Identification of the Remains of the Romanov Family Worksheet Answers

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Mystery Solved: The Identification of the Two Missing Romanov Children Using Deoxyribonucleic acid Analysis

• Odile Thousand. Loreille ,
• Mark J. Wadhams,
• Suni M. Edson,
• Kerry Maynard,
• Carna E. Meyer,
• Harald Niederstätter,
• Cordula Berger,
• Burkhard Berger,
• Anthony B. Falsetti,
• Peter Gill,
• Walther Parson,
• Louis N. Finelli

Mystery Solved: The Identification of the Two Missing Romanov Children Using Dna Assay

• Michael D. Coble,
• Odile M. Loreille,
• Mark J. Wadhams,
• Suni M. Edson,
• Kerry Maynard,
• Carna East. Meyer,
• Harald Niederstätter,
• Cordula Berger,
• Burkhard Berger,
• Anthony B. Falsetti

x

• Published: March xi, 2009
• https://doi.org/ten.1371/journal.pone.0004838

Figures

Abstruse

One of the greatest mysteries for nigh of the twentieth century was the fate of the Romanov family, the last Russian monarchy. Following the abdication of Tsar Nicholas 2, he and his married woman, Alexandra, and their five children were eventually exiled to the metropolis of Yekaterinburg. The family unit, along with iv loyal members of their staff, was held captive by members of the Ural Soviet. According to historical reports, in the early morning hours of July 17, 1918 the entire family along with four loyal members of their staff was executed by a firing team. After a failed attempt to dispose of the remains in an abandoned mine shaft, the bodies were transported to an open field just a few kilometers from the mine shaft. Nine members of the group were buried in one mass grave while two of the children were buried in a separate grave. With the official discovery of the larger mass grave in 1991, and subsequent Dna testing to ostend the identities of the Tsar, the Tsarina, and three of their daughters – dubiety persisted that these remains were in fact those of the Romanov family. In the summertime of 2007, a group of amateur archeologists discovered a collection of remains from the 2d grave approximately 70 meters from the larger grave. We report forensic DNA testing on the remains discovered in 2007 using mitochondrial Deoxyribonucleic acid (mtDNA), autosomal STR, and Y- STR testing. Combined with additional DNA testing of material from the 1991 grave, nosotros have virtually irrefutable prove that the two individuals recovered from the 2007 grave are the two missing children of the Romanov family: the Tsarevich Alexei and i of his sisters.

Citation: Coble MD, Loreille OM, Wadhams MJ, Edson SM, Maynard Yard, Meyer CE, et al. (2009) Mystery Solved: The Identification of the Two Missing Romanov Children Using DNA Analysis. PLoS 1 4(3): e4838. https://doi.org/10.1371/periodical.pone.0004838

Editor: Michael Hofreiter, Max Planck Found for Evolutionary Anthropology, Federal republic of germany

Received: January 28, 2009; Accepted: February 17, 2009; Published: March 11, 2009

This is an open up-access article distributed under the terms of the Creative Commons Public Domain annunciation which stipulates that, in one case placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, congenital upon, or otherwise used by anyone for any lawful purpose.

Funding: This written report was funded past the U.S. Military machine DNA Identification Laboratory and the Innsbruck Medical University, Innsbruck, Austria. The funders had no part in study design, information drove and analysis, determination to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

For over 300 years, the Romanovs ruled the land of Russia. In 1917 following the Bolshevik revolution, the concluding ruling Russian Tsar, Nicholas Ii, abdicated his crown in favor of his brother Grand Knuckles Michael, who declined to accept the throne. Nicholas and his family - his wife, the Tsarina Alexandra, and their v children: Olga, Tatiana, Maria, Anastasia, and the Tsarevich (Crown Prince) Alexei were held in exile in Yekaterinburg, Russia. As well present with the royal family unit were four loyal members of their staff: Dr. Eugene Botkin, the family physician; Alexei Trupp, valet to the Tsar; Anna Demidova, maid to the Tsarina; and Ivan Kharitonov, the family unit cook.

In July of 1918, the Ural Soviets feared an try to rescue the Tsar and his family unit by the White Russian Regular army [1]. A conclusion was made by the Ural Soviets to execute the unabridged family, with the idea that upon hearing of the Tsar's death the will of the people loyal to the Tsar would be cleaved. In the early on morning hours of July 17, 1918 the regal family and their staff were led to the cellar of the Ipatiev House where they were existence held and executed.

In the late 1970s, a local geologist, Dr. Alexander Avdonin was able to locate the mass grave containing the remains of 5 of the seven members of the purple family and their iv servants. Avdonin and a handful of shut friends kept the location of the grave a secret until the autumn of the Soviet Marriage in 1991 [2].

