TITLE: Multi-Proxy Sampling Protocol for Teeth from Archaeological Collections
AUTHORS: Sarah Defant, Pavlína Ingrová, Denisa Zlámalová, Arkadiusz Sołtysiak, Zuzana Hofmanová

[DESCRIPTION]
This protocol introduces a streamlined approach for concurrent sampling for endogenous and microbial ancient DNA (aDNA) and stable isotopes (particularly, but not exclusively, enamel sampling for strontium isotope analysis) from a single archaeological tooth. By combining sampling strategies into a single workflow, the protocol eliminates the risk of inadvertently sampling different individuals. It reduces the need for multiple sampling rounds, optimises the use of each sample, and eliminates the need to collect multiple samples for different analyses. Additionally, it minimises logistical complexity by reducing shipments between institutions and the risk of sample loss during transit. 


[BEFORE_START]
This Protocol is destructive! Every element must undergo thorough documentation prior to sampling, ideally via CT-scans or 3D modelling, but at a minimum through high-resolution photography.

This protocol requires clean-room conditions.

Make sure all equipment and work surfaces have been thoroughly cleaned before starting: 
For all tools and work surfaces used for extracting powder for aDNA analysis: Clean thoroughly with diluted commercial bleach and/or DNA Exitus Plus, followed by UV light exposure.
For all tools used in enamel powder extraction: Clean thoroughly with alcohol, and wipe and/or brush down before use.


 
 


[GUIDELINES]
This Protocol is destructive. 

Every element must undergo thorough documentation prior to sampling, ideally via CT-scans or 3D modelling, but at a minimum through high-resolution photography.

This protocol requires clean-room conditions.

Stated times refer to the standard procedures in the aDNA laboratory at Masaryk University Brno, Czechia. 


[STEPS]
SECTION: Decontamination &amp; Pre-Sampling
1. General Remarks

 


 

SECTION: Decontamination &amp; Pre-Sampling
1.2. 1. All new samples in their original (sample) bags must be cleaned on all sides using diluted bleach and then UV-irradiated for30 min before further use. Be careful when cleaning the bags to avoid removing any sample information/markings on the sample bag. If necessary, document the sample bag (e.g., by photographing it) before decontamination to preserve important information.

2. In the meantime, prepare new, clean, correctly labelled plastic bags and A4 sheets of paper (one per sample). Each sheet should be clearly labelled with the sample ID written top centre.


 

3. Once the new samples have been decontaminated, each sample must be photographed as follows:

Position the tooth (removed from the decontaminated original plastic bag) on the pre-prepared A4 sheet with the sample ID written at the top. Use forceps/pliers or a piece of aluminium foil to handle the tooth. 
Place a scale/ruler directly below the tooth.
Add the original sample label or the original sample bag beneath the scale/ruler to ensure that both the original grave ID and the newly given sample ID are visible in the photograph.
Take one photo showing all components (new sample ID, tooth, scale/ruler, original sample label/sample bag).
Take a second, close-up photograph that includes only the new sample ID, the tooth, and the scale/ruler (excluding the original label or bag).
Rotate the tooth 180° along its vertical axis using forceps/pliers or a piece of aluminium foil, and repeat for this side. 

 

Once the tooth has been photographed from both sides, place it into one of the newly prepared, labelled plastic bags using forceps/pliers or a piece of aluminium foil. 
Dispose of the A4 sheet with the sample ID, the old sample bag (including any old labels), and the aluminium foil. If forceps/pliers were used, these must be decontaminated before handling the next sample. 

 
SECTION: Decontamination &amp; Pre-Sampling
1.4. New bags with the samples inside must be cleaned again with diluted bleach on all sides and UV-irradiated for30 min. 
 
SECTION: Preparation of Sample
2.1. 
 

1. Place a layer of paper towels in the UV chamber. 

2. Prepare small sheets of aluminium foil and place them in the UV chamber.

3. Change gloves.

4. Place the tooth on the aluminium foil and place the labelled bag next to it. 

 

4. UV-irradiate each tooth for 15 min - change gloves before turning the tooth!

5.  After the tooth has been UV-irradiated from both/all sides, change gloves and place each tooth back into its respective bag. 

6. All individual bags should then be placed into a collective bag labelled with the site code. Additionally, the collective bag should be labelled to indicate whether the samples and bags have undergone UV-radiation (e.g. <Site Code>, UV samples ‱ and UV bags ‱)

7. If sampling cannot be undertaken immediately, place the bag with the samples into a decontaminated, sealed container. 

The tooth is now ready for sampling. 

