Table 1.

Authenticity criteria for ancient DNA

Criteria	Importance for authenticity
Reagents and plastic to be used for work with ancient DNA must be checked for the possible presence of amplifiable templates. Since the templates can be present in trace amounts, and thus be amplified in only one out of several samples, multiple checks have to be performed.	Prevents contamination through reagents and disposable materials.
All the manipulations that are used to extract DNA are performed on solutions with no templates using the same solutions. PCR is performed with a double negative control; the normal one (reaction mix with no template) and the reaction mix with the "empty" extract.	Helps detect contamination which could have happened during extraction or during the PCR mix preparation.
Positive controls are usually not used, since they carry the risk of potential contamination.	Prevents contamination.
When possible, several independent extractions of DNA are performed from different areas of the sample.	Helps identify local contamination of the sample itself.
Repeated amplifications of material obtained from the same and from different extractions.	Helps identify sporadic contamination and facilitates the identification of erroneous nucleotides which were included into products amplified from degraded DNA extracts with a small amount of template molecules.
Cloning of amplification products and/or sequencing of multiple clones.	Identifies heterogeneity in the amplified products, which stems from contamination or amplification of degraded DNA with modified nucleotides.
Determination of the number of amplified DNA template molecules (must be determined for each pair of primers, since the number of amplified molecules can vary noticeably depending on the length and the nucleotide content of the amplified fragment, and also on the sensitivity of the specific pair of primers).	Determines the possibility of insertions of nucleotides not present in the original sequence. Extracts which contain only a few or even a single molecule are very much prone to yield erroneous insertions, so it is necessary to perform several amplifications. Extracts which contain at least 1,000 molecules require only one amplification reaction.
Peculiar "molecular behavior," a reverse correlation between the efficiency of amplification and the length of the amplified fragment .	If the sample does not exhibit more intensive amplification of shorter fragments than that of longer fragments, as compared to modern DNA, this indicates that the source of the amplified DNA is contaminated by modern templates.
Biochemical analysis of the level of preservation of macromolecules.	A high level of biochemical preservation of macromolecules indicates a high probability of preserved DNA molecules being found in the sample. This DNA can be analyzed. Thus, the test would support the authenticity of the sequencing results.
Avoid introducing nuclear sequences into mtDNA.	Nuclear DNA has regions homologous to mtDNA, so this fact must be taken into account if mtDNA amplification is used.
Independent confirmation of results in a different laboratory.	This helps identify laboratory contamination of samples or reagents, but it does not rule out contamination that was present in the sample itself (contaminants which were a part of the sample before it arrived at the laboratory, for instance during archeological excavation). This requirement used to be mandatory. Now it has been dropped.