The opening of the sarcophagi, compilation of the skeletons and sampling were carried out with the participation of archaeologists and anthropologists. The procedures were recorded in a written report with photodocumentation. The sealed metal caskets and, in the case of the royal couple, additional inner glass boxes, were opened by a restorer. The innermost wooden cases, which contained the skeletal remains, were placed in new, clean plastic boxes for transportation. The person carrying out this procedure wore a scrub suit, disposable gloves and a facial mask. The skeletons were compiled in a sterile operating room of the National Institute of Oncology. The operating table was prepared with a disposable surgical bed sheet for each skeleton. The sampling was carried out with sterile, surgical tools. The staff wore scrub suits, disposable gloves and facial masks. The bone samples were put in sterile, DNase- and RNase-free 50-ml centrifuge tubes. The participating persons were STR genotyped. Of note is that a number of people had come into direct contact with the skeletons since their discovery in the nineteenth century. During the excavations, transport and investigations, the bones had become contaminated. The remains, especially those of the royal couple, had been investigated several times (1848, 1883, 1967 and 1984) without precautions against DNA contamination. The details of the examined skeletons are summarised in Table 2. The bone samples used for DNA extraction are indicated in online resource 2. The skull and poorly preserved skeletal bones of the King and Queen were consolidated using polyvinyl acetal (Alvar, Shawinigan Chemicals) in 1967. This consolidant proved to be a PCR (polymerase chain reaction) inhibitor in the first DNA extracts of the Göttingen laboratory (see the “DNA extraction” section and online resource 3).
Y-chromosomal STR and autosomal STR analyses of the samples have been performed in parallel and independently in the Department of Pathogenetics of the National Institute of Oncology (Budapest, Hungary) and in the Department of Historical Anthropology and Human Ecology of the Johann-Friedrich-Blumenbach Institute for Zoology and Anthropology (Göttingen, Germany). Overlapping STR marker panels were used to confirm the results and the reliability of both laboratories.
Sample storage, DNA isolation and PCR setup occurred in a dedicated clean-room facility supplied with a HEPA-filter and overpressure. PCR products have never been present in this area. The laboratory personnel wore disposable hooded overalls, facemasks and boot covers. Prior to experimental procedures, the working surface was decontaminated with 10% bleach, washed with Type 1 ultrapure water (Millipore), then UV-irradiated for 20 min. Negative controls were included for every extraction procedure and PCR. The genetic profiles of the laboratory staff were also determined.
The samples were handled in a laboratory that was twice yearly tested and certified by GEDNAP (German DNA Profiling Group). The laboratory routine (Hummel 2003) includes the following main points. The laboratories are separated strictly into a pre- and a post-PCR area, and all samples and laboratory staff pass only in the direction from pre- to post-PCR. The pre-PCR area is entered only by fully genetically typed personnel wearing laboratory coats, hairnets and facemasks. The typing results from the ancient samples were compared to the respective data from the laboratory personnel. All working surfaces and all non-disposables are cleaned with soap (Alconox), bi-distilled water and 70% ethanol before and after the treatment of each sample. Negative controls were included in each PCR batch.
The bone samples used by the laboratories for DNA extraction are summarised in online resource 2.
The surface of the bone samples was wiped with cotton swabs soaked in 0.5% NaOCl (Sigma-Aldrich). The specimens were then dipped in 0.5% NaOCl solution for 15 min and washed three times with Type 1 ultrapure water (Millipore). The bone pieces were dried overnight and UV irradiated on every side for 10 min. We ground the samples with Freezer/Mill (Spex Sampleprep) for 30–60 s. Decalcification of 0.15–0.20 g bone powder was performed in 5 ml of 0.5 M EDTA (pH 8.0) (Sigma-Aldrich) on roller mixer at 4 °C for 72 h. The EDTA solution was changed every 24 h, following centrifugation at 2500g for 15 min. The demineralised pellet was washed in 5 ml of ultrapure water. The DNA was isolated from the pellet using DNA IQ system kit (Promega). We followed the “bone protocol” (Promega) modified with a 3-h-long digestion step. The DNA was eluted in a first 40-μl fraction and a second 20-μl fraction. New DNA extracts for mitochondrial sequencing analysis were also prepared. An additional 10 min 0.8% NaOCl treatment of the II/52 femur powder and 7 min 0.5% NaOCl treatments of the tarsal powders of Béla III and II/52 were applied. The bone powders were washed three times with Type 1 ultrapure water (Millipore) and processed as described above.