8. Dispose of all paper towels and aluminium foil, change gloves, and decontaminate the UV chamber with diluted bleach. 
SECTION: Sampling - Preparation of Workstation / Set up
3. Required Materials 

Precision scale (digital weighing scale)
Drill handpiece with adjustable speed 
Diamond saw wheel
Round Carbide bur 
Round Nose Dental Pliers 
Mandrel/Adapter designed to hold small dental burs (1.6mm diameter) in a handpiece (2.35mm) 
Round diamond coated dental bur
Cylindrical diamond coated dental bur 
Toothbrush
2 larger tubs/plastic containers
Magnifying lamp (to clean enamel pieces)
Diluted commercial bleach solution (1:10) (diluted with UV-irradiated Reverse Osmosis water) 
UV-treated Reverse Osmosis Water (for aDNA sampling)
Deionised Water (for isotope sampling)
DNA Exitus Plus
Alcohol (Isopropyl, Ethanol or Methanol) 
3(-4) labelled 2.0ml Safe-Lock Tubes
10-20ml glass vials with lid
Labels (optional)
Weighing üaper (folded/creased in the middle)
Paper towels
Paper
Waterproof pen (for labelling the tubes)
Pen (for recording the weights)

Not all materials are needed in the working area at once, but they should remain accessible to help streamline the process. 

 

SECTION: Sampling - Calculus (if present)
4. This part of the protocol (Section 4) is based on the procedure outlined in: 
 

If calculus is present, prepare an additional labelled Safe-Lock tube and a second piece of weighing paper, folded in half. 

1. Tare the labelled Safe-Lock tube on the weighing scale (i.e. zero out its weight).

2. Carefully remove any visible calculus deposits using a dental tool such as a sickle scaler. Collect the material directly onto the weighing paper. 

 

3. Transfer the removed calculus to the Safe-Lock tube, weigh it, remember the weight and place it aside. 

4. Place the tools in a tub with commercial bleach solution (1:10).

5. Dispose of the weighing paper. 

6. Change gloves and immediately continue with sampling the pulp chamber: Section 5. 
SECTION: Sampling - Pulp Chamber
5. Cleaning

1. Manually remove any dirt deposits on the tooth using, for example, a periodontal scaler or a dental sickle probe.

2. Wipe the tooth with a small amount of diluted commercial bleach solution (1:10). 
(Note: This step may vary depending on lab protocols. Consult lab guidelines before proceeding!)

3. Wipe the tooth with a small amount of UV-treated Reverse Osmosis water. 
SECTION: Sampling - Enamel
6. 

 


Depending on the state of preservation of the crown after the extraction of powder for aDNA analysis, two different protocols can be followed:
SECTION: Sampling - Enamel
6.1. Cleaning of Sample & Preparations

1. If not yet done, place the tooth crown into a 10-20ml sealable glass vial filled with deionised water.

 


 


2. Place the vial(s) in an ultrasonic bath and sonicate until any adhering soil is removed and the water runs clear. Multiple water changes will be necessary during this process (always use deionised water). 

 

3. While the piece is sonicating, clean the dental drill, workstation and the weighing scale with alcohol (Isopropyl, Ethanol or Methanol) to remove any loose dust and dirt particles. 

4. Prepare a small vial with alcohol where tools can be placed after use.  

5. Label a Safe-Lock tube

6. Place a paper towel on the work station.

7. Place a folded weighing paper outside the workstation, within easy reach. 

8. Remove the enamel piece from glass vial, wipe it gently with a small amount of alcohol, and place it on the prepared work surface. 


 


SECTION: Sampling - Enamel
6.1. 


 



Cleaning of Sample & Preparations

1. If not yet done, place the enamel piece/crown fragment into a 10-20ml sealable glass vial filled with deionised water.


 


2. Place the vial(s) in an ultrasonic bath and sonicate until any adhering soil is removed and the water runs clear. Multiple water changes will be necessary during this process (always use deionised water). 

 

3. While the piece is sonicating, clean the dental drill, workstation and the weighing scale with alcohol (Isopropyl, Ethanol or Methanol) to remove any loose dust and dirt particles. 

4. Label a Safe-Lock tube.

5. Place a piece of aluminium foil or a paper towel on the workstation.

6. Place a folded weighing paper outside the workstation, within easy reach. 

7. Remove the enamel piece from glass vial, wipe it gently with a small amount of alcohol, and place it on the prepared work surface. 


 



SECTION: Sampling - Enamel
6.2.    Sample Procedure


 

1. Attach a diamond bur to the dental drill and wipe down the drill the drill with alcohol again. 

2. Abrade the surface, removing approximately the top 0.01 mm (potentially diagenetically altered surface enamel) and any visible surface discolouration. 

