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).
The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Peißen Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.
The sampling of ancient human remains for the purposes of DNA recovery and analysis is inherently destructive1,2,3,4. The samples themselves are precious specimens and morphological preservation should be preserved wherever possible. As such, it is imperative that sampling practices be optimized to both avoid unnecessary destruction of irreplaceable material and to maximize the probability of success. Current best practice techniques are based on a small cohort of studies limited to either forensic surveys5,6, studies of ancient specimens where the development of optimal sampling is not the direct aim of the study7, or dedicated studies utilizing either non-human remains8 or targeting a very small selection of anatomical sampling locations (used here to denote a specific area of a skeletal element from which bone powder, for use in downstream DNA analyses, was generated)9,10. The sampling protocols presented here were optimized in the first large-scale systematic study of DNA preservation across multiple skeletal elements from multiple individuals11. All samples stemmed from skeletal elements recovered from 11 individuals excavated from the church graveyard of the abandoned medieval settlement of Krakauer Berg near Peißen, Saxony-Anhalt, Germany (see Table 1 for detailed sample demographics) and, as such, may need modification for use with samples outside of this geographical/temporal range.
Individual | Sex | Estimated age at death | 14C dates (CE, Cal 2-sigma) |
KRA001 | Male | 25-35 | 1058-1219 |
KRA002 | Female | 20-22 | 1227-1283 |
KRA003 | Male | 25 | 1059-1223 |
KRA004 | Male | 15 | 1284-1392 |
KRA005 | Male | 10-12 | 1170-1258 |
KRA006 | Female | 30-40 | 1218-1266 |
KRA007 | Female | 25-30 | 1167-1251 |
KRA008 | Male | 20 | 1301-1402 |
KRA009 | Male | Unknown | 1158-1254 |
KRA010 | Male | 25 | 1276-1383 |
KRA011 | Female | 30-45 | 1040-1159 |
Table 1: Genetically determined sex, archaeologically determined estimated age at death, and radiocarbon dating (14C Cal 2-sigma) for all the 11 individuals sampled. This table has been adapted from Parker, C. et al. 202011.
These protocols allow for a relatively straightforward and efficient generation of bone powder from eight anatomical sampling locations across five skeletal elements (including the pars petrosa) with limited laboratory-induced DNA contamination. Of these five skeletal elements, seven anatomical sampling locations found on four skeletal elements have been determined to be viable alternatives to the destructive sampling of the petrous pyramid11,12. These include the cementum, dentin, and pulp chamber of permanent molars; cortical bone gathered from the superior vertebral notch as well as from the body of thoracic vertebrae; cortical bone stemming from the inferior surface of the apical tuft and shaft of the distal phalanges; and the dense cortical bone along the exterior portion of the tali. While there are several widely applied methods for the sampling of the pars petrosa4,12,13,14, dentin, and the dental pulp chamber1,2,15, published methods describing the successful generation of bone powder from the cementum16, vertebral body, inferior vertebral notch, and talus can be difficult to obtain. As such, here we demonstrate optimized sampling protocols for the petrous pyramid (step 3.1); cementum (step 3.2.1), dentin (step 3.2.2), and dental pulp (step 3.2.3) of adult molars; cortical bone of the vertebral body (step 3.3.1) and superior vertebral arch (step 3.3.2); the distal phalanx (step 3.4); and the talus (step 3.5) in order to make the effective use of these skeletal elements for both aDNA and forensic research more widely accessible.
Current practice in ancient human population genetics is to preferentially sample from the pars petrosa (step 2.1) whenever possible. However, the pars petrosa can be a difficult sample to obtain, as it is highly valued for a myriad of skeletal assessments (e.g., population history32, the estimation of fetal age at death33, and sex determination34), and, historically, sampling of the pars petrosa for DNA analysis can be highly destructive3,4 (including the protocol presented here, although new, minimally invasive protocols13,14 have now been widely adopted to alleviate this concern). This is compounded by the fact that, until very recently, a large-scale, systematic study of human DNA recovery across the skeleton had not been attempted11, making finding an appropriate sampling strategy when the petrous pyramid is unavailable challenging.
