Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains
November 30th, 2021
The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).
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.
Overview
This protocol outlines best practice guidelines for collecting bone powder from various anatomical locations on medieval skeletal remains. It emphasizes methods for generating bone powder suitable for DNA extraction, which can also be applied to forensic remains.
Key Study Components
Area of Science
- Neuroscience
- Ancient DNA analysis
- Forensic science
Background
- Existing protocols primarily focus on dentin and petrous pyramid sampling.
- This study introduces alternative anatomical locations for bone powder collection.
- Protocols are designed for both ancient and degraded forensic specimens.
- Emphasis on maintaining a sterile environment during sampling.
Purpose of Study
- To provide detailed protocols for bone powder collection from medieval skeletal remains.
- To enhance DNA extraction methods from various skeletal elements.
- To establish a baseline for future techniques applicable to non-human specimens.
Methods Used
- Sampling performed in a clean room with UV light.
- Use of dental drills for precise bone powder collection.
- Collection from multiple anatomical locations to maximize DNA yield.
- Strict decontamination protocols between samples.
Main Results
- The Pars petrosa location yielded the highest endogenous DNA.
- Other anatomical locations also provided adequate DNA for analysis.
- Low duplication rates in libraries indicate high complexity of samples.
- Teeth and thoracic vertebrae showed good molecular recovery.
Conclusions
- Protocols established are effective for ancient DNA recovery.
- Methods can be adapted for forensic applications.
- Future research can build on these techniques for broader applications.
Frequently Asked Questions
Executive Industry Relevance
Ancient DNA sampling protocols provide a framework for maximizing molecular yield from limited, precious biological specimens while minimizing specimen destruction. This approach supports target validation in discovery research by enabling reliable molecular recovery from challenging sample types. The methodology enhances predictive confidence in downstream genetic analyses by ensuring adequate DNA quality for population genetics applications.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of biological systems through molecular analysis of preserved specimens.
- Operational Value: Reduces sample input requirements while maintaining adequate DNA yield for downstream applications.
- Strategic Value: Supports hypothesis testing in target validation by providing access to molecular data from rare or limited specimens.
Screening & Assay Development
- Scientific Value: Provides standardized biological samples suitable for assay development and optimization.
- Operational Value: Ensures reproducibility through strict decontamination protocols and controlled sampling environments.
- Strategic Value: Enables reliable compound evaluation by delivering consistent, high-quality DNA inputs for screening workflows.
Translational & Preclinical Research
- Scientific Value: Supports translational biomarker discovery by enabling molecular analysis across diverse specimen types.
- Operational Value: Facilitates continuity from discovery through preclinical validation with standardized sampling approaches.
- Strategic Value: Informs risk-adjusted advancement decisions by providing molecular data quality metrics.
Pipeline & Workflow Integration
The protocol integrates into discovery workflows by providing standardized methods for biological sample preparation that support hypothesis testing and molecular analysis.
- Discovery Biology: Supports hypothesis testing through reliable molecular recovery from anatomical sampling locations.
- Screening: Enables assay readiness by delivering standardized bone powder samples suitable for DNA extraction.
- Analytics: Provides quantitative DNA yield measurements that help teams compare sample quality and suitability for downstream applications.
- Translational Research: Connects discovery to preclinical continuity through consistent molecular recovery across specimen types.
- Enterprise Reuse: Establishes a reusable capability for biological sample preparation applicable across multiple research initiatives.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in molecular analyses through adequate DNA yield and quality.
- Operational Value: Standardization and reproducibility via strict decontamination and controlled sampling protocols.
- Strategic Value: Better go/no-go decisions through reliable molecular data generation from limited specimens.
- Portfolio Impact: Risk-adjusted prioritization based on molecular recovery potential from anatomical sampling locations.
Implementation Considerations
- Requires expertise in sterile technique and contamination control for ancient DNA work.
- Needs dental drilling equipment and UV-equipped clean room infrastructure.
- Demands cross-team standardization of decontamination protocols between samples.
- Requires adaptation considerations when applying to different specimen preservation states.
- Involves practical limitations related to specimen availability and anatomical accessibility.