3. Remove any traces of dentine from the inner surface. This should ideally be done under a magnifying lamp.  
 

 

4. Wipe down the enamel piece with alcohol

5. Place the enamel piece on weighing paper and weigh it (minimum 10 mg ). Be sure to tare (zero out) the weight of the weighing paper first.           

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               
6. Place the enamel piece in the Safe-Lock tube and record the weight both on the tube and on a separate piece of paper.
SECTION: Sampling - Enamel
6.2. Surface Preparation/Cleaning

1. Attach a small dental diamond bur to the dental drill and wipe down the drill with alcohol again. 

2. Abrade the surface where you want to sample powder from, ensuring a slightly larger surface area is cleaned. Remove approximately the top 0.01 mm (potentially diagenetically altered surface enamel) and any visible surface discolouration. 


 


 

3. Place the crown aside. 

4. Remove the diamond bur and place into a vial with alcohol outside of the workbox. 

5. Carefully dispose of the paper towel.

6. Clean the dental drill and work surface with alcohol to remove any remaining particles.  

SECTION: Cleaning &amp; Decontamination
7. 
 

SECTION: Post-Sampling
8. Check that all tubes are properly labelled with the following information:
Sample ID
Date
Initials of Person who sampled
Weight

 


Check that all the samples are correctly documented/recorded on a sheet of paper that can be taken outside of the lab. 

Separate the main bone/tooth powder sample from the backup powder.

Place all main samples in a labelled, decontaminated plastic bag with the Site/Location and the Date of Sampling written on it. 

Place each back-up sample in an individual decontaminated plastic bag, and place all these plastic bags in another decontaminated plastic bag, clearly labelling it with Site/Location, Date, and "Back-Up". 

Store samples in a light-protected box in a cool, dry place until further processing (e.g., inside a fridge).
SECTION: Cleaning &amp; Decontamination
7. 
 

SECTION: Sampling - Enamel
6.4. Sampling Procedure

1. Place a piece of aluminium foil on the work surface and position the weighing paper in the middle of it.

2. Attach a different small dental diamond bur to the dental drill. 

3. Wipe down the crown with a small amount of alcohol to remove any loose particles, then place it on the work surface. 

4. Hold the tooth near the centre of the weighing paper and carefully remove enamel powder from the entire surface/growth axis, from the cusp to the CEJ, ensuring you are working perpendicular to the crown. 


 

 

 

5. Collect 10-20 mg  of enamel powder (no less than 10mg!). If there is more, keep it as a backup. 

 

6. Pour the powder into the Safe-Lock tube and record the weight. 

 

SECTION: Post-Sampling
8. Check that all tubes are properly labelled with the following information:
Sample ID
Date
Initials of Person who sampled
Weight

 


Check that all the samples are correctly documented/recorded on a sheet of paper that can be taken outside of the lab. 

Separate the main bone/tooth powder sample from the backup powder.

Place all main samples in a labelled, decontaminated plastic bag with the Site/Location and the Date of Sampling written on it. 

Place each back-up sample in an individual decontaminated plastic bag, and place all these plastic bags in another decontaminated plastic bag, clearly labelling it with Site/Location, Date, and "Back-Up". 

Store samples in a light-protected box in a cool, dry place until further processing (e.g., inside a fridge).

SECTION: Cleaning &amp; Decontamination
7.1. Ultrasonicate with alcohol by placing the items into a glass vial filled with alcohol:
Diamond drill bits used for enamel powder extraction
Glass vial used to clean the tooth crown/enamel piece 
Any other tools used in the process of extracting enamel powder

 

Soak the following items in diluted commercial bleach solution (1:10) for at least 15 min , brush them with an UV-irradiated toothbrush (soaked in diluted bleach solution (1:10)), and rinse with UV-irradiated Reverse Osmosis water (do this in the tubs which were prepared in Section 3). 
Diamond wheel attachment used to separate root from crown
Carbide bur 
Pliers
Any other tools that came in direct contact with the sample

Throw away
Aluminium foil
Paper towels 
Paper plates (if used)
Weighing paper
A4 paper 
Gloves
Any remaining unused tooth powder

SECTION: Cleaning &amp; Decontamination
7.1. Ultrasonicate with alcohol by placing the items into a glass vial filled with alcohol:
Diamond drill bits used for enamel powder extraction
Glass vial used to clean the tooth crown/enamel piece 
Any other tools used in the process of extracting enamel powder