Initially, the DNA of all ten samples was extracted by two different extraction methods: “QiaVac MinElute Standard” and “EZ1” (online resource 3). Because some samples, in particular those of King Béla III and Queen Anna, contained too many inhibiting substances to enable successful amplification, two new extraction methods: “QiaVac MinElute Short” (online resource 3) and “QiaVac MinElute Organic” were developed. The latter was most successful for most samples and is described below. The surface of each bone fragment was decontaminated by incubation for 15 min in commercially available bleach (6% NaOCl) followed by a 15-min rinse in bi-distilled water. The samples were dried overnight at 37 °C, then crushed in a steel mortar and powdered in a ball mill (Retsch). Approximately 0.25 g of bone powder was incubated with rotation with 3900 μl of EDTA (0.5 M; pH 8.0) and 100 μl of Proteinase K (600 mAnson-U/ml) at 37 °C for 18 h. Following the 18-h incubation, an additional 50 μl of Proteinase K (600 mAnson-U/ml) was added, and the samples were rotated for 1 h at 56 °C. The lysate was centrifuged for 3 min at 3300. The supernatant was mixed with 3 ml of phenol by inverting for 6 min. For phase separation, the samples were placed for 10 min at 56 °C. The organic phase was removed, and the samples were mixed with 4.5 ml of chloroform by inverting for 6 min. The phases were separated as described above. The aqueous phase was mixed with 16 ml of PB buffer (Qiagen) and 100 μl of sodium acetate buffer (3 M; pH 5.2), centrifuged for 3 min at 3300g and transferred to MinElute columns with large-volume funnels on a QIAvac 24 Plus vacuum system (both Qiagen). The lysate was pulled through by vacuum, followed by three washing steps with 700 μl of PE buffer (Qiagen). The MinElute columns were centrifuged for 1 min at 15,700g and then dried at room temperature with open lids for 20 min. DNA elution was performed three times with 20 μl of warm RNase-free water (Qiagen).
Y-chromosomal and autosomal STR analyses
All the STR amplifications were performed in a GeneAmp 9700 thermal cycler (Applied Biosystems). We applied 7 μl of DNA from the first elutes in the commercially available STR kits. PCRs were composed and run according to the kit protocols with 34 cycles.
AmpFlSTR Yfiler kit (Applied Biosystems) was used to analyse the Y-chromosomal STRs.
We used AmpFlSTR MiniFiler (Applied Biosystems), Investigator Hexaplex ESS and Investigator ESSplex Plus (both Qiagen) kits as well as self-designed tetraplex PCR to amplify autosomal STRs. Our self-designed tetraplex PCR included the markers D2S441, vWA, D10S1248 and TH01 (online resource 4). These PCR mixes contained 5 μl of DNA extract, 1× GoTaq Flexi Buffer (Promega), 2 mM MgCl2, 0.2 mM dNTPs, 3.2 μg of BSA, 2.5 U GoTaq Hot Start Polymerase (Promega) and the PCR primers (online resource 4) in a final volume of 20 μl. The cycling conditions were 1 cycle of 2 min at 95 °C, 45 cycles of 30 s at 94 °C, 1 min at 58 °C, 1 min at 72 °C and a final extension of 50 min at 60 °C. Simplex reactions targeting D2S441 and D3S1358 (online resource 4) (Urquhart et al. 1995; Krenke et al. 2002) were also run under the above conditions.
The PCR fragments were separated on a 3130 Genetic Analyzer (Applied Biosystems) using POP7 in a 36-cm capillary array. We evaluated the results using the GeneMapper Software v.4.0 (Applied Biosystems).
The amplification of Y-chromosomal STRs was carried out using the Powerplex Y kit (Promega) and a lab-internal decaplex Y-miniSTR-kit (for primer sequences see online resource 4). The decaplex reaction setup contained 1× Qiagen Multiplex PCR Master Mix plus, 0.25 μl of ammonium sulphate (3 M) and 2.25 μl of primer set. The reactions were performed using 3–4 μl of DNA extract in a final volume of 25 μl. The cycling was performed in a Mastercycle (Eppendorf) and consisted of 5 min at 95 °C; 10 cycles of 1 min at 94 °C, 1.5 min at 62 °C and 1 min at 70 °C; 30 cycles of 1 min at 90 °C, 1.5 min at 59 °C and 1 min at 70 °C. At the end of the cycling, a final elongation of 45 min at 60 °C was added.
