U.S. Department of Justice Federal Bureau of Investigation Washington
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U.S. Department of Justice Federal Bureau of Investigation Washington, D.C. 20535-0001 June 4, 2018 Next Generation Sequencing Analysis of Hairs from Alexandra Feodorovna, Empress of Russia and Louise of Hesse-Kassel, Queen consort of Denmark. Synopsis In June 2017, Captain Peter Sarandinaki, the founder of the Scientific Expedition to Account for the Romanov Children (SEARCH) foundation provided two sets of hairs to the FBI Laboratory to perform deoxyribonucleic acid (DNA) analysis to provide additional information regarding the potential sources of the hairs. The DNA Support Unit (DSU) of the FBI Laboratory performed this testing under the terms of a Memorandum of Understanding (MOU) executed between the FBI and SEARCH. The first set of hairs was identified to the FBI Laboratory as having come from a Fabergé locket bought by antique expert Nikolai Bachmakov. The heart shaped locket contained a picture of Alexandra Feodorovna, Empress of Russia and the wife of Nicholas II Romanov, the last Tsar of the Russian Empire, as well as a lock of hair. Because specialized tools are required to open such lockets, Mr. Bachmakov assumed that the locket had been closed for decades and that the hairs may have belonged to one of the Romanov’s children. The second set of hairs was found in a picture frame that Mr. Bachmakov bought from a living relative of the Romanov family. The frame contained a picture of Louise of Hesse-Kassel, Queen consort of Denmark, the maternal grandmother of Tsar Nicholas II as well as a large lock of light hairs believed to be from the Queen. Mitochondrial DNA (mtDNA) was successfully isolated and sequenced from hairs from both the Fabergé locket and the frame using next generation sequencing (NGS). The sequence of the mitochondrial genome (mtGenome) obtained from the Fabergé locket hairs was identical to the published mtGenome of Empress Alexandra Romanov. The hairs are therefore consistent with being from a maternal relative of the Empress. It is noted, however that because mtGenome sequence information cannot be used to differentiate among maternal relatives, it is not possible to confirm that the hairs are from a single individual. Additionally, the mtGenome sequence obtained from the frame hairs matched the published sequence of Tsar Nicholas Romanov, to include the point heteroplasmy at position 16169 that characterizes the Tsar’s maternal lineage. Based on these results, and assuming the lock originates from a single individual, these hairs are consistent with being from Louise of Hesse-Kassell, Queen of Denmark or another individual maternally related to Tsar Nicholas II. Background In 2012, Mr. Nikolai Bachmakov, a renowned expert in antique restoration, bought a pre-1899 Fabergé locket at an antique show in Florida. Using specialized tools, Mr. Bachmakov successfully opened the locket. On one side was a small black and white photograph of Empress Alexandra Romanov (1872-1918), wife of Tsar Nicholas II of Russia and, on the other side, a lock of light hairs that he assumed belonged to one of their five children (Appendix, Figure 1). In 2012, in an effort to address his question, Mr. Bachmakov sent a few hairs from the locket to a commercial laboratory for mitochondrial DNA testing. The laboratory obtained no results. In March 2017, Mr. Bachmakov was introduced to Captain Peter Sarandinaki, the founder of the Scientific Expedition to Account for the Romanov Children (SEARCH) foundation. This non-profit organization was originally founded to locate the missing remains of two Romanov children, Tsarevich Alexis and his sister, Grand Duchess Maria. When he learned about the hairs found in the Fabergé locket, Capt. Sarandinaki told Mr. Bachmakov about the FBI Laboratory’s ancient DNA work and, with Mr. Bachmakov’s consent, he contacted the FBI DNA Support Unit on March 19, 2017 requesting the Laboratory’s assistance with DNA testing. A Memorandum of Understanding was then executed between the FBI Laboratory and SEARCH to perform this testing. At the time, Mr. Bachmakov was also in possession of a frame that contained a 67 mm x 43 mm picture of Louise of Hesse-Kassel (1817-1898), wife of King Christian IX of Denmark and maternal grandmother of Tsar Nicholas II. Positioned on top of the picture in the frame was a lock of light hairs that had presumably belonged to Louise (Appendix, Figure 2). The frame had originally belonged to Maria Feodorovna (princess Dagmar of Denmark), one of Louise’s daughters. It was subsequently transferred to descendants of the Romanov family (Appendix, Figure 3) who were in possession of it until 2017 when it was purchased by Mr. Bachmakov. DNA Testing On June 15, 2017, Capt. Sarandinaki provided a few hairs