 

Soak the following items in diluted commercial bleach solution (1:10) for at least 15 min , brush them with an UV-irradiated toothbrush (soaked in diluted bleach solution (1:10)), and rinse with UV-irradiated Reverse Osmosis water (do this in the tubs which were prepared in Section 3). 
Diamond wheel attachment used to separate root from crown
Carbide bur 
Pliers
Any other tools that came in direct contact with the sample

Throw away
Aluminium foil
Paper towels 
Paper plates (if used)
Weighing paper
A4 paper 
Gloves
Any remaining unused tooth powder

SECTION: Cleaning &amp; Decontamination
7.3. Wipe down with DNA-Exitus Plus
Handheld drill & station
Weighing scale
Ultrasonicator
Camera
Any other electronic components used in the process
Dried ultrasonicated strontium tools

Clean with diluted commercial bleach solution (1:10):
Any surface or item that came into contact with the sample, especially the work surface
Any non-electronic tools that came into contact with the sample (e.g., pens, bags, etc.)
SECTION: Cleaning &amp; Decontamination
7.2. CHANGE GLOVES
SECTION: Cleaning &amp; Decontamination
7.2. CHANGE GLOVES
SECTION: Cleaning &amp; Decontamination
7.3. Wipe down with DNA-Exitus Plus
Handheld drill & station
Weighing scale
Ultrasonicator
Camera
Any other electronic components used in the process
Dried ultrasonicated strontium tools

Clean with diluted commercial bleach solution (1:10):
Any surface or item that came into contact with the sample, especially the work surface
Any non-electronic tools that came into contact with the sample (e.g., pens, bags, etc.)

SECTION: Sampling - Enamel
6.3. CHANGE GLOVES
SECTION: Sampling - Pulp Chamber
5.1. Separating the Roots from the Crown


 


1. Hold the tooth securely with pliers over a piece of aluminium foil or a paper plate. 

2. Carefully separate the crown from the roots just below the Cemento-Enamel Junction (CEJ) using a round diamond saw wheel at high speed.

 
 

3. Gently tap the tooth on the aluminium foil or paper plate to dislodge and remove any loose powder. 

4. Remove the diamond saw wheel and place it into diluted commercial bleach solution (1:10) for decontamination.
SECTION: Sampling - Pulp Chamber
5.2. CHANGE GLOVES
SECTION: Sampling - Pulp Chamber
5.3. Extraction of Tooth Powder 

1. Attach a round carbide bur to the handpiece of the drill. 
 
2. Reduce the drill speed to low-medium. 

3. Place the Safe-Lock tube on the weighing scale and tare (zero out) the weight of the tube. 

4. Place the creased weighing paper in the centre of the A4 paper or aluminium foil, ensuring the paper plate or second piece of aluminium foil is pushed to the back of the working surface. 

5. Hold the roots or crown between the pliers over the centre of the weighing paper. Carefully and slowly drill into the inner pulp chamber of both the roots and crown, collecting the resulting powder on the weighing paper. 

 

 


6. Carefully transfer the powder into the tube. Creasing the paper will assist in transferring the powder, particularly if it is statically loaded. 

 

 

7. Collect enough powder for a main sample (30-50 mg) and a back-up (minimum 7 mg, ideally at least 25 mg ). 

 


SECTION: Sampling - Preparation of Workstation / Set up
3.2. Preparation for sampling the tooth

Start this part with a fresh pair of gloves! 

1. Label 2.0ml Safe-Lock tubes.
The number of tubes required depends on the specific laboratory procedures. If samples are to be shipped to another lab for further processing, it is advisable to label at least two tubes and retain one as a backup. If dental calculus is present, prepare at least one additional tube. 

2. Prepare paper towels outside of the working bench/sampling box.

3. Place a sheet of A4 paper or aluminium foil on the work surface.

4. Place another layer—aluminium foil, paper towel, or paper plate—on top of the first sheet, according to respective lab protocols.

5. Fold 1 or 2 weighing papers in half (use 1 if no calculus is present, 2 if calculus is present):
    -   Place them inside the working box with the opening facing away from you.

6. Insert a diamond saw wheel into the drill handpiece.

7. Lay out the pliers and any additional tools (e.g. dental scaler, if there is calculus present) on the work surface. 

8. Keep a round carbide dental bur outside the working space, within reach but at a safe distance—preferably stored in a closed container. 


