Computational protocol: Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia

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Protocol publication

[…] The two hypervariable regions HVI and HVII of the mtDNA were amplified by PCR in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The primers F15997/R16401 for HVI and F29/R408 for HVII [] were used. PCR was carried out in a 25-µl reaction mixture following Zupanič Pajnič et al. [], but for the bone samples, 0.8 µl of AmpliTaq Gold DNA Polymerase (Applied Biosystems) was used, BSA (Sigma; final concentration 50 ng/µl) was added, and the number of cycles was increased to 38. Prior to sequencing, the PCR products were purified using Centricon 100 spin dialysis columns (Millipore Corporation) following the manufacturer's recommendations. Sequencing reactions were performed in a Biometra UNO Thermoblock in both orientations in order to verify the accuracy of base-calling. In cases of length heteroplasmy in the poly-C strand, the polymorphisms behind the C-stretch in the forward sequencing reaction and before the C-stretch in the reverse reaction were confirmed by repeating amplification and sequencing reactions according to the recommendations []. Following Bandelt and Parson [] in cases of heteroplasmic length variants, the dominant variants were reported. The primers used for sequencing the PCR products were the same as for the amplification. Sequencing reactions were carried out using 6 µl ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit, v 1.1 (Applied Biosystems), 8 µl purified PCR product as a template, 2 µl 5 µM sequencing primer, and 4 µl sterile distilled water for each sample. The sequencing conditions and products purification have been described by Zupanič Pajnič et al. []. Then, 20 µl of Hi-Di™ formamide (Applied Biosystems) was added to 15 to 20 µl of purified sequencing product, heat denatured, and snap cooled on ice. Automated DNA sequencing was carried out on an ABI PRISM™ 3130 Genetic Analyser (Applied Biosystems) using the 3130 Performance Optimized Polymer POP 4 (Applied Biosystems) and Data Collection v 3.0 Software (Applied Biosystems). The denatured samples were electrokinetically injected for 10 s at 1.2 kV into a 36-cm capillary array. Electrophoresis was run at 15 kV and 60°C with the UltraSeq36 POP4 sequencing module. The analysis of mtDNA sequencing data was performed using AB DNA Sequencing Analysis Software v 5.2 (Applied Biosystems). The sequences were aligned and compared with the Anderson sequence [] from 16030 to 16381 for the HVI region and from 55 to 388 for the HVII region using BioEdit software. […]

Pipeline specifications

Software tools Sequencing Analysis Software, BioEdit
Application Sanger sequencing
Organisms Homo sapiens