The protocols presented here help to alleviate that challenge by providing a set of optimized procedures for DNA sampling from archaeological/forensic skeletal remains including the pars petrosa as well as seven alternate anatomical sampling locations across four additional skeletal elements. The critical steps included are all intended to minimize the possibility of DNA loss/damage due to either inefficient sampling (steps 2.1.6 and 3.2.1.3) or overheating of samples during drilling/cutting (step 3.1.6). Additionally, it has been noted throughout the protocol that it may be necessary to modify/omit the pre-treatment steps to ensure the best performance in highly degraded samples. It should also be noted that even among the selected elements presented here, there remain several possible alternative sampling techniques (particularly for the pars petrosa13,14), as well as ample room for further optimization of the underexploited anatomical sampling locations presented here (i.e., the talus: step 2.5 and the vertebrae: step 2.3).
It is also important to keep in mind that these protocols have been designed and tested using ancient juvenile-adult remains of high quality (good morphological preservation) for the purposes of endogenous human DNA analyses. The results presented may not extend to more highly degraded materials, other preservation contexts, infant remains, non-human remains, or studies of pathogens or the microbiome, as a greater exploration into the use of these protocols in additional contexts is still needed. Additionally, the alternative skeletal elements presented here (the teeth, vertebrae, distal phalanx, and tali) may be challenging to assign to a single individual among commingled remains, necessitating sampling from multiple elements to ensure a single origin. Despite these limitations, making these protocols widely available can help alleviate some of the heterogeneity surrounding sample selection and processing by providing a generalized and quantitatively optimized framework for use in a wide range of future aDNA/forensic studies on human remains.
The authors have nothing to disclose.
The authors would like to thank the laboratory staff of the Max Planck Institute for the Science of Human History for their help in the development and implementation of these protocols. This work would not have been possible without the input and hard work of Dr. Guido Brandt, Dr. Elizabeth Nelson, Antje Wissegot, and Franziska Aron. This study was funded by the Max Planck Society, the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program under grant agreements No 771234 – PALEoRIDER (WH, ABR) and Starting Grant No. 805268 CoDisEASe (to KIB).
#16 Dental Drill Bit | NTI | H1-016-HP | example drilling bit |
0.6 mm scroll saw blade | Fisher Scientific | 50-949-097 | blade for Jewellers Saw |
22mm diamond cutting wheel | Kahla | SKU 806 104 358 514 220 | Dremel cutting attachment |
Commercial Bleach | Fisher Scientific | NC1818018 | |
Control Company Ultra-Clean Supreme Aluminum Foil | Fisher Scientific | 15-078-29X | |
DNA LoBind Tubes (2 mL) | Eppendorf | 22431048 | |
Dremel 225-01 Flex Shaft Attachment | Dremel | 225-01 | Dremel flexible extension |
Dremel 4300 Rotary Tool | Dremel | 4300 | Example drill |
Dremel collet and nut kit | Dremel | 4485 | Adapters for various Dremel tool attachments/bits |
Eagle 33 Gallon Red Biohazard Waste Bag | Fisher Scientific | 17-988-501 | |
Eppendorf DNA LoBind 2 mL microcentrifuge tube | Fisher Scientific | 13-698-792 | |
Ethanol (Molecular Biology Grade) | Millipore Sigma | 1.08543 | |
FDA approved level 2 Surgical Mask | Fisher Scientific | 50-206-0397 | PPE |
Fisherbrand Comfort Nitrile Gloves | Fisher Scientific | 19-041-171X | PPE |
Fisherbrand Safety Glasses | Fisher Scientific | 19-130-208X | PPE |
Granger Stationary Vise | Fisher Scientific | NC1336173 | benchtop vise |
Invitrogen UltraPure DNase/Rnase free distilled water | Fisher Scientific | 10-977-023 | |
Jewellers Saw | Fisher Scientific | 50-949-231 | |
Kimwipes | Sigma-Aldritch | Z188956 | |
Labconco Purifier Logic Biosafety cabinet | Fisher Scientific | 30-368-1101 | |
LookOut DNA Erase | Millipore Sigma | L9042-1L | |
Medium weighing boat | Heathrow Scientific | HS120223 | |
MSC 10pc plier/clamp set | Fisher Scientific | 50-129-5352 | Miscellaneous clamps/vise grips for securely holding samples while drilling/cutting |
Sartorius Quintix Semi-Micro Balance | Fisher Scientific | 14-560-019 | enclosed balance |
Tyvek coveralls with hood | Fisher Scientific | 01-361-7X | PPE |
Weigh paper | Heathrow Scientific | HS120116 |