 

9. Place the tooth on the prepared sheet of paper, aluminium foil, or paper plate—ideally without touching it directly. 
SECTION: Sampling - Pulp Chamber
5.4. Weighing the Sample 

1. Move the weighing scale to your work surface.

2. Place the weighing paper in the centre of the weighing scale and tare (zero out) its weight.

3. Carefully pour enough powder for the main sample onto the paper (ideally 50 mg ). Record the weight, then transfer the powder into the second labelled tube and place aside. 

 

4. Pour the remaining (back-up) powder onto the weighing paper, record the weight, and transfer it back into the labelled tube. This back-up sample should be no less than 7 mg .
SECTION: Sampling - Pulp Chamber
5.5. Post-DNA sampling & Cleaning 

1. Carefully place the roots back into the plastic bag using a piece of aluminium foil or pliers, then remove from the workstation. 

 

2. Optional if enamel is sampled: 
Place the crown/crown pieces, or enamel pieces (depending on condition of the crown after separation) into a glass vial filled with deionised water. (Tip: not all pieces of the crown need to be placed in deionised water—only enough to extract the desired amount of enamel powder). If enamel is not sampled, place the crown back into the plastic bag.

3. Place the round carbide bur, pliers, and any other tools used during the process into the prepared container with diluted commercial bleach solution (1:10) for decontamination. 

 

4. Dispose of any disposable equipment (e.g., aluminium foil, paper, weighing paper, paper plates) along with your gloves, ideally wrapping the material inside your gloves before disposal.

SECTION: Cleaning &amp; Decontamination
7.5. At this point, a new sample can be started: Refer back to Step 3.2. 
SECTION: Sampling - Pulp Chamber
5.6. CHANGE GLOVES
SECTION: Sampling - Pulp Chamber
5.7. Record the sample weights on the labelled tubes using a waterproof pen, and also note them on a separate sheet of paper. 
SECTION: Post-Sampling Documentation
9. Once all teeth from one site have been sampled, make sure to take photos from all sides on a neutral background with a scale/ruler (refer back to Section 1: Pre-Sampling Documentation).  
SECTION: Decontamination &amp; Pre-Sampling
1.1. Required Materials

DNA Exitus Plus
Diluted commercial bleach solution (1:10) (diluted with UV-irradiated Reverse Osmosis water)
Polyethylene clear plastic bags
A4 paper
Waterproof pen
Aluminium foil
Forceps/Pliers
Paper towels
Scale/Ruler
Camera 
SECTION: Preparation of Sample
2. Required Materials

Diluted commercial bleach solution (1:10) (diluted with UV-irradiated Reverse Osmosis water)
Forceps/Pliers
Polyethylene clear plastic bags
Waterproof pen
Aluminium foil
Paper towels
SECTION: Post-Sampling Documentation
9. Once all teeth from one site have been sampled, make sure to take photos from all sides on a neutral background with a scale/ruler (refer back to Section 1: Pre-Sampling Documentation). 
SECTION: Decontamination &amp; Pre-Sampling
1.3. Change gloves after each sample to avoid contamination. 
SECTION: Decontamination &amp; Pre-Sampling
1.5. All individual sample bags should then be placed into a single collective bag labelled with the site code. Additionally, the collective bag should be labelled to indicate whether the bags (and later the samples - see Section 2) have undergone UV-irradiation (e.g. <Site Code>, UV bags ‱,  UV samples). 

If the process needs to be paused at this stage, place the bag containing the samples into a decontaminated, sealed container. You may then resume the procedure at a later time.
SECTION: Sampling - Preparation of Workstation / Set up
3.1. Preparation for decontamination after sampling

1. Prepare one plastic tub/container with diluted commercial bleach solution (1:10) away from the working bench. This will be used to submerge all tools after sampling.

2. Place a UV-irradiated toothbrush into the bleach solution (1:10) tub, to be used later for cleaning the tools.

3. Prepare a smaller plastic tub/container with UV-treated Reverse Osmosis water, also away from the working bench. This will be used to rinse the tools after bleach submersion, helping to prevent corrosion.

4. Set up a drying area with paper towels, where tools can be left to dry after decontamination.
SECTION: Cleaning &amp; Decontamination
7.4. CHANGE GLOVES
SECTION: Cleaning &amp; Decontamination
7.6. At the end of the sampling day, place all portable equipment in a UV chamber and irradiate for several hours overnight. 
SECTION: Cleaning &amp; Decontamination
7.4. CHANGE GLOVES
SECTION: Cleaning &amp; Decontamination
7.5. At this point, a new sample can be started: Refer back to Step 3.2.
SECTION: Cleaning &amp; Decontamination
7.6. At the end of the sampling day, place all portable equipment in a UV chamber and irradiate for several hours overnight.