An official recovery and forensic anthropological investigation was conducted on the nine skeletons disinterred from the mass grave. DNA testing of the remains recovered in 1991 was conducted by Dr. Peter Gill, formerly of the Forensic Science Service (FSS) and Dr. Pavel Ivanov, a Russian geneticist [3]. Nuclear Dna testing of 5 STR markers confirmed the sex of the skeletons and established a familial relationship amidst the remains of the Tsar, the Tsarina and iii of their daughters recovered from the grave. Previous mtDNA testing (outlined in Figure S1) confirmed a maternal relationship betwixt HRH Prince Philip, the Duke of Edinburgh, the Tsarina, and her three daughters. The Duke of Fife and Princess Xenia Cheremeteff Sfiri, maternal relatives of Nicholas were used to reassociate the putative remains of the Tsar. A single point heteroplasmy at position 16169 (C/T = "Y") was observed in the mtDNA sequence of the Tsar, whereas his maternal relatives were fixed for 16169 T. To confirm the authenticity of the heteroplasmy, DNA testing conducted at the Military machine DNA Identification Laboratory (AFDIL) compared the mtDNA haplotype from the remains of 1000 Duke Georgij Romanov (d. 1899), brother of Tsar Nicholas II [4]. Both Tsar Nicholas II and Grand Duke Georgij Romanov shared the same point heteroplasmy at 16169 – but in differing ratios. The Tsar was mostly C/t while his blood brother was mostly T/c.

Despite the overwhelming forensic prove, doubts pertaining to the authenticity of the remains persisted [v]. Skeptics pointed to the two children missing from the mass grave - Alexei and one of his sisters - as evidence that the bodies plant in the mass grave were not the Romanov family. The identity of the missing princess was the source of a loftier profile disagreement betwixt Russian and US forensic anthropologists: the Russians were convinced that Maria was missing from the mass grave, while the American experts believed that Anastasia was missing [2]. Rather than bring closure to the about 70 twelvemonth mystery of the fate of the Romanovs, identification of only five of the seven family unit members continued to fuel speculation that somehow these 2 miraculously escaped the bullets of the executioners and made their way out of Russian federation.

After the discovery of the "first" mass grave, several attempts were fabricated in the ensuing years to find the "second" grave, which was believed to be relatively nearby (P. Sarandinaki, personal communication). In the summer of 2007, a group of amateur archeologists discovered a few bone fragments approximately 70 meters from the beginning grave. Following an official archeological earthworks conducted by Dr. Sergei Pogorelov, Deputy Director of the Sverdlovsk Region'southward Archaeological Institute, a prepare of 44 bone fragments and teeth were advisedly recovered from the site.

After a thorough assay of the remains past both Russian and US anthropologists, the scientific conclusions were the following:

• Based on duplicative anatomical units such as the midline portion of the occipital, no less than, or a minimum of ii people were nowadays among the recovered remains.
• 1 person nowadays amidst the remains was of female sex, based on conspicuously visible sciatic notch dimensions, with a biological or developmental age of approximately 15–19 years.
• The sexual activity of the other person was probably male, again based on the incipient breadth of the sciatic notch, and the biological age ranged from 12–15 years.
• Given the limited fragmented textile coupled with the lack of representative diagnostic anatomy, it was not possible to determine the racial or bequeathed type or estimate living stature from the remains.
• Three silver amalgam fillings discovered on the crowns of two molars recovered from the grave suggest that at least one person was of an aloof status.
• The overall age of the burial site was most likely greater than 60 years former based on culturally diagnostic material found contextually with the bones.

In tardily 2007, the Russian regime invited a team of scientists to conduct independent Deoxyribonucleic acid testing of the remains from the second grave. Nosotros present the results from mtDNA, autosomal STR and Y-STR testing of these remains at two contained laboratories highly specialized in ancient Dna (aDNA) studies: the Armed services DNA Identification Laboratory (AFDIL, Rockville, Maryland, USA) and the Institute for Legal Medicine (GMI, Innsbruck, Austria). We likewise nowadays the results of a new analysis of the remains from the first mass grave attributed to Tsar Nicholas II, his married woman Alexandra, Olga, Tatiana and a tertiary daughter who could be either Anastasia or Maria. The DNA analysis of all three genetic systems confirms that the samples tested from the second grave are one female and 1 male kid of Tsar Nicholas Ii and Tsarina Alexandra, solving the mystery of the missing Romanov children.

