News and ViewsExcavation protocol of bone remains for Neandertal DNA analysis in El Sidrón Cave (Asturias, Spain)
Introduction
The retrieval of ancient DNA from human fossils is gradually becoming a common practice, and many papers focus on methodologies for maximizing DNA extraction and analysis (e.g., Richards et al., 1995, Handt et al., 1996, O'Rourke et al., 1996, Hofreiter et al., 2001, Gilbert et al., 2005b, Malmström et al., 2005). DNA extraction has been mostly described for previously discovered fossils that have been stored at museums, sometimes for a long time. Recently, Pruvost et al. (2007) reported that freshly excavated and unwashed bones yield twice as many authentic DNA sequences as bones treated with standard excavation procedures. But, specific methodological guidelines for excavating fossil remains have barely been approached. The site of El Sidrón (Fortea et al., 2003, Rosas et al., 2006), in which the high density of human remains guarantees a certain chance of finding new fossils in successive field seasons, allows us to describe new methodological approaches to avoid both DNA destruction and contamination.
Until recently, contamination of Neandertal samples was relatively easy to address since Neandertal mitochondrial sequences differ substantially from those of modern humans (Krings et al., 1997, Krings et al., 2000, Ovchinnikov et al., 2000, Schmitz et al., 2002, Serre et al., 2004, Beauval et al., 2005, Lalueza-Fox et al., 2005, Lalueza-Fox et al., 2006, Caramelli et al., 2006, Orlando et al., 2006, Krause et al., 2007b). However, contamination remains an issue for mtDNA extraction from Upper Paleolithic modern human remains (Caramelli et al., 2003), and new metagenomic techniques are producing massive amounts of data from putative Neandertal nuclear DNA which is supposedly identical or nearly identical to potential modern human contaminants (Green et al., 2006, Noonan et al., 2006). Recently it has been suggested (Wall and Kim, 2007) that the metagenomic data from Vindija Vi33.16 (Green et al., 2006) included substantial modern contamination. These concerns necessitate the development of new methodological protocols even in Neandertal excavations.
Contamination of samples by exogenous DNA remains a major problem facing ancient human DNA studies (Pääbo et al., 2004; Willerslev and Cooper, 2005). The degradation of endogenous DNA over long periods of time results in the tendency for ancient samples to preferentially yield contaminant DNA, whether derived by handling of the specimens or from DNA previously generated in the same laboratory (Handt et al., 1996, Pääbo et al., 2004). Probably the most difficult contamination to control occurs prior to preparation of the specimen for genetic analysis (Pääbo et al., 2004, Gilbert et al., 2005a, Willerslev and Cooper, 2005, Pruvost et al., 2007). For example, a large proportion of excavated archaeological material, including human bone, is handled, washed with water, and labeled during or immediately after excavation. High levels of pre-laboratory derived DNA contamination in human remains have been described, even when standard sample decontamination guidelines to remove such contaminants have been followed (e.g., Richards et al., 1995, Handt et al., 1996, Hofreiter et al., 2001, Gilbert et al., 2005b, Malmström et al., 2005). The exact mechanism of this contamination by handling is still poorly understood, but the transport of DNA molecules from the exogenous source to the interior of the specimen seems to correlate with the moisture content of the sample (Gilbert et al., 2005b). In a study of Neolithic remains it was possible to demonstrate that the people involved in the initial washing steps were more likely to contaminate the specimens than were the people involved in the later laboratory analysis (Sampietro et al., 2006, Sampietro et al., 2007).
One possible guideline to control pre-laboratory contaminants (Sampietro et al., 2006) is to type every person involved in the manipulation and study of the remains; this can be achieved with mtDNA studies, however, in the new genomic approaches the amount of genetic data required precludes such typing. In addition, it is impossible to type all handlers of Neandertal remains that were excavated long ago; for instance, the first genomic analysis was undertaken with Vindija Vi33.16, a bone retrieved in 1980 for whom all handlers are unlikely to be known or available. Additionally, older contaminants might not be easily distinguishable from endogenous sequences, since older contaminants will also tend to degrade, depending on the storage conditions (Sampietro et al., 2006).
Currently, the best way to overcome the problems of contamination of ancient samples is to minimize the initial source of contamination; that is at the time of excavation itself. We have developed an anti-contamination protocol for the Neandertal site of El Sidrón (Spain) (Fortea et al., 2003, Rosas et al., 2006), and we have tested its effectiveness through DNA analysis of samples retrieved before and after the protocol was implemented.
Section snippets
Materials and methods
The El Sidrón is a karstic system located in the region of Asturias, northern Spain (Fig. 1). A significant archaeo-paleontological record with 1,400 Neandertal fossil remains (labeled SD) and 358 lithic artifacts has been recovered at the site since 2000 (Fortea et al., 2003, Rosas et al., 2006). During the first seasons, the remains were excavated using standard archaeological methods, with no special precautions relating to washing and handling. In 2004 it was possible to retrieve short
Results
Interestingly, everyone involved in the retrieval and study of the El Sidrón remains has different mtDNA haplotypes (Table 1). The two archaeologists display substitutions in the region typed for SD-441 in two overlapping fragments (16230–16262 and 16244–16278, numbered according to the human reference sequence from Anderson et al., 1981). Their DNA can be observed in around 1–10% of the resulting clones of this pre-protocol specimen. However, the overwhelming majority of the contaminant
Discussion
Our mtDNA results from the remains at El Sidrón suggest that the extraction protocol designed to prevent DNA contamination from handling has significantly increased the ratio of endogenous sequences retrieved. In the only two cases in which it was possible to attribute the contaminant sequences to a particular researcher, both were the archaeologists that handled and washed the remains at the site prior to the implementation of the excavation protocols. This finding corroborates previous
Acknowledgements
We thank A. Bernardo, A. B. Díez, J. Fernández de la Vega, C. Goitia, D. Santamaría, A. Soto, J. Trenor and N. Zardaín for their efforts and interest in the field and laboratory work. We are grateful to A. Estalrrich, C. Martínez-Maza, M. Bastir, and S. García-Vargas for managing the fossil collection. This research was supported by Principado de Asturias-Universidad de Oviedo Grant CN-04-152 and a MEC grant (CGL2006-03987) to C.L.-F.
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