Minimally-invasive sampling of pars petrosa (os temporale) for ancient DNA extraction v2

Abstract

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 minimally-invasive way by drilling from the outside. 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).

Full text

DEC 11, 2020
Minimally-invasive sampling of
pars petrosa
(
os
temporale
) for ancient DNA extraction V.2
In 2 collections
Eleftheria Orfanou , Marie Himmel , Franziska Aron ,
Wolfgang Haak
Department of Archaeogenetics, Max Planck Institute for the Science of
Human History;
Friedrich-Schiller Universität Jena
WarinnerGroup MPI EVA Archaeogenetics
Eleftheria Orfanou
DOI:
dx.doi.org/10.17504/protocol
s.io.bqd8ms9w
Protocol Citation: Eleftheria
Orfanou, Marie Himmel,
Franziska Aron, Wolfgang
Haak 2020. Minimally-invasive
sampling of pars petrosa (os
temporale) for ancient DNA
extraction. protocols.io
https://dx.doi.org/10.17504/p
rotocols.io.bqd8ms9wVersion
created by Eleftheria Orfanou
License: This is an open
access protocol distributed
under the terms of
the Creative Commons
Attribution License, which
permits unrestricted use,
distribution, and reproduction
in any medium, provided the
original author and source are
credited
Protocol status: Working
We use this protocol and it's
working
Created: Dec 04, 2020
Last Modified: Dec 11, 2020
PROTOCOL integer ID:
45216
1 1 2
1
1
2
ABSTRACT
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 minimally-invasive way by drilling from the outside.
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).
VERSION 2
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Keywords: ancient DNA,
petrous pyramid, sampling,
non-invasive method,
archaeogenetics, archaeology
GUIDELINES
Worki ng i n a n Ancient DNA L a boratoryWorki ng i n a n Ancient DNA L a boratory
-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
- two 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.
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MATERIALS
Lab materi a l sLab materi a l s
Safe-Lock Tubes 2 ml Biopur (preferably packed individually) Eppe ndorfEppendorf C a tal ogCata l og
##00301215970030121597
DNA AWAY® 4000 ml C a rl RothCarl Roth Cata l og #Cata l og # X996.2X996.2
Weighing Paper MN 226 block of 100 sheets 9 x 115 cm M A C H EREY-NAGELMAC H EREY -NAGEL
Gm bH & Co. KGGm bH & Co. KG Catalog #Catalog #186002186002
Precision forceps 18/10 steel extra sharp bent points without ridges
L=105mm V W R S cienti ficVWR Scie ntifi c Catalog #Catalog #232-0008232- 0008
Weighing pans ROTILABO® blue antistatic 330 ml 140 mm 140 mm C a rlCarl
RothRoth Cata l og #Cata l og # 2159.2 2159.2
Lab equ i pmentLab e quipm ent
PCR work bench (e.g. AirClean Dead Air PCR Werkbank, 48´´)
UV irradiation box or cross linker (e.g. Vilber Lourmat Bio-Link BLX-254)
Air extraction/vacuuming unit
Drill Lab Handpiece (e.g. K-POWERgrip™ Installation Lab Handpieces from Kavo
Dental Excellence; SKU: 10022916)
Rounded dental drill bit (e.g. NT1 from Kahla; SKU: H1-016 HP)
Balance (e.g. Ohaus Adventurer balance AX1502)
Anti-static instrument (e.g. Zerostat 3 from Zerostat; SKU: SAFAZ108812)
Aluminium foil (lab grade or sterile preferred)
Paper towels
Polyethylene clear plastic bags
Marking or masking tape
Camera
Ge neral Reagent sGe neral Reagent s
Solution of household bleach (2-6% NaClO, then diluted to a working solution
concentration of 0.2-0.5% NaClO)
Thermofisher DNA AWAY
UV-irradiated and deionised tap water
SAFETY WARNINGS
Re a gentsRe a gents
Household bleach solution
(2-6%) diluted to a working concentration of
0.2-0.5 % NaClO in total.
- H290 May be corrosive to metals.
- H314 Causes severe skin burns and eye damage.
- H411 Toxic to aquatic life with long lasting effects.
- EUH206 Warning! Do not use together with other products. May release
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dangerous gases (chlorine).
Remove from surface after recommended incubation time with water-
soaked tissue.
DNA AWAY
- H290 May be corrosive to metals.
- H314 Causes severe skin burns and eye damage.
Note: Both bleach solutions and DNA AWAY are used for
decontamination. DNA AWAY is less corrosive than bleach and should
be prefered for decontamination of sensitive equipments such as
surfaces of electric devices.
EquipmentEquipment
UV radiation
- UV radiation can damage eyes and can be carcinogenic in contact with
skin. Do not look directly at unshielded UV radiation. Do not expose
unprotected skin to UV radiation.
- UV emitters generate ozone during operation. Use only in ventilated
rooms.
Usage of sharp tools
Always hold the sample with pliers while cutting and drilling to avoid
injuries
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BEFORE START INSTRUCT IONS
Planni ngPlanni ng
The sampling procedure of any skeletal element for DNA extraction should be
performed ideally in a dedicated sampling room. This should notnot 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.
EquipmentEquipment
Make sure all necessary equipment is available (see Materials).
Docum ent a tionDocumentati on
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.
1
Safety information
Place a sheet of aluminium foil under the hood.
Everything that comes in contact with the sample needs to be decontaminated in order to
avoid cross-contamination between samples. Change gloves regularly, especially between
different samples.
Workstation preparation
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Place weighing paper, drill bits, and forceps on a sheet of aluminum foil on an easily accessible
clean surface outside of the hood. Alternatively, you may place them within 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 Biopur tubes
Weighing trays/pans (optional)
Clean and labelled sample bags according to the amount of samples
Small pieces of aluminium foil for wrapping the bone
Small tape strips to fix the aluminium foil while covering the bone
Paper towels
Diluted commercial bleach solution (1:10)
UV-irradiated filtered/deionised water
Figure 1. Set up of the workstation
Preparation of sample
15m