Results

Quantification

DNA quantification gave values greater than 4000 mitochondrial genome equivalent per microliter (mtGEs/µl) for the 143 bp mtDNA target except for bone sample 4.44 which independent less than 100 mtGEs/µl and bone sample v.21 which contained 2923 mtGEs/µl (details in Table S1). No indication of the presence of PCR inhibitors was observed. Quantification of nuclear DNA produced concentrations between 11 and 615 pg/µl for all samples. No detectable quantification results were observed in any reagent blank control for either the mtDNA or the nuclear Dna targets.

Mitochondrial Deoxyribonucleic acid Testing

We starting time analyzed the remains discovered in 2007 (Table one). Nosotros obtained total control region profiles [16024-576] for three samples (144.1, 146.one and 147). Sequences of all iii samples between 16024 and 576 were confirmed by three independent teams to be: 16111T, 16357C, 16519C, 263G, 315.1C, 524.1A and 524.2C. The mutual 16519 C variant and an Air conditioning doublet insertion in the HVIII Air-conditioning repeat region are newly characterized variants exterior of HVI/HVII for these samples compared to the original mtDNA testing [3] where these regions were not sequenced. For samples 140, 141, 143 and 145, we analyzed HVI [16024-16391] and HVII [35-369]. Samples sequenced for HVI and HVII were confirmed for 16111T, 16357C, 263G, and 315.1C. Two samples (139 and 142) failed to yield reproducible information.

Second, full control region [16024-576] profiles were generated from the remains of the Tsarina and iii of her daughters recovered originally from the showtime grave (Table 2). Successful amplifications were obtained for all skeletal elements, with amplicons as big as 444 bp (GMI) or 440 bp (AFDIL). Sequences from all individuals confirmed previously published results in HVI and HVII and matched the sequence haplotype obtained with the recently discovered skeletal remains [three].

To assess the frequency of this sequence, we first focused on the German database (n = 513 samples) inside the EDNAP mtDNA Population Database (EMPOP; [6]) since Tsarina Alexandra was a German princess. We found no exact matches either in the German database or within the 3,340 W Eurasia sequences in EMPOP. Finally, nosotros searched the Tsarina's haplotype using a global mtDNA database of 23,627 individuals (4,839 individuals in the The states SWGDAM mtDNA database and 18,788 individuals from an internal AFDIL Research Section database equally of 01/16/09) and found no match making this haplotype a rare sequence.

The sequence of the total command region [16024-576] was also determined from the remains of Tsar Nicholas II (a molar from skeleton #4) and matched the published data of HVI and HVII from Gill et al. [iii] and Ivanov et al. [4]: 16126C, 16169Y, 16294T, 16296T, 16519C, 73G, 263G, 315.1C with the transition at position 16519 newly characterized in the control region. The point heteroplasmy at position 16169 was present with C existence the major component over T (Figure 1).

To appraise the frequency of the haplotype from the Tsar, we showtime focused on the Denmark database (n = 209 samples) within the EMPOP database since the Tsar'south mother was a Danish princess. We establish no exact matches in the Danish database. Among the 3,340 West Eurasian sequences in EMPOP, 3 sequences matched (0.09%) when 16169C (matching the rCRS) was considered. A search of the AFDIL+SWGDAM mtDNA database revealed 19 matches (nineteen/23,627 = 0.08%) to the Tsar's haplotype when 16169C was considered. No matches to the Tsar's haplotype+16169T were observed. The relative frequency of the Tsar'due south mtDNA haplotype was considered to exist rare.

Autosomal STR Testing

The best preserved fragments were identified by an anthropological inspection and two compact bone fragments, each coming from a femoral bone (146.1 and 147), were selected for nuclear STR testing. An anthropologist (ABF) determined that fragment 147 likely belonged to a female based on the general size and shape of the femoral caput and angulations of the femoral neck.

The STR results for samples 146.i and 147 are shown in Table 3. Each allele was replicated at least seven times past both laboratories. The results of the sex activity-typing marker amelogenin revealed that sample 146.ane was from a male person and confirmed that sample 147 was from a female (Figures S2 and S3). We plant no show of contamination amidst our STR profiles. In fact, of the 1458 alleles amplified above our reporting threshold at AFDIL (100 RFUs for heterozygote alleles and 200 RFUs for homozygous alleles) among all autosomal and Y-STRs, only half-dozen alleles (0.iv%) would exist considered as spurious or drib-in artifacts. Half of these spurious alleles occurred at stutter positions of the accurate allele – indicating that they were most likely generated by preferential amplification during the early rounds of PCR.

