Were incubated at 65uC for 12 h under agitation at 300 rpm. The supernatant was transferred to a new 15-ml tube, and isolation of DNA was Calyculin A performed using 
the Wizard genomic purification kit (Promega Corporation) according to the manufacturer’s protocol with minor modifications. The supernatant was mixed with 2 ml nuclei lysis solution followed by incubation for 3 h at room temperature.Sequencing of mtDNAThe PCR products were purified using two methods, the QIAquickH PCR purification kit (Qiagen, Hilden, Germany) and with Exonuclease I (ExoI) (Thermo scientific, Waltham, MA, USA) and FastAP thermosensitive Alkaline Phosphatase (Thermo scientific) in a mixture. Sanger dideoxy sequencing was performed using the ABI PRISMHBig DyeTM terminator Cycle Sequencing Ready Reaction kit v3.3 (Applied Biosystems). Sequencing reactions were run on an ABI Prism 3730 instrument (Applied Biosystems) and the sequencing data was analysed using the Sequencher 4.5 software package (Gene Codes Corporation, Ann Arbor, MI, USA). The obtained mtDNA sequences were?Identification of Carin GoringTable 1. Primer sequences and cycling conditions used for amplification.Name IFb-16128 MedChemExpress POR-8 IR-16348 IIFa-45 IIR-287 15971 16410 15971 R17 Amelogenin F Amelogenin R doi:10.1371/journal.pone.0044366.t59 Primer sequence GGTACCATAAATACTTGACCACCT GACTGTAATGTGCTATGTACGGTAAA ATGCATTTGGTATTTTCGTCTG TTGTTATGATGTCTGTGTGGAAAG TTAACTCCACCATTAGCACC GAGGATGGTGGTCAAGGGAC TTAACTCCACCATTAGCACC CCC GTG AGT GGT TAA TAG GGT CCCTGGGCTCTGTAAAGAATAGT ACTAGAGCTTAAACTGGGAAGCTGDNA region HVIFragment size 221 bpHVII243 bpHVI440 bpHVI616 bpChr X and Y106 bp (XX) 112 bp (XY)compared to a reference sequence, the revised Cambridge reference sequence (rCRS), and deviations were reported 1317923 as sequence differences to rCRS with Genbank accession number NC_012920 [16,17]. The mtDNA database EMPOP (www. empop.org) was used to estimate the frequency of a particular mtDNA sequence. When comparing two mtDNA sequences, at least two differences between them are required for a conclusive exclusion [15].Sex determinationA DNA-based sex determination of the skeletal 
remains was performed, based on analysis of the amelogenin gene (AMEL). The gene is located both on the X chromosome (AMELX) and the male-specific Y chromosome (AMELY) and a common target for sex determination in forensic DNA analyses is a six bp deletion on the X chromosome [18]. The amelogenin region was amplified using 0.2 mM of each primer (Table 1), 10 ml DNA extract, 16PCR Gold Buffer (Applied Biosystems), 0.2 mM dNTPs, 1.5 mM MgCl2 (Applied Biosystems), 10 Glycerol, 0.16 mg/ ml BSA, and 5 U AmpliTaqGoldTM (Applied Biosystems) in a total reaction volume of 30 ml. The cycling conditions were 10 minutes at 95uC, followed by 45 cycles of 30 seconds at 95uC, 45 seconds at 55uC, 60 seconds at 72uC, and a final extension step of 7 minutes at 72uC. The PCR products were sequenced using the Pyrosequencing technology, which is based on sequencing by synthesis, where incorporation of nucleotides results in generation of light [19]. Purification of templates and generation of single stranded products were performed according to the SQA template preparation protocol using the PSQ 96 Sample Preparation Kit and Streptavidin SepharoseTM High Performance beads (Quiagen, Hilden, Germany). The PSQTM96 SQA reagent kit was used for sequencing, and the reactions were run on a PSQ 96MA instrument using the SQA analysis in the PSQ 96MA (version 2.1) software. The