TITLE: Wedge sampling of pars petrosa (os temporale) for ancient DNA extraction
AUTHORS: Raphaela Stahl, Lena Semerau, Eleftheria Orfanou, Marie Himmel, Franziska Aron, Wolfgang Haak

[DESCRIPTION]
This protocol describes how to obtain bone powder from the pars petrosa of disarticulated ossis temporalis, specifically from the dense parts around the cochlea, in a more precise way by cutting a wedge into the bone before drilling. 

The pars petrosa has been shown to consistently yield high amounts of aDNA (Pinhasi et al. 2015 PLoS One, doi: 10.1371/journal.pone.0129102).

[BEFORE_START]
Planning
The sampling procedure of any skeletal element for DNA extraction should be performed ideally in a dedicated sampling room. This should not happen in the buffer preparation room.

Sampling of each pars petrosa takes around 20-40 minutes, but this can vary depending on the nature of the sample.

Equipment
Make sure all necessary equipment is available (see Materials).

Documentation
This protocol is destructive, therefore for all samples consultation and permission with curators should be performed prior running this protocol.

All anatomical and morphological features of the pars petrosa should be documented (e.g. pre-sampling photos, CT-scans) before sampling. 

If petrous bones from both sides are available, sample only from one side, in order not to cause alterations to the external morphology of both. 

[GUIDELINES]
Working in an Ancient DNA Laboratory
-All steps of the protocol should take place in a clean room facility specifically designed for ancient DNA.
-The researcher performing lab work should wear correspondingly suitable lab-wear, such as:
 - full-body suit with hood (e.g. Tyvek)
 - hairnet
 - face mask
 - three pairs of clean gloves
 - clean shoes 
 - protective glasses

- Sample processing should be carried out in separated work benches with integrated UV irradiation (e.g. Dead Air PCR work bench)
- Surfaces and equipment should be regularly decontaminated with e.g. bleach solution or Thermofisher's DNA AWAY (or similar) and irradiated with UV.


Please see the following for more detailed guidance: 

Llamas, B. et al., 2017. From the field to the laboratory: Controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era. STAR: Science & Technology of Archaeological Research, 3(1), pp.1–14. Available at: https://doi.org/10.1080/20548923.2016.1258824.


[STEPS]
SECTION: Workstation preparation
1. 
 


Place a sheet of aluminium foil under the hood.

Place weighing paper, drill bits, and a pair of tooth pliers or forceps on a second sheet of aluminium foil on an easily accessible clean surface outside of the hood. Alternatively, you may place them within the hood but be sure to cover them with aluminum foil or a paper towel to protect them from dust produced during sampling.

You will also need:
2 ml Safe-Lock Biopure tubes
Weighing trays/pans
Clean and labelled sample bags according to the amount of samples
Small pieces of aluminium foil for wrapping the bone (optional)
Small tape strips to fix the aluminium foil while covering the bone (optional)
Paper towels
Diluted commercial bleach solution (1:10)
UV-irradiated filtered/deionised water

 


SECTION: Preparation of sample
2. Place a sheet of aluminium foil in the UV chamber.

Place the pars petrosa on the aluminium foil and place the labelled sample bag next to it
 (to avoid mixing up samples in case you work on more than one). 

UV- irradiate the bone for 15 min on each side.
 

 


SECTION: Preparation of sample
3. Take a new 2 ml tube and label it with the sample ID on top of the lid and on the side of the tube. Tare the precision scale with the tube placed in the center.

Label a new sample bag with the sample ID to be used after sampling. 
 

SECTION: Preparation of sample
4. Take a weighing tray with a new pair of gloves.

Prepare a sheet of weighing paper by creasing it mid-way and place it under the area where you want to sample.
 


SECTION: Sampling procedure
5. 
Wedge sampling of pars petrosa
SECTION: Sampling procedure
5.1. Familiarize yourself with the anatomy of the petrous bone (e.g., Pinhasi et al. 2015). Locate the dense parts around the cochlea (b & c) as shown below in Figure 2. 

Ideally you will target area c (orange) shown in Figure 2, which is located roughly halfway between the jugular fossa (bottom) and the petrous crest (top). 

 

 
 
To make this desired area more accessible for drilling, it is easier to turn the pars petrosa in a different angle (see Figure 2a). Locating the densest part of the bone around the cochlea is simplified by using the wedge cutting method for sampling. Hence the petrous bone is cut in half, while holding the bone fraction with a tooth plier at the temporal bone. 


 
Therefore a round diamond saw blade is used to cut starting from the apex partis petrosae, following the margo superior partis petrosae and will stop doing so shortly after the internal acoustic opening (meatus acusticus internus). (Figure 2a)

See below a picture after cutting out this wedge. (Figure 3)

  
 

SECTION: Sampling procedure
5.2. To prevent residual dust/dirt from the outer surface and crevices to contaminate your freshly drilled bone powder, you can wrap the sample with aluminium foil and/or tape. Leave the part that you want to drill unwrapped. (Figure 4).

 

SECTION: Sampling procedure
5.3. Drill at low speed (and high torque) within the cochlear region in parallel to the auditory canal.

Collect clean bone powder on the weighing paper. Of note, it is possible that you will drill into internal canals and porosities that can be filled with soil, despite your bone being cut open for better visibility. If so, follow our Pro-Tip below.

Empty the drilling hole by carefully tipping it out onto the collection paper.
 

 

SECTION: Weighing of bone powder
6. Transfer the clean powder bone into a labelled 2 ml Safe-Lock Biopure tube. The folded weighing paper will help guiding the powder into the tube.

Close the tube, wipe it with a damp paper towel and weigh the bone powder. Make sure that this exact tube was used before to tare the scale. 

 

 

Note down the weight in mg on the side of the tube. Please refrain from labeling the lid with this information, it'll be too hard to read afterwards. Of course labeling the lid with the sample ID is recommended.

Store the bone powder at-20 °C until further processing.
SECTION: Weighing of bone powder
7. Take photos after sampling for documentation. 
Put bone back into a new, UV irradiated, labelled bag.
SECTION: Decontamination
8. 
 

Carefully clean the workspace and the equipment before sampling the next sample:
SECTION: Decontamination
8.1. Throw away disposable material such as aluminium foil and weighing tray/pan.
Clean surfaces and tools such as pliers with bleach solution
Use less aggressive DNA decontamination reagents (e.g. DNA away) for sensitive material such as electronic devices (e.g. handpiece of the drill, precision balance). 
Wipe off bleach with water-wet paper towels afterwards to prevent corrosion; air dry.
SECTION: Decontamination
8.2. Drill bits and saw blades can be reused after careful cleaning: 
incubate them in bleach solution (1:10 dilution) for at least 2 minutes. 
      You can use a UV irradiated and bleached toothbrush for brushing the saw blade. 
Clean with UV irradiated water to remove all bleach and let it dry.

 

SECTION: Decontamination
9.     (or in case of UV irradiation of multiple samples at once,   )and repeat the sampling procedure with the next sample.