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Introduction
Ancient DNA (aDNA) analysis has been carried out on various ancient samples of human, animal, and microorganic origin [1-5]. Since its introduction in the 1980s, it has grown in both application and utility by answering many questions in the archaeological and anthropological sciences (Fig. 1) [6-12]. Having now become almost routine, aDNA analysis is considered by scientists to be an indispensable research tool (Fig. 2) [5].
There have also been technical difficulties, however, concerning authenticity. At the earliest stage, aDNA analysis commonly depended on polymerase chain reaction (PCR) to amplify small amounts of endogenous DNA in specimens. The problem was that, whereas ancient samples contain extremely low levels of endogenous DNA, the nucleic acids amplified by PCR are commonly exogenous [13], which means that PCR does not necessarily amplify target aDNA but rather, might detect modern DNA that had contaminated ancient samples [14].
To resolve this issue, in the 2000s, researchers speculated that strict adherence to prescribed research protocols could reduce the likelihood of modern DNA contamination of ancient samples to a minimum [7, 15]. The protocols include the use of disposable protective clothing, cloning and sequencing, independent replication of experiments, ultraviolet irradiation, blank controls, treatment of uracil-N-glycosylase, and physical isolation of pre-PCR facilities, among still others (Table 1, Fig. 3) [14]. Nevertheless, these cannot be considered an ultimate solution to the authenticity problem.
Review
A novel aDNA analysis technique
Fortunately, with the high-throughput sequencing advancements over the past decades, aDNA research was revolutionized, remarkably bolstering aDNA analysis authenticity. Specifically, by next-generation sequencing (NGS), ultra-short fragments of ancient genomes can be more reliably analyzed, even in cases where the amount of contaminant modern DNA far exceeds that of endogenous aDNA in samples. NGS came to rapidly replace PCR, thereby potentiating more consistently successful aDNA analysis outcomes [16-25]. Thanks to all of this, whole-genome sequencing and large-scale population-level studies via high-resolution paleogenomic analysis of ancient samples have been rapidly increasing in number [8, 26-28]. There is also optimism that paleogenomics will very soon offer revelations on phenotypic changes and histories of infectious diseases [8].
In fact, most aDNA research in the 2020s has been based on new techniques such as NGS. In archaeological and anthropological research, however, NGS relative to PCR is not so simple or cheap to apply to any ancient samples. Actually, NGS application remains a technical barrier for most archaeologists.
Moreover, cases considered important by archaeologists might not be regarded as equally significant by genetic scientists, who seek to uncover molecular histories of animals, evolution, and migration on the grand scale. Thus, ancient samples reflective of the more particular and antiquarian interests of archaeologists often are regarded by genetic scientists as unworthy of NGS implementation with its expense and complexity.
Cost-effectiveness and other practical considerations in aDNA research
When a new, powerful tool superior to the established technological mode of research emerges, should the old one be completely discarded? The extent to which PCR has been eliminated from aDNA study is due mostly to the belief that it is not sufficiently reliable for highly degraded ancient samples. And yet, PCR has not been eliminated in the other fields of biological research. In the case of forensic science for instance, PCR is still widely used to obtain genetic profiles from highly degraded samples. Certainly, older technologies and techniques should not necessarily be considered obsolete and unusable.
We note that in clinical medicine, a multiplicity of diagnostic-radiological tools continues to be employed. Comparing simple X-ray with computed tomography (CT) and magnetic resonance imaging (MRI), there is no disagreement that CT and MRI are much more powerful tools; however, the clinical value of simple X-ray, even today, cannot be underestimated. Simple X-ray is cheaper than CT and MRI, and, in certain circumstances, can yield invaluable findings not easily obtainable by either of those newer modalities. Overall, X-ray’s cost savings and efficiency for diagnosis of certain disorders [29] make it, still, one of the most widely applicable diagnostic tools in the clinical setting.
Similarly, we need to reconsider the role of PCR in aDNA analysis. Of course, PCR, when applied to ancient samples, is likely to produce erroneous outcomes more frequently than NGS. Nonetheless, PCR has advantages over NGS. PCR as compared with NGS is simple, time-efficient, easily interpretable, and above all, very cheap. Also, considering that aDNA decays continuously over time [30], is it not astute, where immediate NGS application is not possible or practical, to make preemptive use of PCR?
Note, too, that in the field of archaeology, as already noted, there are many ancient samples that would not draw other scientists’ immediate attention, and for which, aDNA analysis could not be conducted in the short term. For those cases, conducting PCR analysis immediately might well be better than no analysis at all. Indeed, as in clinical medicine, multiple diagnostic tools could be used complementarily: PCR could be coordinated with NGS for aDNA analysis purposes (Table 2, Fig. 4) [31, 32].
Conclusion
Numerous ancient samples are discovered every year at archaeological sites, and the aDNA therein rapidly decomposes over time. Unfortunately, genetic analysis is not so readily or easily applied to those samples, given the financial, time and effort costs incurred in NGS work. In some such cases too, genetic workup is just skipped. As for PCR, whereas it is not a perfect tool for aDNA analysis, it can, in consideration of the aspects of cost-effectiveness, immediacy and other conveniences, be very useful as a support tool in aDNA analysis, provided that the possibility of misdiagnosis is kept well in mind.
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