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Biological Sexing of a 4000-Year-Old Egyptian Mummy Head to Assess the Potential of Nuclear DNA Recovery From the Most Damaged and Limited Forensic Specimens

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Biological Sexing of a 4000-Year-Old Egyptian Mummy Head to Assess the Potential of Nuclear DNA Recovery From the Most Damaged and Limited Forensic Specimens

Odile Loreille et al. Genes (Basel).

Abstract

High throughput sequencing (HTS) has been used for a number of years in the field of paleogenomics to facilitate the recovery of small DNA fragments from ancient specimens. Recently, these techniques have also been applied in forensics, where they have been used for the recovery of mitochondrial DNA sequences from samples where traditional PCR-based assays fail because of the very short length of endogenous DNA molecules. Here, we describe the biological sexing of a ~4000-year-old Egyptian mummy using shotgun sequencing and two established methods of biological sex determination (RX and RY), by way of mitochondrial genome analysis as a means of sequence data authentication. This particular case of historical interest increases the potential utility of HTS techniques for forensic purposes by demonstrating that data from the more discriminatory nuclear genome can be recovered from the most damaged specimens, even in cases where mitochondrial DNA cannot be recovered with current PCR-based forensic technologies. Although additional work remains to be done before nuclear DNA recovered via these methods can be used routinely in operational casework for individual identification purposes, these results indicate substantial promise for the retrieval of probative individually identifying DNA data from the most limited and degraded forensic specimens.

Keywords: Egypt; ancient DNA; high throughput sequencing; hybridization capture; mitochondrial genome; mtGenome; mummy; sexing.

Conflict of interest statement

Names of commercial manufacturers are provided for identification purposes only and inclusion does not imply endorsement of the manufacturer, or its products or services by the FBI. The views expressed are those of the authors and do not necessarily reflect the official policy or position of the FBI or the U.S. Government. This is FBI Laboratory publication #18-13. The views and conclusions contained in this document are those of the author and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the DHS or S&T. In no event shall DHS, NBACC, S&T, or Battelle National Biodefense Institute have any responsibility or liability for any use, misuse, inability to use, or reliance upon the information contained herein. DHS does not endorse any products or commercial services mentioned in this publication.

Figures

Figure 1
Figure 1
Head of the Djehutynakht mummy (2010–1961 BC). Photograph © 2018 Museum of Fine Arts, Boston, USA.
Figure 2
Figure 2
Sequencing strategy in this study. Molecular work performed at the Federal Bureau of Investigation (FBI) Laboratory is in blue, while work performed at Harvard Medical School (HMS) is in pink. Cap1-Lib2 has both colors because the extraction and library preparation of Lib2 was done at the HMS, while the hybridization capture took place at the FBI. CAP-LIB1 is the result of merging data from runs I and II, while CAP-LIB2 is the result of merging runs III and IV. Lib1 and Lib2 were shotgun-sequenced together on the HiSeq at National Bioforensic Analysis Center (NBFAC, run V).
Figure 3
Figure 3
(a) Size distribution of the reads that aligned to the mtGenome in CAP-LIB1. Reads ≤35 bp or >70 bp (shown in orange) were removed from the final data to avoid any impact from nonspecific [34] or contaminating reads. The length with the greatest number of reads was 47 bp. (b) Size distribution of the reads that aligned to the mtGenome in CAP-LIB2. Only reads >35bp and ≤70 bp (shown in blue) were retained in the final data. The length with the greatest number of reads was 38 bp.
Figure 4
Figure 4
Distribution of 40,910 reads over the entire mtGenome. Sequence coverage at each position ranged from 5× to 247× (average 108×).

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