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2Place 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 00:15:00 on each side.
Safety information
Note
If possible, avoid touching the bone directly. Use precision forceps or something similar to
handle each bone individually.
Ti p:Ti p: It is possible to UV irradiate multiple bones at once in the same UV chamber. Ensure
each bone is on a separate bit of foil, and keep the corresponding labelled sample bag next to
it to prevent sample mix up.
3Take a new 2 ml tube and label it accordingly. If this is the beginning of the sampling session, tare
the precision scale with the tube placed in the center.
Label a new sample bag with the sample ID.
Safety information
To avoid contamination, tubes should remain closed except when the bone powder is added.
4Remove the bone from the UV chamber by using a paper towel with a small amount of household
bleach solution.
Wipe the surface of the bone region you want to sample with a bleach-dampened paper towel
and then carefully clean the bone with a water-dampened paper towel to remove excess bleach.
Use another sheet to wipe the bone dry.
15m
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Note
If the bone was not washed after excavation this can be a tedious step. Make sure you
brush/shake off as much of the residual soil as possible before starting.
5Take 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.
Note
Bone powder can be statically charged and difficult to transfer to tubes. By slightly folding the
weighing paper, it makes it easier to move the bone powder from the weighing paper into the
tube.
6
Minimally-invasive sampling of
pars petrosa
6.1 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). However, since this protocol
does not cut the petrous bone in half, it will be easier to orient the bone so that your are looking
at the posterior part, which contains the internal acoustic opening (
meatus acusticus internus
).
To target the cochlear region you will have to position the drill a few millimetres laterally (the
outer side where the temporale is/would be attached) and choose an angle so that the drill
hole will not penetrate the auditory canal (Figure 3). Of note, the auditory canal also leads
laterally/dorsally outwards, so it is advisable to drill in a parallel line.
Sampling procedure
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CITATION
Ron Pinhasi, Daniel Fernandes, Kendra Sirak, Mario Novak, Sarah Connell, Songül Alpaslan-
Roodenberg, Fokke Gerritsen, Vyacheslav Moiseyev, Andrey Gromov, Pál Raczky, Alexandra Anders,
Michael Pietrusewsky, Gary Rollefson, Marija Jovanovic, Hiep Trinhhoang, Guy Bar-Oz, Marc
Oxenham, Hirofumi Matsumura, Michael Hofreiter (2015). Optimal Ancient DNA Yields from the Inner
Ear Part of the Human Petrous Bone. PLoS ONE.
LINK
https://doi.org/10.1371/journal.pone.0129102
Figure 2. Dense parts around cochlea (b & cb & c). Part cc provides higher endogenous aDNA
yields than part bb and aa.
(Pinhasi et al. 2015).
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Figure 3. Before (top) and after (bottom) sampling of pars petrosa.
6.2 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. For ease of orientation, mark the position of the internal
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auditory meatus with a permanent marker (Figure 4).
Figure 4. Wrapping the pars petrosa with aluminium foil and marking of the auditory canal.
6.3 Use the drill to clean (abrade) the surface of sample window at low speed.
Discard or set aside the powder from this cleaning step and place a new weighing tray and/or
weighing paper under the sample.
6.4 Drill at low speed (and high torque) from the outside towards the cochlear region in parallel to
the auditory canal.
Collect
clean
bone powder on the weighing paper. Of note, it is very likely that you will drill into
internal canals and porosities that can be filled with soil. If so, stop and try to reorient your
drilling angle.
Empty the drilling hole by carefully tipping it out onto the collection paper.
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Note
Safety information
Pro-Tip:Pro-Tip: If residual dirt/soil is mixed in with your collected clean bone powder, you can
physically separate them by carefully tipping the edges of the weighing paper repeatedly.
In doing so, the different weights and particle sizes will easily separate. Discard or set
aside the dirt.
Be careful when unwrapping the bone, the vibration of the drilling will loosen residual
dirt/soil and bone powder, which will accumulate in the wrapping.
7Transfer the clean powder bone into a labelled 2 ml Safe-Lock Biopur tube. The folded weighing
paper will help guide the powder into the tube.
Close the tube, wipe it with a damp paper towel and weigh the tube.
Safety information
Note
Write down the weight in mg on the cap and on the side of the tube.
Store the bone powder at -20 °C until further processing.
Bone powder can become statically charged. In such case, consider using an anti-static gun
and apply to the weighing paper and tube.
Ideally you wish to collect around 30 mg to 50 mg of powder for extraction (if using
our protocol for ancient DNA Extraction). If you have more than 50 mg then store it as
back-up material in a separate tube.
8Take photos after sampling for documentation.
Put bone back into a new, UV irradiated, labelled bag.
Weighing of bone powder
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9
Safety information
Carefully clean the workspace and the equipment before sampling the next sample:
Everything that came in contact with the sample needs to be cleaned/decontaminated in
order to avoid cross-contamination between samples.
9.1 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.
9.2 Drill bits 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.
Note
Check conditions of drill bits after ~5 uses and replace with new if necessary.
10 (or in case of UV irradiation of multiple samples at once, )and repeat the sampling
procedure with the next sample.
Decontamination
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