The Russian government, which invited Coble to lead an independent investigation of the remains in 2007, gave him and his colleagues access to 44 bone fragments from the tzar and his family for comparison.
Coble and colleagues first tested mitochondrial DNA—which is passed down through mothers—and found a match between the children's remains, those of the Tzarina Alexandra, and a living maternal relative of Prince Philip, Duke of Edinburgh.
Next, a "large-scale paternity test" of DNA inherited from each parent showed that 15 genetic markers were consistent with samples from the remains, said Coble, whose research is described in the journal PLoS One.
Lastly, DNA from a living relative and distant cousin, Andrew Romanov, was found to share 17 Y-chromosome markers with the male child.
The lab's data confirms previous DNA research of the Romanov remains published in Proceedings of the National Academy of Sciences earlier this year by Evgeny Rogaev of the University of Massachusetts Medical School.
Rogaev and colleagues had compared a bloodstain taken from the tzar's shirt during an 1891 assassination attempt with samples from living descendants and bone fragments from the boy's remains. They all matched.
End of a Sad Chapter
For retired U.S. sea captain Peter Sarandinaki, the findings have fulfilled his quest to "end a very sad chapter in Russian history."
Sarandinaki, whose great-grandfather was a lieutenant general in the Tzarist army in the early 1900s, founded the SEARCH Foundation to find the children's remains.
"I am excited and relieved that the two missing remains were found and identified as those of Alexei Romanov [and one of his sisters]," he said.
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