The STR analysis revealed the presence of two split up individuals and a very loftier degree of allele sharing was noted amid the two profiles, suggesting that the individuals were closely related. The Sibship Alphabetize (SI) was calculated by determining the likelihood ratio (LR) of the hypothesis (H₁) that samples 146.1 and 147 are siblings compared to the alternative hypothesis that these samples belong to 2 unrelated individuals (H₂). The SI was adamant to be five.6 meg in favor of H₁. In other words, the Deoxyribonucleic acid evidence is 5.half-dozen meg times more likely if samples 146.1 and 147 were siblings rather than if these samples were from 2 unrelated individuals.

To ostend that these ii siblings were also related to the Romanov remains recovered from the get-go mass grave, we conducted STR testing on skeletal elements representing the other five members of the purple family unit (Tabular array ii). The results are shown in Table 3. The profiles for vWA matched the data published past Gill et al. [3] from the quadruplex markers tested [seven]. For the TH01 locus, nosotros obtained alleles vii and ix.iii for the Tsar, identified equally a seven/10 genotype from Gill et al. [iii]. Similarly, for one of the daughters, the TH01 genotype was previously eight/10 and is now 8/nine.3. In the Gill et al. publication [three], it was the practice at the time to combine the 9.3 and 10 alleles for TH01 and to use the '10' designation as the standard nomenclature for both. The Dna Commission of the International Society for Forensic Haemogenetics (ISFH) afterwards recommended the utilize of "ix.3" to draw the microvariant allele in TH01 [viii]. After the year 2000, the 9.3 allele designation at TH01 was utilized for all profiles sent to the UK national DNA database. This minor (historical) difference in the classification has no issue on our comparisons. All of the genotypes at vWA and TH01 were fully concordant among the skeletons from the first grave.

All of the additional microsatellites tested in this study confirmed the parental relationship between the skeletal remains of Tsar Nicholas II and Alexandra and the other remains tested in this study. All of the alleles from iii daughters from the first grave can be explained by half-allele sharing with the profiles from Nicholas and Alexandra. Importantly, the two skeletal remains from the newly discovered grave prove the aforementioned one-half allele sharing genotypes with both Nicholas and Alexandra equally putative parents. When nosotros calculated the LR of the hypothesis (H_ane) that samples 146.1 and 147 are the children of Tsar Nicholas Two and Tsarina Alexandra (and siblings of the three princesses from grave 1) compared to the alternative hypothesis that these samples are individuals completely unrelated to the Romanov family (H₂), nosotros institute that the DNA show is 4.36 trillion times more than likely if sample 147 is a daughter of Tsar Nicholas Two and Tsarina Alexandra, and over eighty trillion times more than likely if sample 146.1 is a son of Tsar Nicholas II and Tsarina Alexandra than if these samples were from two unrelated individuals.

Y-STR Testing

Finally, to compare the contour of the Tsar and his son to a paternally living descendant of the Romanov family unit, nosotros conducted Y-STR testing on the skeletal material. Nosotros first generated a 17 Y-STR loci profile from sample 146.1 and and then from a molar of the Tsar. Finally, in a separate laboratory, nosotros generated the profile of Prince Andrew Andreevich Romanov, a distantly related cousin of Tsar Nicholas Ii (Figure 2). An instance of 4 of the markers is shown in Figure three. Nosotros observed an exact lucifer betwixt all 3 men over all 17 markers (Tabular array 4). To determine the significance of this match, we searched the Y-STR haplotype against a database of 4,163 individuals (http://usystrdatabase.org/) and found no match. A search of the YHRD database (http://www.yhrd.org) was conducted and no match was observed between the 17 locus profile and the 10,243 haplotypes including at to the lowest degree two,068 individuals from the Eurasian Metapopulation.

Effigy 2. Romanov paternal lineage used for Y-STR testing.

DNA testing for 17 Y-STR markers was conducted on the remains from Tsar Nicholas II and his son, the Tsarevich Alexei (sample 146.i in the 2d grave). A distantly related cousin, Prince Andrew Andreevich Romanov of San Francisco, California, was used as a living relative to compare to the skeletal material.

https://doi.org/10.1371/journal.pone.0004838.g002

Figure iii. An example of 4 Y-STR markers from the iii Romanov relatives.

Each panel is a screenshot from the blue dye channel of Y-Filer (Practical Biosystems, Foster City, CA). The top panel was developed from the skeletal remains of Alexei, the heart console was developed from a tooth sample from Tsar Nicholas II, and the bottom panel was developed from Prince Andrew Andreevich Romanov. The loci are (from L to R): DYS456 (16 repeats), DYS389I (xiii repeats), DYS390 (24 repeats), and DYS389II (29 repeats).

https://doi.org/ten.1371/periodical.pone.0004838.g003

Give-and-take

The truthful fate of the Romanov family was unknown to all except for a handful of people for nearly 70 years. Gill et al. [iii] conducted the original Deoxyribonucleic acid testing after the preliminary anthropological investigations from the first grave.