from the Fabergé locket as well as a few hairs from the frame to the FBI Laboratory. For purposes of clarity, from here forward hairs from the locket will be referred to as “Lo-hairs” (i.e. Fabergé locket hairs) and hairs from the frame will be referred to as “Fr-hairs” (i.e. frame hairs). DNA was extracted from all hairs with a protocol that favors the recovery of very small nucleic acids. Six Fr-hairs (4 to 5cm) were co-extracted in October 2017 and five Lo-hairs of various sizes (1.5 cm, 2.5 cm, 3.5 cm, 4 cm and 4.5 cm) were co-extracted in January 2018. All DNA extractions were accompanied by two extraction controls (reagents only; referred to as a “reagent blank” or RB). Each DNA extract and RB was converted into a double indexed Illumina library and shotgun sequenced. In some cases, libraries were also enriched in mitochondrial DNA using an in-solution hybridization capture and subsequently sequenced. Results DNA analyses for Lo-hairs Shotgun sequencing of the library produced 841,245 unique human sequences. 97.56% (820,689) were nuclear DNA sequences and the other 2.44% (20,556) were mtDNA sequences. The mitochondrial DNA enriched libraries produced 1,814,081 human sequences, of which 98.4% (1,784,498) were mtDNA sequences. After removing duplicate reads, 54,245 unique mtDNA sequences remained, and the mtGenome was entirely sequenced with coverage ranging between 202 and 511 sequences (average 286.42x), see Figure 1. Figure 1: Sequence distribution over the mtGenome after hybridization capture. The final mtGenome profile for the Lo-hairs can be found in Table 1. The sequence belongs to haplogroup H1af2 and is 100% concordant with the published sequence of Empress Alexandra [GenBank # FJ656214]. This reference sequence was obtained using skeletal remains found in 1991 on the Koptikovsky Road, close to Ekaterinburg, Russia and identified as those of Empress Alexandra Romanov (Gill et al., 1994, Nature Genet 6 (2): 130-135 - Coble et al., 2009; PLoS One 4 (3): e4838 - Rogaev et al., PNAS 106 (13): 5258-5263). Reference Reference Allele Count Coverage Frequency Position 263 A G 257 259 99.2 315.1 - C 255 382 66.8 524.1 - C 228 403 56.6 524.2 - A 228 403 56.6 750 A G 257 259 99.2 1438 A G 233 238 97.9 3010 G A 262 262 100.0 4137 C T 260 261 99.6 4769 A G 222 226 98.2 8860 A G 307 311 98.7 15326 A G 309 311 99.4 16111 C T 383 386 99.2 16357 T C 287 304 94.4 16519 T C 250 253 98.8 Table 1: mtGenome profile of Lo-hairs. Positions in italics refers to positions where length heteroplasmy was detected. These variants are always reported with lower frequencies than regular single nucleotide polymorphism variants. DNA analyses for Fr-hairs Shotgun sequencing produced 533,498 unique human sequences. 98.93% (527,816) were nuclear DNA sequences and the other 1.07% (5,682) were mtDNA sequences. The mitochondrial DNA enriched libraries produced 8,310,139 human sequences, of which 96.35% (8,006,451) were mtDNA. Following the removal of sequence duplicates, 54,384 unique mtDNA sequences remained. The entire mtGenome was recovered with coverage ranging between 22 and 392 sequences (average 142.17x; see Figure 2). Figure 2: Sequence distribution over the mtGenome after hybridization capture. The mtGenome sequence of the Fr-hairs can be found in Table 2. The profile belongs to haplogroup T2a1a and is identical to the published sequence of Tsar Nicholas [GenBank # FJ656215]. This reference sequence was obtained using skeletal remains found in 1991 on the Koptikovsky Road, close to Ekaterinburg, Russia and identified as those of Tsar Nicholas Romanov (Gill et al., 1994, Nature Genet 6 (2): 130-135 - Ivanov et al., 1996, Nature Genet 12 (4):417-420 - Coble et al., 2009, PLoS one 4 (3): e4838 - Rogaev et al., PNAS 106 (13): 5258- 5263). The point heteroplasmy at position 16169 that is known to occur in the Tsar’s maternal lineage is also present in the Fr-hairs, at a frequency of 74.4%T (see Figure 3). Reference Reference Allele Count Coverage Frequency Position 73 A G 120 122 98.4 263 A G 211 221 95.5 315 - C 141 271 52.0 709 G A 136 136 100.0 750 A G 129 136 94.9 1438 A G 98 100 98.0 1842 A G 57 58 98.3 1888 G A 94 94 100.0 2706 A G 107 110 97.3 2850 T C 82 84 97.6 4216 T C 86 86 100.0 4769 A G 44 44 100.0 4917 A G 114 120 95.0 6257 G A 147 149 98.7 7022 T C 89 90 98.9 7028 C T 88 89 98.9 8697 G A 75 77 97.4 8860 A G 160 166 96.4 10463 T C 55 55 100.0 11251 A G 153 156 98.1 11719 G A 126 129 97.7 11812 A G 115 115 100.0 13368 G A 111 114 97.4 13965 T C 140 140 100.0 14233 A G 121 121 100.0 14687 A G 145 147 98.6 14766 C T 80 83 96.4 14905 G A 116 119 97.5 15326 A G 211 219 96.3 15452 C A 132 135 97.8 15607 A G 123 129 95.3 15928 G A 150 162 92.6 16126 T C 139 139 100.0 16169 C T 64 86 74.4 16294 C T 46 46 100.0 16296 C T 46 46 100.0 16519 T C 110 111 99.1 Table 2: mtGenome profile of Fr-hairs.