Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

These protocols serve as best practice guidelines for bone powder generation for use in the downstream DNA extraction across a range of skeletal elements. There are a few published ancient DNA sampling protocols for locations other than the dentin and the petrous pyramid. Today, we'll show the detailed sampling methods for three alternatives.

These methods also show great potential for use with degraded forensic remains, but also as a baseline for developing similar techniques with non-human specimens. Perform all sampling in a dedicated clean room, under a UV light-equipped PCR hood or biosafety cabinet. Turn off the airflow and spread sterile aluminum foil across the bench top to catch any stray bone powder or fragments.

Place a sheet of weigh paper into a sterile weighing tray. Hold the decontaminated molar by the enamel with the root facing down over a weighing tray, using a handheld clamp. Equip a dental drill with a diamond-edged circular cutting wheel.

With the drill set to a medium speed setting, lightly touch the edge of the drill to the root at an angle of approximately 20 degrees. Scrape the sample downward into the tray to collect the yellow, outermost material from the root. Stop collection once the lighter material of the dentin becomes visible.

Transfer the powder to a two milliliter LoBind Safe-Lock tube, and record this collected mass of powder using an enclosed balance with an accuracy of at least 0.01 milligram. Transfer the powder from the weigh paper to a two milliliter LoBind Safe-Lock tube for extraction. Store this tube at 20 degrees Celsius.

Ensure to recover all the bone fragments and as much powder as possible. Change the foil between each sampling, disposing of the used foil in an autoclaveable biohazard bag. Decontaminate the working area and spread sterile aluminum foil across the bench top every time before moving to the next bone.

Place a small sheet of weigh paper into a standard weighing tray. Remove and discard the outermost layer of the cortical bone of the superior vertebral arch using a dental drill. Then, secure the vertebrae in a hand clamp, with the spinous process outward and superior aspect down.

While holding the vertebra, drill upwards into the center of the V-shaped notch, formed by the fusion of the spinous process with the lamellae with low speed and high torque. Cease drilling when there is a noticeable drop in resistance. Change the drilling position slightly, and repeat until 50 to 100 milligrams of bone powder is collected.

Transfer the bone powder from the weigh paper to a two milliliter LoBind tube for extraction, and store it at 20 degrees Celsius. Ensure all bone fragments and as much powder as possible is recovered before disposing of a foil. Decontaminate the working area and prepare for sample collection, as previously described.

Place a small sheet of weighing paper into a standard weighing tray. Hold the talus dome upward, and medial surface toward the collector, over the weighing tray. Scrape cortical bone to a depth of approximately one millimeter from the neck of the talus using a dental drill with a low-gauge bit set to low speed and high torque.

Change the drilling position and repeat this until approximately 50 to 100 milligrams of bone powder is collected. Transfer this bone powder from the weigh paper to a two milliliter LoBind tube for extraction. Store the powder at 20 degrees Celsius indefinitely.

Store the remaining bone and excess powder in a dry, temperature-controlled sterile environment at 25 degrees Celsius. Dispose of all waste in autoclave biohazard bags or receptacles. Sterilize or decontaminate all reusable equipment and expose it to UV between each sampling.

The Pars petrosa yielded higher endogenous DNA than the other 23 anatomical sampling locations surveyed. The seven additional anatomical sampling locations presented in this protocol produced the next highest yields. The alternative locations consistently produced DNA yields adequate for standard population genetics analyses.

Duplication rates in libraries stemming from all anatomical sampling locations were low, indicating high complexity. The teeth, as well as thoracic vertebrae showed adequate molecular recovery. One of the most challenging aspects of bone sampling is the drilling process, such as how to hold a drill, where to drill, and when to stop drilling.