The veracity of the results were later challenged by Knight et al. [9] who doubted the authenticity of sequences generated from the nested PCR strategy. Knight argued that the amplicon sizes were unusually long, and therefore the results were unreliable. Hofreiter et al. [10] and Gill and Hagelberg [eleven] accept offered a rebuttal to the opinions made by Knight et al. [9]. All the same, Knight et al. [12] insisted: "To the contrary, only a 221-bp amplicon could exist produced (mayhap from endogenous degraded DNA template), but not a 400-bp nested product…. [the] results in (Gill et al.) are non plausible". Information technology is generally our experience that highly degraded mtDNA templates are often just amplified with 270 bp amplicons or less. Notwithstanding, given this unique opportunity to re-examination the verbal fabric originally evaluated by Gill et al. [iii], we were successfully able to amplify 444 bp and 440 bp fragments using a classic aDNA distension strategy (increased wheel number, additional BSA, and additional polymerase), see Figure 4. Not just was it possible to amplify up to 444 bp of mtDNA, we also successfully amplified a number of high molecular weight alleles from the nuclear STRs tested (nether ∼375 bp for autosomal markers and under ∼335 bp for the Y-chromosomal markers). It is very likely that the extremely cold climate in Yekaterinburg, where the basis is typically frozen from September until Apr, provided an ideal environment to preserve the remains.

Figure 4. Screenshots of the 16111 C-T variant and 16357 T-C variants from three samples sequenced from fragments amplified for 440 bp.

Legend for the gel: L = Ladder, RB = Reagent Blank, 7.49 = Sample from Tsarina Alexandra, 146.1 = Sample from Alexei, five.21 = Sample from Tatiana, NC = Negative Control, PC = Positive Control, Fifty = Ladder.

https://doi.org/10.1371/journal.pone.0004838.g004

Some other issue which generated doubt about the early on Dna testing was the point heteroplasmy at np 16169 in the mtDNA sequence of the Tsar. At the fourth dimension this was a contentious finding. In the early on to mid-1990s, point heteroplasmy was believed to exist an extremely rare phenomenon and was not easily explained as the presence of two different mtDNA haplotypes within an individual. Contained testing of samples was extremely important to provide the necessary confidence that the results were valid. The mtDNA results from the declared Tsar were thus independently confirmed in the laboratory of Erika Hagelberg at Cambridge University, a co-author on the 1994 publication [three]. In improver, the 16169 T/C point heteroplasmy was confirmed by AFDIL in bone samples from Tsar Nicholas Ii and his brother, Grand Duke Georgij [four]. Finally the heteroplasmy was detected and confirmed once again in this study by both AFDIL and GMI (Effigy 1).

Today, the existence of heteroplasmy is understood to be relatively common, although occurrence at the specific 16169 position is itself rare [13]. Among our internal AFDIL mtDNA database of xviii,788 haplotypes, we have observed three instances (0.016%) of point heteroplasmy at np 16169. Only one sample of Western European ancestry shares the aforementioned haplogroup T* as the Tsar, just differs from Nicholas at seven nucleotides within the control region. Consequently, multiple observations of this rare heteroplasmic event can be considered to be a very powerful indicator of relatedness.

Two questions posed by Gilbert et al. [14] to assess the results of an aDNA written report is for the researcher, reader, and reviewer to ask the questions, "What data is presented here that makes the results and/or conclusions believable?" and "Is there whatever reason to not believe this?" We used a three-pronged DNA mark organization to develop our results.

The mtDNA results alone can be considered conclusive. The new samples matched exactly the mtDNA data of Tsarina Alexandra (and the HVI and HVII data of a living relative, HRH Prince Philip), indicating that these samples were maternally related to her. If one includes the anthropological data almost these samples: specifically that i of the samples recovered from the second grave was most probable the femur of a young adult female (sample 147), we can conclude that these samples were from the missing children of the Tsarina since the femora from the Tsarina and her three other children were recovered and deemed for in the first grave.

Autosomal STR genotypes were developed to form a family pedigree of the Romanov family. The Dna profiles of the two samples from the 2d grave fit perfectly into the family unit tree of the Tsar and Tsarina with all of the alleles of the two samples explained past Mendelian inheritance.

A 17-marker Y-STR haplotype from the remains of Tsar Nicholas II matched exactly to the Y-STR haplotype from femur of the male sample (sample 146.i) found in the second grave. The aforementioned 17-marker haplotype was also observed to match a living Romanov relative.

