This article reports the findings and methodology of a comparative study designed to examine both cloned and direct sequences amplified from ∼3,500 year-old ancient northern fur seal DNA extracts.
The challenges associated with the retrieval and authentication of ancient DNA (aDNA) evidence are primarily due to post-mortem damage, which makes ancient samples prone to contamination from “modern” DNA sources. The necessity for authentication of results has led many aDNA researchers to adopt methods considered to be “gold standards” in the field, including cloning aDNA amplicons instead of directly sequencing them; however, no standardized protocol has emerged regarding the necessary number of clones to sequence, how a consensus sequence is most appropriately derived, or how results should be reported in the literature. In addition, there has been no systematic demonstration of the degree to which direct sequences are affected by damage or whether direct sequencing would provide disparate results from a consensus of clones. In addressing this issue, the current study used majority rules and the Consensus Confidence Program to generate consensus sequences for each individual from the cloned sequences, which exhibited damage at 31 of 139 base pairs across all clones. In no instance did the consensus of clones differ from the direct sequence. This study demonstrates that, when appropriate, cloning need not be the default method, but instead, should be used as a measure of authentication on a case-by-case basis, especially when this practice adds time and cost to studies where it may be superfluous. (publisher abstract modified)