Amplifications with the Powerplex Y kit (Promega) were performed with 1–3 μl of DNA extract. The cycling consisted of 11 min at 94 °C, 1 min at 96 °C, 10 cycles of 1 min at 94 °C, 1 min at 60 °C, 1.5 min at 70 °C and 30 cycles of 1 min at 90 °C, 1 min at 58 °C and 1.5 min at 70 °C. At the end of the cycling, a final elongation of 30 min at 60 °C was added.
Self-designed heptaplex and decaplex miniSTR assays and the commercially available Investigator ESSplex SE plus and Investigator ESSplex SE QS kits (both Qiagen) were used for amplification of the autosomal STRs. The self-designed heptaplex miniSTR assay was used as described in Seidenberg et al. (2012), except that amelogenin was labelled with 6-FAM. The amplifications were performed for 40 or 45 cycles in a Mastercycler (Eppendorf) using 0.1–5 μl of ancient DNA extracts. The decaplex miniSTR assay was used as described by Fehren-Schmitz et al. (2015) with 40 cycles using 0.5–5 μl of ancient DNA extracts. Further amplifications were performed using the Investigator ESSplex SE plus and the Investigator ESSplex SE QS (both Qiagen). The PCR reactions used 0.1–5 μl of DNA extracts and ran for 40 cycles in a Mastercycler (Eppendorf).
The PCR products were checked for quality and quantity on a 2.5% agarose gel. Afterwards, the products were separated on a 3500 Genetic Analyzer (Applied Biosystems) using POP7 in 50-cm capillaries. The 3500 series Data Collection Software v2.0 was used for data collection, and the GeneMapper Software v.5.0 was used for allele determination.
Consensus profiles were generated for the samples at each laboratory. We considered an allele valid if it was present in at least two independent replicates. In the case of incomplete profiles, the results of the two laboratories were pooled before establishing a consensus profile.
Y haplogroups were statistically predicted by Athey’s haplogroup predictor (http://www.hprg.com/hapest5/) (Athey 2005).
We used the “Familias 3” software (http://familias.no) (Kling et al. 2014) to perform kinship analysis based on the autosomal STR data of the persons and the population allele frequencies (Molnár et al. 2011; Rak et al. 2010).
Y-profiles were searched against the Y Chromosome Haplotype Reference Database (YHRD) (http://yhrd.org) (Willuweit and Roewer 2015) and US Y-STR Database (https://www.usystrdatabase.org) (Fatolitis and Ballantyne 2008).
Mitochondrial DNA analysis
The complete mitochondrial control region (the hypervariable region) was determined in the samples of Béla III (tarsal), Anna of Antioch (rib) and II/52 (tarsal, femur). The control region was covered by ten overlapping PCRs using primers described by Eichmann and Parson (2008). We used the second elutes of DNA extracts as PCR templates. New DNA extracts were also prepared following an additional NaOCl treatment to eliminate residual mitochondrial DNA contamination (see the “DNA extraction—Budapest” section). The PCR mixes contained 2–4 μl of DNA extract, 1× Phire Reaction Buffer (Thermo Scientific), 0.2 mM dNTPs, 0.4 μl of Phire Hot Start II DNA Polymerase (Thermo Scientific) and 0.2 μM of each primer in a final volume of 20 μl. The cycling conditions are given in online resource 5. The PCR products were purified with ExoSAP-IT (Affymetrix) and sequenced using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems). The reaction products were purified with BigDye XTerminator Purification Kit and resolved on ABI PRISM 3130 Genetic Analyzer (Applied Biosystems). Data analysis was performed by the Sequencing Analysis v5.2 software. The SNPs were identified by comparison to the revised Cambridge Reference Sequence (rCRS). Sequences were confirmed by at least two different amplification products. We used the EMPOP database (http://empop.online) (Parson and Dür 2007) and HaploGrep2 (https://haplogrep.uibk.ac.at/) (Kloss-Brandstätter et al. 2011) to infer the mitochondrial haplogroups.