After examining mitochondrial sequences, autosomal STR and Y-STR profiles all linking the remains to living relatives of the Romanov, we likewise compared our STR results from Tsar Nicholas Two with a profile developed past the Sverdlovsk Regional Forensic Bureau (Yekaterinburg) from a claret stain on a shirt worn by Nicholas when he was a immature man. On Apr 29, 1891 while touring the city of Otsu, Nihon the Tsarevich Nicholas Romanov was attacked by a Japanese policeman during an attempted bump-off [fifteen]. Nicholas sustained ii blows to the side of his head from a saber used past the aggressor before the aggressor was subdued. Fortunately, Nicholas survived the assail and the encarmine shirt he wore that day was returned to Russia equally a relic of the attack. Eventually, the shirt was placed in storage at the Hermitage Museum in Saint petersburg. In the summertime of 2008, the Sverdlovsk Regional Forensic Agency took three samples from blood stains on the shirt. One of the samples gave full autosomal and Y-STR profiles [16]. The other two samples gave partial DNA profiles for both autosomal and Y-STRs, with all of the alleles in the partial profile shared with the alleles from the full profile. We compared our DNA profiles from the tooth of Nicholas II to the blood stain contour and institute complete concordance at all loci. For the first time, in that location is now a link between the ante-mortem prove Deoxyribonucleic acid profile from Nicholas II to the post-mortem skeletal remains from the first grave.

Taken together, all of the results and conclusions agree with the hypothesis that the samples recovered from grave ii are the missing children of Tsar Nicholas II and Tsarina Alexandra. It should be mentioned that a well publicized debate [2] over which daughter, Maria (according to Russian experts) or Anastasia (co-ordinate to US experts), has been recovered from the second grave cannot exist settled based upon the Deoxyribonucleic acid results reported here. In the absence of a DNA reference from each sister, we tin simply conclusively identify Alexei – the just son of Nicholas and Alexandra.

For virtually ninety years the fate of the Romanov family was shrouded in mystery. It wasn't until several years later on the execution of the family that the Soviet regime acknowledged the death of all of the Romanovs. Speculation grew that some of the family unit escaped the executioners and found their mode out of Russia. The most famous claimant was Anna Anderson, a Smooth peasant who convinced many that she was Anastasia [2]. With the discovery of the beginning grave, and subsequent Deoxyribonucleic acid testing, Anna Anderson was exposed as an imposter [17]. In fact, since 1918 over 200 people accept claimed to be one of the v Romanov children (http://www.romanov-memorial.com/pretenders.htm). Here we are able to give a total account of all of the Romanov family and can conclude that none of the family unit survived the execution in the early morning hours of July 17, 1918.

Materials Tested

Fabric from the grave discovered in 2007.

Fragments from 10 samples out of 44 were selected for DNA analysis: nine os fragments (cranial, pelvic, scapular, or femoral) and 1-half of the crown portion of a molar. It was determined that the tooth fragment would likely produce a DNA contour; even so, we decided to preserve the textile rather than destroy it during testing. Two of the 9 samples (146.1 and 147) were divided and analyzed by 3 independent teams: the AFDIL (research section), the AFDIL (mitochondrial casework section) and the GMI laboratory. The mitochondrial casework section likewise analyzed the remaining seven samples and focused only on mtDNA testing for these samples post-obit their standard operational protocols.

Material from the grave excavated in 1991.

The remains of the Tsar and his family were laid to rest at the Cathedral of Saint Peter and Saint Paul in St. Petersburg, Russia in 2001. Fortunately, the Sverdlovsk Regional Forensic Bureau Laboratory (Yekaterinburg) predictable the possibility of future DNA testing, and preserved a limited number of fragments from each skeleton. At to the lowest degree two to 3 samples per private (os and/or teeth) were brought to the AFDIL and to the GMI laboratories for Deoxyribonucleic acid analysis. For convention, we followed the naming of the skeletons of the regal family according to the Russian anthropological/facial reconstruction studies from the mid-1990s: Skeleton #3 = Olga; Skeleton #4 = Tsar Nicholas II; Skeleton #5 = Tatiana; Skeleton #6 = Anastasia (or Maria); and Skelton #7 = Tsarina Alexandra (Tabular array 2).

Methods

DNA extractions and analysis were performed past three contained teams, all specialized in aDNA studies and working in adequate facilities. Specialists from the mitochondrial casework section of the AFDIL (MJW, SME, KM) worked in an American Society of Criminal offense Lab Directors (ASCLD) accredited laboratory and focused on mitochondrial Dna analysis, following their standard operating protocols. 1 specialist from the research section (OML) used a split laboratory devoted to aDNA studies and focused on STR analysis. Finally, the GMI team (HN, CB, BB) used an ISO 17025 accredited laboratory to replicate mtDNA and STR analysis. In all laboratories, precautions to monitor contagion by using controls throughout the process and isolation of pre-and post PCR areas were observed at all times. Material and equipment were cleaned using a 10% bleach solution and UV irradiated at 254 nm in a cantankerous-linker for 10 to 45 minutes.

