Guidelines for handling biological excavation findings in order to avoid DNA contamination in ancient DNA analyses.

The following guidelines are based on the study of Llamas 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, 1-14, DOI: 10.1080/20548923.2016.1258824, which can be downloaded from here.

The petrous bone, part of the temporal bone, is the hardest and most dense bone in the mammal body. DNA preservation in petrous bones and teeth reveals that the endogenous DNA content in petrous bone and teeth is high. Recent studies have confirmed that sampling of petrous bone and teeth is a good practice since they preserve aDNA extremely well, even when the samples are from warmer climates like Africa, the Near East etc. We note, that morphological and biomolecular analyses of tooth crowns do not have to be compromised by DNA sampling, if only sampling the root, and petrous bones can be sampled with careful drilling without affecting the external temporal bone, which is the part mainly used in morphological studies.

Precautions that can potentially make a big difference:

Disposable gloves: Wear disposable medical gloves during excavation and when handling specimens to protect samples from human DNA contaminants. Change gloves between specimens, after touching hair or face, or after touching communal items (e.g. trowels, pens). Wearing two pairs of gloves and changing the outer one regularly is particularly efficient, because putting gloves on sweaty hands may be challenging.

Do not wash specimens with water: Water contains contaminating bacterial DNA, and can deeply penetrate into the sample and cause unwanted hydrolytic damage to the endogenous DNA. Light brushing of specimens with a dry brush is preferred to washing with water. Sampling of the surrounding matrix can also be useful for microbial studies, to identify and filter out environmental DNA sequences from the archaeological sample sequencing data.

Storage: Samples should either be completely dry to avoid further contamination with microbial DNA (microbial growth) and damage (hydrolysis), or stored in a cold, dry place as soon as possible (e.g. deep freezer).

Optional precautions for maximal effect:

Protective gear: In addition to gloves, other (optional) disposable protective gear may include surgical mask, hair cover/net, and sleeves to cover the arms (Figure 1). If budget and conditions allow, a clean disposable surgical gown or body suit and goggles are desirable.

Protect the site: Protect the site from dust, rain and direct sunlight to limit hydrolysis, irradiation, and further contamination with environmental DNA once the specimen is exposed.

Dedicated trained staff: If possible, assign one or two members of the excavation team to be formally trained and solely responsible for collecting the ‘contaminating-modern-DNA-free’ samples for aDNA analysis. This should limit the introduction of contaminating DNA from multiple individuals, and can therefore be detected as a systematic ubiquitous signal during computational analyses of the sequencing data.

Clean tools: Clean tools (e.g. trowels, dental picks, and brushes) with ≥3% bleach between samples, and dry them prior use on the next sample. The oxidative power of bleach will degrade contaminant DNA.

Keep records: Document soil types, wet/dry conditions, associated biological materials, details about visible treatments to the remains (e.g. artificial mummification), and people who handle the specimens. This metadata can be extremely helpful when designing the aDNA study, or when interpreting the results,

In situ sampling: In situ sampling is preferable if conditions allow. In this situation, the following procedures should be implemented:

If the skeletal material is articulated and the orientation of a body can be identified, the skeletal remains to be sampled for aDNA analysis should be freed from the soil and collected first, before excavating the rest of the skeleton.

If several specimens are mingled (e.g. mass grave or collective burial), unearthed skulls should be protected while excavating until all of the individuals have been identified. Samples that are unequivocally assigned to each individual can then be collected (most likely teeth or cranial elements).

Mummy bundles efficiently protect human remains against contaminating DNA from modern humans. In that specific case, in situ sampling should be avoided. Instead, it is recommended to open the bundles and to proceed with sampling in dedicated facilities, such as the archaeology laboratory or the museum.

Storage conditions: If the sample is too wet or frozen in permafrost, immediate storage in a freezer is highly recommended. Repeated humidity changes and freeze and thaw cycles should be minimised/avoided, as condensing water and its crystallisation will promote diagenesis. If freezing the sample and preserving the cold chain is not possible, wrap wet remains in clean paper towel and let dry on a clean surface (out of direct sunlight) to prevent mould formation.

Store samples individually in a clean, dry, airtight container (e.g. plastic Ziploc bag or tube) to avoid cross-contamination by contact with other samples.

Clearly label the sample container with date, site location, sample type, name of excavators, etc.

Chemical treatment: The use of preservative agents, hardeners, varnish glue, adhesive tape, or any chemicals such as sodium fluoride should be avoided, as they can inhibit experimental enzymatic reactions or introduce contaminating DNA.

Museum specimens: Specimens stored in museums or in other facilities are extremely valuable for aDNA research, despite the potential lack of contextual information, storage at room temperature, and extensive manipulation with bare hands. When sampling museum specimens, we strongly advise the routine implementation and use of protective gear and appropriate sampling protocols as outlined above.