generated lig.Were incubated at 65uC for 12 h under agitation at 300 rpm. The supernatant was transferred to a new 15-ml tube, and isolation of DNA was performed using the Wizard genomic purification kit (Promega Corporation) according to the manufacturer’s protocol with minor modifications. The supernatant was mixed with 2 ml nuclei lysis solution followed by incubation for 3 h at room temperature.Sequencing of mtDNAThe PCR products were purified using two methods, the QIAquickH PCR purification kit (Qiagen, Hilden, Germany) and with Exonuclease I (ExoI) (Thermo scientific, Waltham, MA, USA) and FastAP thermosensitive Alkaline Phosphatase (Thermo scientific) in a mixture. Sanger dideoxy sequencing was performed using the ABI PRISMHBig DyeTM terminator Cycle Sequencing Ready Reaction kit v3.3 (Applied Biosystems). Sequencing reactions were run on an ABI Prism 3730 instrument (Applied Biosystems) and the sequencing data was analysed using the Sequencher 4.5 software package (Gene Codes Corporation, Ann Arbor, MI, USA). The obtained mtDNA sequences were?Identification of Carin GoringTable 1. Primer sequences and cycling conditions used for amplification.Name IFb-16128 IR-16348 IIFa-45 IIR-287 15971 16410 15971 R17 Amelogenin F Amelogenin R doi:10.1371/journal.pone.0044366.t59 Primer sequence GGTACCATAAATACTTGACCACCT GACTGTAATGTGCTATGTACGGTAAA ATGCATTTGGTATTTTCGTCTG TTGTTATGATGTCTGTGTGGAAAG TTAACTCCACCATTAGCACC GAGGATGGTGGTCAAGGGAC TTAACTCCACCATTAGCACC CCC GTG AGT GGT TAA TAG GGT CCCTGGGCTCTGTAAAGAATAGT ACTAGAGCTTAAACTGGGAAGCTGDNA region HVIFragment size 221 bpHVII243 bpHVI440 bpHVI616 bpChr X and Y106 bp (XX) 112 bp (XY)compared to a reference sequence, the revised Cambridge reference sequence (rCRS), and deviations were reported 1317923 as sequence differences to rCRS with Genbank accession number NC_012920 [16,17]. The mtDNA database EMPOP (www. empop.org) was used to estimate the frequency of a particular mtDNA sequence. When comparing two mtDNA sequences, at least two differences between them are required for a conclusive exclusion [15].Sex determinationA DNA-based sex determination of the skeletal remains was performed, based on analysis of the amelogenin gene (AMEL). The gene is located both on the X chromosome (AMELX) and the male-specific Y chromosome (AMELY) and a common target for sex determination in forensic DNA analyses is a six bp deletion on the X chromosome [18]. The amelogenin region was amplified using 0.2 mM of each primer (Table 1), 10 ml DNA extract, 16PCR Gold Buffer (Applied Biosystems), 0.2 mM dNTPs, 1.5 mM MgCl2 (Applied Biosystems), 10 Glycerol, 0.16 mg/ ml BSA, and 5 U AmpliTaqGoldTM (Applied Biosystems) in a total reaction volume of 30 ml. The cycling conditions were 10 minutes at 95uC, followed by 45 cycles of 30 seconds at 95uC, 45 seconds at 55uC, 60 seconds at 72uC, and a final extension step of 7 minutes at 72uC. The PCR products were sequenced using the Pyrosequencing technology, which is based on sequencing by synthesis, where incorporation of nucleotides results in generation of light [19]. Purification of templates and generation of single stranded products were performed according to the SQA template preparation protocol using the PSQ 96 Sample Preparation Kit and Streptavidin SepharoseTM High Performance beads (Quiagen, Hilden, Germany). The PSQTM96 SQA reagent kit was used for sequencing, and the reactions were run on a PSQ 96MA instrument using the SQA analysis in the PSQ 96MA (version 2.1) software. The generated lig.