Experiments performed in all three laboratories were witnessed by ii Russian scientists from the Sverdlovsk Regional Forensic Bureau Laboratory (Yekaterinburg): Tamara Tsitovich and Natalia Bandurenko at AFDIL; and Elena Trynova and Elena Vylegzhanina at GMI.

Grooming of the samples

At the AFDIL, all the samples were beginning extensively sanded with an aluminum oxide sanding rock fastened to a dremel tool (Dremel, Racine, WI), sonicated in Deoxyribonucleic acid free h2o and accented ethanol then placed in a sterilized smoke hood to air-dry overnight. The next day, the samples were powdered with a cleaned, DNA-gratis stainless steel Waring MC2 blender cup (Waring, Torrington, CT).

At the GMI, sample pre-treatment comprised extensive mechanical cleaning of the surface of the bones and teeth with UV-irradiated sandpaper and/or sterile scalpel blades, followed past a 20 min soak in sodium hypochlorite solution (≥4% active chlorine), and ane washing step each in sterile water and absolute ethanol. The cleaned samples were dried in a laminar menses hood under a constant air-stream over nighttime and a terminal 15 min UV-irradiation step was applied. The dried samples were powdered by means of a sterile dental drill.

DNA extractions

The GMI grouping and the mtDNA casework section extracted the Deoxyribonucleic acid according to Loreille et al. [xviii] but the GMI team added a terminal purification step using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The AFDIL research scientist used a slightly modified protocol that avoids organic extraction [19]. Between 100 and 400 mg of fine bone powder every bit well equally a "reagent blank" were incubated and gently shaken in 3 ml of extraction buffer (EDTA 0.5 M, 0.5% lauryl-sarcosinate) and 100 µl of proteinase K (20 mg/ml) overnight at 56°C. The tubes were centrifuged for iii minutes at 4000 g, the extraction buffer transferred into a Centricon xxx (Millipore Corp., Bedford, MA) and and then concentrated until the book had decreased to 100 ul. The solution was transferred into a clean tube and purified using the MinElute PCR Purification Kit (Qiagen, Valencia, CA). The final book varied between 25 µl and 100 µl.

DNA quantification

The mitochondrial Dna content of each sample tested at the GMI was determined by quantitative real-time PCR following the protocol detailed in Niederstätter et al. [20]. Nuclear DNA was quantified using the human-specific AluYb8 assay described in Walker et al. [21], including an internal amplification positive control to test for the presence of PCR inhibitors in the DNA extracts.

Mitochondrial DNA assay

At the GMI, v overlapping "midi"-amplicons ranging from 282 to 444 bp [22] in two multiplex PCR assays and carefully selected "mini"-amplicons [23] were amplified and sequenced with the PCR primers to generate a consensus sequence displaying full double-strand sequence coverage of the mtDNA control region. Sequencing was performed according to Berger and Parson [22] and Eichmann and Parson [23].

The AFDIL casework group amplified mtDNA using a redundant amplification strategy described in Edson et al. [24]. Hypervariable Regions one (HVI), two (HVII), and three (HVIII) as well every bit mini-variable region ane (MVR1) were amplified for each sample as template availability and quality immune. The size of the amplicons varied from 126 to 440 bp. All post-PCR products, including controls, were purified using ExoSAP-IT™ (USB Co., Cleveland, OH) and the purified templates were sequenced with the Big Dye Terminator Bike sequencing kit (Applied Biosystems, Foster City, CA). Sequencing products were purified using Performa® DTR Ultra 96-well plates (Edge BioSystems, Gaithersburg, Md) and dried down in an evaporator/concentrator centrifuge. Formamide/EDTA (three∶one) was used to rehydrate the product prior to loading on an Applied Biosystems 3130twoscore Genetic Analyzer. The multiple sequences were aligned and compiled using the Sequencher software v4.7 (GeneCodes, Ann Arbor, Michigan). Differences from the revised Cambridge Reference Sequence [25], [26] were determined and used for mtDNA database searches.

