Provenance and ethics
The human remains were excavated from the Nuwayrat necropolis near Beni Hasan, Egypt. They were donated between 1902 and 1904 by the Egyptian Antiquities Service to the members of the Beni Hasan excavation committee and subsequently donated to the Institute of Archaeology, University of Liverpool and exported under the John Garstang export permit. The human remains were then donated to the World Museum (previously the Liverpool City Museum) in 1950. Sampling permit was granted by the World Museum.
Ancient DNA extraction, library preparation and sequencing
Sampling and DNA extraction of seven permanent teeth belonging to an individual from Nuwayrat were carried out in dedicated ancient DNA facilities at Liverpool John Moores University. Library preparation and sequencing were carried out at The Francis Crick Institute (Supplementary Table 1). Before subsampling, the teeth were decontaminated by wiping with 1% sodium hypochlorite, followed by wiping with molecular biology grade water and ethanol. Approximately 44–66 mg of cementum-enriched powder was extracted from each tooth using a Dremel drill at the lowest possible rotations per minute (5,000 rpm).
DNA was extracted using 1 ml of extraction buffer consisting of 0.45 ml of 0.5 M EDTA (pH 8.0) and 10 μl of 10 mg ml−1 proteinase K per 50 mg of bone powder. The mixture was incubated overnight (approximately 18 h) at 37 °C and purified on the High Pure Viral Nucleic Acid Large Volume Kit (Roche) using a binding buffer described in ref. 63 and QIAGEN buffer PE. DNA was eluted in approximately 100 μl of QIAGEN elution buffer.
Extracts were turned into single-stranded DNA libraries39 (without treatment to remove uracils), double-indexed64 and then underwent paired-end sequencing on a HiSeq 4000 to approximately seven million reads per library for initial screening (Supplementary Table 1). All samples were processed alongside negative lysate and extraction controls and positive and negative library controls. On the basis of the assessment of the initial sequencing results, two libraries were selected for extra rounds of deeper sequencing on the NovaSeq 6000 and NovaSeq X platforms, following the selection of fragments greater than 35 bp using polyacrylamide gel electrophoresis65 for the library built from the NUE001b5e1 extract (Supplementary Table 1), with a resulting total of 8.3 billion 2 × 100 sequence pairs.
Radiocarbon dating
New radiocarbon dating was generated for the individual that yielded DNA from Nuwayrat (NUE001) by Beta Analytic using accelerator mass spectrometry. We directly dated the upper-left third molar (NUE001b3) and lower-left first premolar (NUE001b5), both of which yielded DNA that was deep sequenced (Supplementary Table 1). The results are reported in Supplementary Table 2. The femur of this individual was previously radiocarbon dated66,67 (Supplementary Information section 1 and Supplementary Table 2). All dates were calibrated using OxCal v.4.4.4 (ref. 68) with atmospheric data in IntCal20 (ref. 69). We also combined the three independent dates using the R_Combine() function in OxCal70 (Supplementary Table 2). We rounded the calibrated dates outwards to 10 years unless error terms were smaller than ±25 bp, in which case we rounded outwards to 5 years71.
Isotope analysis
Dental collagen and enamel were extracted from the lower-left second molar. Dentine collagen was extracted for carbon (δ13C) and nitrogen (δ15N) isotope analysis following a modified Longin method72,73. Mass spectrometry was performed using a Flash 1112 series elemental analyser coupled with a Finnigan DELTA V Advantage (Thermo Fisher Scientific) using established protocols74. Analytical precision (1σ) of the in-house calibrated standards74 were 0.08 and 0.07 for δ13C and δ15N, respectively.
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