultaneous genotyping of SNPs from the single copy loci (46 autosomal and 29 Y chromosomal markers) side by side with SNPs from the mitochondrial genome (49 markers) that appears in up to thousands of copies per cell in certain tissue types. All the autosomal SNPs (from the SNPforID Consortium) included in the multiplex assay are unlinked and are distributed widely across autosomes, enabling genetic fingerprints to be distinguished. Mitochondrial DNA and Y chromosome polymorphisms that define haplogroups common in European populations are included to allow for maternity and paternity testing and for the analysis of genetic genealogies. After assay optimization we estimated the accuracy (99.83%) and call rate (99.66%) of the protocol on 17 mother–father–child/ children families and five internal control DNAs. In addition, 79 unrelated Estonian and Swedish DNA samples were genotyped and the accuracy of mtDNA and Y chromosome haplogroup inference by the multiplex method was assessed using conventional genotyping methods and direct sequencing. ß 2009 Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Department of Biotechnology, IMCB, University of Tartu, 23 Riia St., 51010 Tartu, Estonia. Tel.: +372 7 375 029; fax: +372 7 420 286. E-mail address: karlakrj@ut.ee (K. Krjuts ˇkov). G Model FSIGEN-464; No of Pages 6 Please cite this article in press as: K. Krjuts ˇkov, et al., Evaluation of the 124-plex SNP typing microarray for forensic testing, Forensic Sci. Int. Genet. (2009), doi:10.1016/j.fsigen.2009.04.007 Contents lists available at ScienceDirect Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig 1872-4973/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2009.04.007