In order to carry out intra-sample comparisons, we identified the three distinct areas for DNA extraction (Fig 1):

part A: bone at the apex of the petrous pyramid, which is largely trabecular (spongy).

part B: dense white bone, most commonly found surrounding the inner ear; depending on the preservation of the sample and natural variability (see S2 Fig) it can exist also in the area between the semi-circular canals, the outer ear, and the mastoid process.

part C: dense bone of the otic capsule (inner ear) which consists of the cochlea, vestibule, and three semi-circular canals, it surrounds the membranous osseous labyrinth and houses the organs of hearing and equilibrium in living organisms. In contrast to the whitish part B, it is of a yellowish-to-green range of hues.

While isolation and identification of part A is easily achieved due to the obvious porosity of the trabecular bone, separation of parts B and C requires precise work, since the inner ear (part C) is normally encapsuled in the dense white bone (part B). To isolate these parts, we combined the use of a Dremel disk saw and a sandblaster (Renfert Classic Basic). The latter allows for precise separation of the bone by controlling the output pressure, which in turn greatly helps in the identification of the inner ear (C) part. In attempting to identify part C, it is often easiest to first locate the superior semicircular canal before any sample processing occurs, which is easily identifiable on the unprocessed petrous bone by the arcuate eminence on the superior aspect of the bone.
In order to conduct intra-petrous comparisons on our archaeological samples, we first identified and isolated part A, and removed it from the rest of the petrous bone located in a UV cabinet. We then removed the dense white bone (part B) surrounding the otic capsule (part C) and then proceeded into clearing it of the remaining surrounding white bone (S1 and S2 Figs). All three parts were transferred to individual sample boats and put inside a UV chamber individually where they were decontaminated for 10 minutes on each side. Each part was then ground to very fine powder (~5 μm) using a mixer mill (Retsch MM400) and aliquots of 150 mg were recovered to proceed with DNA extraction. To minimize modern contamination, all these steps were done in a dedicated lab for preparation of ancient bone samples, with the researchers using full cover suits, double gloves, hair nets and face masks. All non-disposal equipment and work surfaces were cleaned and decontaminated with DNA-ExitusPlus and ethanol throughout the sample preparation process, and then subjected to UV radiation for at least 30 minutes.
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