1. Introduction

On 18 October 2009, the Museum of Fine Arts, Boston (MFA) opened an exhibition called The Secrets of Tomb 10A: Egypt 2000 BC . One of the most intriguing items in the collection was a mummified human head discovered over a century ago in the necropolis of Deir el-Bersha (also known as Dayr al-Barshā). The site is located on the east bank of the Nile River in close proximity to the city of Mallawi, approximately 250 km south of Cairo. Deir el-Bersha is known for tombs cut into cliffs of limestone that date back to the Old Kingdom (2686–2181 BC), the First Intermediate Period (about 2100–2040 BC) and the Middle Kingdom (MK; 2040–approx. 1640 BC). During the MK 11th and 12th Dynasties (2040–1783 BC), it served as the chief cemetery for the governors or regional lords (a.k.a. nomarchs) of the 15th Upper Egyptian Nome (a.k.a. the Hare Nome).

2, In 1915, Deir el-Bersha was excavated by a joint MFA-Harvard University team directed by George A. Reisner with the assistance of Hanford L. Story and Said Ahmed Said [ 1 ]. On 23 April, Reisner’s men began clearing the burial shaft from tomb number 10A. Six days later, at the bottom of a 30-foot pit, they discovered the burial chamber of an early MK governor named Djehutynakht. It is not yet clear whether he is Djehutynakht IV, son of Ahanakht I, or Djehutynakht V, son of Nehri I [ 1 3 ]. Both were nomarchs of the Hare Nome, and while they shared the same name (which means Thoth [the main local deity] is Strong), there is no evidence they were related. Tomb 10A contained a second occupant: the governor’s wife, who was also called Djehutynakht. Although the tomb had been plundered in antiquity and most of the valuable jewels stolen, many objects were left behind. In fact, the recovered contents of Tomb 10A are considered one of the largest burial assemblages of the MK ever discovered. The funerary equipment includes pottery, canopic jars, models showing men and women in different daily life activities, nearly 60 model boats, and a famous, exquisitely carved and painted processional group composed of a priest and four offering bearers, known as the “Bersha procession” [ 4 ]. Befitting their high status, the governor and his wife were both buried in finely decorated rectangular wooden coffins placed within larger coffins, all made of thick cedar of Lebanon boards. Upon discovery, the coffins of the governor were nearly intact with the exception of the head end that had been removed by tomb robbers. The intricate carvings and paintings on the governor’s outer coffin make it an unparalleled masterpiece of MK art (see pictures in [ 5 ]).

When the tomb was looted, the mummies of the governor and his wife were damaged by thieves in search of fine jewelry. A torso, originally attributed to Lady Djehutynakht, was found in the far corner of the burial chamber, but was recently argued to belong to the governor after re-excavation of Tomb 10A [ 6 ]. A mummified head, which could neither be attributed to the governor nor to his wife, was found atop the governor’s coffin ( Figure 1 ).

In an effort to learn more, the head was analyzed with computerized tomography (CT) in 2005. The CT scanning of the head revealed extensive bilateral post-mortem alterations of the facial bones [ 7 ]. The absence of these bones, together with the lack of comparative data on ancient Egyptian skulls, preclude definitive morphological sex determination; however, the presence of large mastoid processes, robust occipital and temporal regions, and pronounced gonial flaring of the mandible, suggest that the skull more likely belonged to a man [ 8 ], ( Figure S1 ). In order to unequivocally determine the biological sex of the individual, the MFA collaborated with the Federal Bureau of Investigation (FBI) Laboratory to perform DNA analysis.

At the time the FBI was contacted, the ancient DNA community had largely given up on the testing of ancient Egyptian human remains. Though DNA extraction and amplification from ancient Egyptian samples had been attempted in the early days of paleogenetics, these initial attempts either resulted in failure (4500-year-old human femurs [ 9 ]) or yielded data that turned out to be the product of modern DNA contamination (2400-year-old mummy [ 10 ] and 1600-year-old sacred monkey bones from the Saqqara Baboon Galleries [ 11 ]), both contaminated with modern human DNA). These early failures prompted studies on DNA survival [ 9 12 ]. Together with older research showing that DNA degradation (depurination in particular) is primarily influenced by temperature, pH, oxygen, and water [ 13 ], these analyses suggested that Egyptian environmental conditions likely cause DNA to degrade to fragments smaller than 100 base pairs (bp) in just a few hundred years [ 14 15 ].

Currently, forensic DNA testing at the FBI, as well as in nearly all global operational laboratories, is based on targeted PCR amplification of fragments ranging from 90–1200 bp in size, followed by size-based capillary electrophoresis for short tandem repeats (STRs) or Sanger sequencing for mitochondrial DNA (mtDNA). These approaches have served the forensic community well over the past twenty years. They present limitations, however, to both the quality and quantity of genetic data that can be recovered from the most challenging specimens. Recently, a number of commercial high throughput sequencing (HTS) assays, designed specifically for forensic applications, have become available [ 16 17 ]. While these assays overcome many of the limitations of traditional capillary electrophoresis-based forensic DNA analyses, they are still based on targeted amplification of defined genomic regions. As a result, their utility is limited to samples harboring DNA fragments large enough for PCR amplification.

One of the primary advantages of HTS is that DNA fragments of very short size can be recovered and sequenced, thus obviating the need for targeted PCR. Here, we exploit this feature and describe the use of shotgun sequencing to determine the biological sex of a 4000-year-old Egyptian mummy.