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

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.

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

“…sequencing error, PCR error, DNA damage, pseudogenes) could be consistently distinguished from authentic signal. We have employed methods for doing exactly that in other NGS studies [23,56,57]. However, these analyses have been largely manual, which is not suitable to a production environment.…”

Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

“…The first genetic marker analyzed in human paleogenetic studies was mitochondrial DNA (mtDNA) because of its higher copy number in the cell than nuclear DNA. Probe hybridization assays used biotinylated DNA or RNA probes targeting the two hypervariable segments of the mtDNA control region (CR) ( Briggs et al, 2009 ; Krause et al, 2010 ; Maricic et al, 2010 ; Enk et al, 2013 ; Kihana et al, 2013 ; Templeton et al, 2013 ; Eduardoff et al, 2017 ; Loreille et al, 2018 ). Another uniparental marker, the Y-chromosome DNA (Y-DNA), was also used to study aDNA.…”

Sex estimation using sexually dimorphic amelogenin protein fragments in human enamel

“…An alternative method of sex estimation involves the detection of DNA markers from the X-and Y-chromosome (Loreille et al, 2018;Madel et al, 2016;Salido et al, 1992;Skoglund et al, 2013). Historically the most prominent methods rely on the detection of the amelogenin gene family, which has isoforms on both the X-and Y-chromosome (AMELX and AMELY) (Ballantyne, 2013;Garvin et al, 2012;Madel et al, 2016).…”

“…Historically the most prominent methods rely on the detection of the amelogenin gene family, which has isoforms on both the X-and Y-chromosome (AMELX and AMELY) (Ballantyne, 2013;Garvin et al, 2012;Madel et al, 2016). Other methods measure the relative number of Xand Y-chromosome reads from massively parallel sequencing runs of DNA retained in samples (Loreille et al, 2018;Mittnik et al, 2016;Skoglund et al, 2013). Detection of these markers therefore can accurately infer the karyotype of sex chromosomes and can be applied in even highly compromised samples (Loreille et al, 2018;Mittnik et al, 2016).…”

“…Other methods measure the relative number of Xand Y-chromosome reads from massively parallel sequencing runs of DNA retained in samples (Loreille et al, 2018;Mittnik et al, 2016;Skoglund et al, 2013). Detection of these markers therefore can accurately infer the karyotype of sex chromosomes and can be applied in even highly compromised samples (Loreille et al, 2018;Mittnik et al, 2016). They are predicated however on the presence of at least small amounts of endogenous DNA template, which may be missing due to degradation of the relatively fragile DNA molecule (Allentoft et al, 2012;Lindahl, 1996;Ottoni et al, 2017).…”