A Blind Testing Design for Authenticating Ancient DNA Sequences

Reproducibility is a serious concern among researchers of ancient DNA. We designed a blind testing procedure to evaluate laboratory accuracy and authenticity of ancient DNA obtained from closely related extant and extinct species. Soft tissue and bones of fossil and contemporary museum proboscideans were collected and identified based on morphology by one researcher, and other researchers carried out DNA testing on the samples, which were assigned anonymous numbers. DNA extracted using three principal isolation methods served as template in PCR amplifications of a segment of the cytochrome b gene (mitochondrial genome), and the PCR product was directly sequenced and analyzed. The results show that such a blind testing design performed in one laboratory, when coupled with phylogenetic analysis, can nonarbitrarily test the consistency and reliability of ancient DNA results. Such reproducible results obtained from the blind testing can increase confidence in the authenticity of ancient sequences obtained from postmortem specimens and avoid bias in phylogenetic analysis. A blind testing design may be applicable as an alternative to confirm ancient DNA results in one laboratory when independent testing by two laboratories is not available.