Validation of the MiniFiler™ Kit in archaeological samples

1. Introduction and aims 

The forensic MiniFiler™ Kit has been designed to amplify nuclear DNA from critical samples. Amplification success is incremented through three different strategies: increasing the sensibility, decreasing the length of the amplicons and bypassing inhibition problems [2]. We pretend to evaluate the usefulness of the forensic MiniFiler™ Kit (Applied Biosystems) for ancient DNA (aDNA). In this study, we tested the MiniFiler™ Kit within a wide range of ancient Spanish samples, from different ages and locations, all of them preserved under different taphonomic conditions.

In the archaeogenetic field, obtaining nuclear DNA information could lead to confirm or reject archaeological hypothesis, to establish relationships between individuals belonging to the same burial site, to determine the sex or to identify different samples belonging to the same individual.

2. Materials and methods 

We studied 6 Neolithic samples from the “Can Sadurní” Cave (Barcelona), 14 Bronze Age samples from 4 different archaeological sites of the Castilla y León Region (North West Spain), 15 Medieval samples from “El Cuellar” (Segovia, Central Spain) and 5 samples of the XVIIIth century from Murcia (South East Spain).

DNA cleaning, grinding, extractions and amplifications were performed in an ancient DNA specialised laboratory, following the recommended criteria of authenticity [3].

DNA extractions were performed over bones and teeth through a silica-based method by Rohland and Hofreiter, 2007 [4]. Amplifications were carried out according to the manufacturer's recommendations. The MiniFiler™ Kit is designed to amplify 8 autosomal STR loci (D13S317, D7S820, D2S1338, D21S11, D16S539, D18S51, CSF1PO, FGA) and the gender-identification region Amelogenin. Amplified STRs markers were separated on a 3730 DNA Analyzer (Applied Biosystems).

In order to validate results, a minimum of 2 independent MiniFiler™ PCRs was performed. For samples from “El Cuellar” it was possible to increase the reliability of results by sampling at list 2 teeth or bones per individual. More precisely, the 15 samples from “El Cuellar” belong to 7 different individuals: 5 adults and 2 infants.

Kinship relationship between samples which yielded partial or complete genetic profiles was estimated using the Familias 1.81 software.

3. Results and discussion 

Results are summarised in Table 1 and Fig. 1.

Table 1. Samples studied (origin, number of samples and individuals) and results obtained in terms of reproducibility.

	Samples origin	No. individuals	No. samples	% replicated results	% not replicated results	% no results
	El Cuellar	7	14	82.54	3.17	14.29
	Cogotas Culture	14	14	3.97	16.67	79.37
	Can Sadurní	8	8	2.78	31.94	65.28
	Murcia	5	5	0.00	0.00	100.00
		34	41	22.32	12.95	64.73
		Total		Average

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Fig. 1. Histograms representing the relative percentages of reproducible results in correspondence to the archaeological site studied.

We obtained very satisfactory results with the well-preserved – and in some cases mummified – samples from “El Cuellar” (Middle Ages), buried in sarcophagus. It was possible to obtain the complete genetic profile from all the adult samples as well as partial profiles from 2 newborn infants. Amelogenin marker allowed us to establish the gender for all the 7 samples. We compared the gender results with the historical, archaeological and anthropological data. The individual buried in a sarcophagus with a female name inscription was classified as masculine skeleton through anthropometric criteria. However, genetic analysis gave the opposite result. A double replication per each sample supported the grave inscription. Globally, for this site, it was possible to obtain a high percentage of replicated results: 82.54% of replication per marker and per individual (see Table 1). Genetic information obtained was practically complete and allowed us to estimate paternity and brotherhood indexes between some of the individuals. Statistical analysis of the kinship relationship between 2 individuals, a mummy and a skeleton, from “El Cuellar” gave a relatively high statistical probability (99.98%). This result supports a previous archaeological hypothesis and demonstrates the importance and power of interdisciplinary studies in archaeogenetics.

Regarding the most recent samples from Murcia, we did not obtain positive results, most likely due to the burial conditions, with a high percentage of humidity, which increases the DNA degradation [1].

Neolithic and Bronze Age samples yielded only very partial profiles, with a low rate of replication in amplifications. In both cases, we studied apparently well-preserved teeth. However, due to their older age, it is important to take into account a longer exposure to the taphonomic environment and subsequently a higher DNA degradation.

4. Conclusions 

This study demonstrates the usefulness of the MiniFiler™ Kit in the field of ancient DNA, specifically in the case of well-preserved historical samples, such as the case of “El Cuellar” samples. Results from prehistoric materials (Cogotas Culture and Can Sadurní Cave) did not show neither sufficient reproducibility nor markers enough of the total set comprised in MiniFiler™ Kit.

Role of funding 

This study was funded by a MICINN project (CGL2006-07828/BOS) and a CAM-UCM project (CCG08-UCM/BIO-3938).

Conflict of interest 

None.