Subsampling bones and teeth for ancient DNA extraction v2

This protocol descibes how to subsample non-human mammalian bones and teeth for ancient DNA extraction. This is an invasive/destructive protocol. Version 2 updates the author list. We specify this as a non-human protocol mainly due to the surface cleaning approach applied here: In human studies, the outer layer of bone/tooth is (usually) removed with the drill to get rid of any potential contaminant human DNA on the surface, whereas in this protocol we target the outer layers and thus do not remove them with the drill, but instead do a less conservative cleaning with bleach and ethanol. The advantage of working with non-human mammals is that we can tolerate some human DNA contamination from the surface of the specimen (though still not ideal), as any sequencing reads originating from contaminant human DNA can be removed bioinformatically. In this protocol, we first discuss what to consider when subsampling bones and teeth (e.g. which bones or which areas of the tooth are best to sample for aDNA), then we describe the procedure for subsampling in an ideal setting (i.e. a dedicated aDNA clean lab), and finally we discuss some considerations when subsampling in a non-ideal setting (e.g. at a museum or in the field). At the end of this protocol, you will have ~50 mg of bone/tooth powder in a 1.5 or 2 mL Eppendorf tube ready to be used for DNA extraction. It is assumed that the extracted DNA will then be sequenced using next-generation sequencing technology. Optional: We also address subsampling for collagen extraction to be used for radiocarbon dating and/or stable isotope analysis, in which case ~250-1000 mg of bone/tooth subsample is obtained. See the Materials for a list of equipment and reagents required. Key references: Alberti, F., et al., Optimized DNA sampling of ancient bones using Computed Tomography scans. Molecular Ecology Resources, 2018. 18(6): p. 1196-1208. https://doi.org/10.1111/1755-0998.12911 Damgaard, P.B., et al., Improving access to endogenous DNA in ancient bones and teeth. Scientific Reports, 2015. 5(11184): p. 11184. https://doi.org/10.1038/srep11184 Adler, C.J., et al., Survival and recovery of DNA from ancient teeth and bones. Journal of Archaeological Science, 2011. 38(5): p. 956-964. https://doi.org/10.1016/j.jas.2010.11.010 Hansen, H.B., et al., Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum. PLOS ONE, 2017. 12(1): p. e0170940. https://doi.org/10.1371/journal.pone.0170940