Table 3.

Methodical approaches specific to analyses of degraded DNA from historical specimens [45]

Stage of analysis	Special procedures	Reagents and methods
Preparation of historic samples	Independent analysis in specialized laboratories in IOGene (Moscow) and University of Massachusetts Medical School (Worchester, USA).	Physical and chemical cleaning of small bone fragments, crushing or drilling to obtain bone powder.
Extraction of DNA from bone remains	All the experimental procedures were performed in sterile PCR-hoods, in accordance with standards for ancient DNA research, keeping to all the safety precautions so as not to contaminate the samples by modern DNA.	DNA was extracted from ~170≤700 mg of cleaned bone material treated by 0.5 M EDTA and proteinase K and was then purified by a QIAquick PCR purification kit (Qiagen) in accordance with the manufacturer's protocol with slight modifications.
Extraction of DNA from archive spots of blood	The biological material was obtained from 4 different blood stains. At least 3 swabs were taken from each spot. In order to minimize contamination, DNA was extracted only from the 2nd and 3rd swabs of each spot.	DNA was extracted with the QIAamp DNA Mini Kit (Qiagen) in accordance with the manufacturer's protocol ("DNA Purification from Dried Blood Spots") with several modifications.
Quantative DNA analysis		The total DNA was quantified by the Quant-iT™ PicoGreen® Assay kit (Invitrogen), human specific DNA was quantified by the Plexor® HY assay kit (Promega) and the 7500 Real-Time PCR System (Applied Biosystems).
Sequencing HVR1 and HVR2 of mtDNA from historic samples	Possible contamination by foreign DNA was monitored by using negative controls (amplification of "empty" extracts and PCR without addition of the template).	mtDNA fragments were amplified as short overlapping fragments. The PCR products were then extracted from the agarose gel using a QIAquick Gel Extraction kit or a MinElute Gel Extraction kit. For additional studies, the PCR products of samples from the second burial site were cloned.
Sequence analysis of the complete mitochondrial genome, extracted from bone remains.	Since the DNA was highly degraded, short overlapping fragments sized 164-383 b.p. were obtained, covering the whole mitochondrial genome.	Because the amount of DNA was initially so small, multiplex amplification was performed using 88 pairs of specially developed primers grouped into 3 kits, and then the products of this PCR were amplified with individual primer pairs. The PCR products were then sequenced using three different strategies
Analysis of the mtDNA extracted from the blood stains on Nicholas the Second's shirt.	Up to 5 or 7 repeated PCR reactions were conducted for some of the SNPs.	Since the quality of preservation in the blood stains was unknown, a kit of primers was developed for the amplification of short (64≤109 b.p.) DNA fragments, which would include very rare SNPs identified in the previous analysis of Skeleton №4 (the putative skeleton of Nicholas the Second).
Extraction and analysis of DNA from modern samples.	All the procedures involved in analyzing modern DNA were performed in other buildings, which were located some distance away from the ancient DNA laboratories. All the living relatives who took part in the study gave their written consent.	DNA obtained from buccal swabs or drops of blood was extracted using standard protocols. PCR was performed using a kit of primers for amplifying longer fragments.
Assembly of fragments into a continuous sequence of nucleotides (contigs).		The sequences were assembled using Seqman software, DNASTAR, and the revised Cambridge reference sequence (rCRS, accession number AC_000021) as a standard.
Sex identification.		Sex was identified using the standard method, amplification of a fragment of the amelogenin gene using several commercial kits: AmpF≤STR® MiniFiler™ (Applied Biosystems) and PowerPlex S5 (Promega). Specially developed primers for the amplification of short fragments specific to the X- and Y-chromosomes were also used.
Analysis of nuclear STR markers.	During the initial study, mtDNA or nuclear DNA extracts that consisted of a mix of individual profiles were discarded from further analysis. Each sample from various extracts was serially amplified. Homozygous loci were considered authentic if multiple independent amplifications confirmed a certain allele for the autosome STR-marker.	The following kits were used in order to obtain autosomal STR profiles from bone samples of the first and second burial sites: AmpF≤STR® MiniFiler™ PCR Amplification Kit (Applied Biosystems) and PowerPlex S5 System (Promega), developed especially for analyzing degraded DNA.
STR-profiles of the Y chromosome.		The AmpF≤STR® Yfiler™ (Applied Biosystems) kit was used, according to the manufacturer's protocol with slight modifications for work with degraded DNA.
Electrophoresis analysis	In order to increase the signal intensity and lower "noise" in the STR-profiles, the products of multiplex amplification were sometimes purified according to the method for genotyping low-copy DNA templates using Qiagen MiniElute PCR purification kit.	Electrophoretic analysis was performed with a 96-capillary sequencer 3730xl DNA Analyzer (Applied Biosystems). The results were analyzed using GeneMapper ® ID software v3.2 (Applied Biosystems).