Autosomal and Y-STR analysis

STR testing was conducted with several standard commercially available kits. The GMI and the AFDIL both used the AmpFlSTR Identifiler, AmpFlSTR MiniFiler, and AmpFlSTR Yfiler PCR amplification kits (Applied Biosystems). For the analysis of bone sample 147, GMI also used the AmpFlSTR SEFiler distension kit (Applied Biosystems). The GMI followed the protocols recommended by the manufacturer and besides tested AmpFlSTR Identifiler and AmpFlSTR Yfiler using 34 cycles. The AFDIL used a depression copy number approach [27] with the AmpFlSTR Identifiler and AmpFlSTR Yfiler PCR amplification kits and used twice the recommended AmpliTaq concentration and six additional PCR cycles [18], [28]. PCR distension with the AmpFlSTR MiniFiler kit at AFDIL was performed according to the manufacturer'south protocol. When necessary, both AFDIL and GMI used markers from an in-business firm miniSTR assay [29], [30]. All STR and Y-STR amplification products were analyzed on either a 3100 or a 3130xl Genetic Analyzer (Applied Biosystems). Analysis of the data was performed using GeneScan software v3.7 and Genotyper v3.7NT or Genemapper® v3.2. Fragment sizing was performed by means of an internal size standard (GeneScan-500 LIZ) and the amplicons were compared with the provided allelic ladder (AmpFlSTR Identifiler, AmpFlSTR MiniFiler and AmpFlSTR Yfiler allelic ladders) for unambiguous allele designation.

Data Analysis and Statistical Calculations

To preserve the independence of the testing performed at each laboratory; no data was transferred to either laboratory during the testing period. One time the testing was completed at AFDIL and the GMI, electropherograms of the information and tabulated results were sent to an independent scientist (PG) for confirmation and concordance of the data.

Nosotros evaluated the weight of the autosomal STR evidence using a likelihood ratio (LR) where two competing hypotheses are evaluated:

The likelihood approach above evaluates two competing scenarios. In the numerator, we evaluate the probability (Pr) of the Deoxyribonucleic acid Evidence (E) given the hypothesis (H₁) that the remains belong to the missing children of the Romanovs. In the denominator of the LR, nosotros evaluate the probability of the Deoxyribonucleic acid bear witness given the alternative (nil) hypothesis (H₂) that these remains were not from the missing Romanov children, but were derived from 2 randomly sampled, unrelated individuals. The LR for autosomal STRs was calculated using the software programme DNAView™ (Charles Brenner, Oakland, CA) through a customized interface developed for AFDIL called LISA (Laboratory Information Systems Application, FTI Inc., Fairfax, VA).

Supporting Information

Tabular array S1.

Quantification results of the samples tested at GMI. MtGE: mitochondrial genome equivalent. Samples 147 and iv.51 were both extracted twice independentely. The values shown in the table represent the Deoxyribonucleic acid concentration of each extract.

https://doi.org/10.1371/journal.pone.0004838.s004

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Acknowledgments

The authors would similar to thank Captain Peter Sarandinaki, of the Southward.E.A.R.C.H Foundation; Alexey Zacharin for linguistic support; Dr. Richard Scheithauer, (Manager, GMI), Bettina Zimmermann (GMI); Dr. John Butler, Becky Hill, and Margaret Kline of NIST for miniSTR primers; Dr. Florabel Mullick and Paul Stone (AFIP); Dr. Craig Mallak (AFMES); Jodi Irwin, Rebecca Simply, Melissa Scheible, Kim Sturk, Jessica Saunier and Toni Diegoli (AFDIL Inquiry Section); James Canik and Dr. Brion Smith (AFDIL Administration); Suzie Barritt-Ross, Demris Lee, and Shelley Johnson (AFDIL Casework); Elena Trynova, Tamara Tsitovich, Elena Vylegzhanina, Natalia Bandurenko, Dr. Vladimir Gromov, and Dr. Nikolay Nevolin of the Sverdlovsk Regional Forensic Bureau Laboratory (Yekaterinburg); Dr. Sergey Nikitin (Moscow); and Vladimir Soloviev, Senior Investigator from the Prosecutor Office of the Russian federation. The authors would like to thank the five reviewers for their helpful comments on this manuscript.

Disclaimer

The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or equally views of the U.S. Department of Defense, the U.S. Section of the Army, or the Military machine Establish of Pathology. Commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely every bit possible. In no case does such identification imply a recommendation or endorsement by the U.S. Department of Defense, the U.South. Department of the Regular army, or the Armed Forces Institute of Pathology nor does it imply that whatsoever of the materials, instruments or equipment identified are necessarily the best available for the purpose.

Writer Contributions

Conceived and designed the experiments: MDC OML MJW HN CB BB WP LNF. Performed the experiments: OML MJW HN CB BB SME KM CEM WP. Analyzed the data: MDC OML MJW HN CB BB SME KM CEM ABF PG WP. Wrote the newspaper: MDC OML ABF PG WP.

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