Review articleProgress in forensic bone DNA analysis: Lessons learned from ancient DNA
Introduction
The aim of this review is to share knowledge, techniques and experience with regard to DNA analyses of skeletal samples between the ancient DNA (aDNA) community and the forensic community. Here, aDNA refers to organic material, such as bone, teeth, tissue, hair, dental calculus or coprolites, from organisms that were either recovered from archaeologic or paleontological sites or stored in museums without the aim of preserving their DNA (i.e., classical museum specimens, such as skins or skeletons). Crucial steps in aDNA studies include sampling, laboratory setup, authentication and contamination issues [1], [2], recovery of intact DNA, potential repair of damaged DNA [3], [4] and subsequent fragment analysis, and Sanger sequencing or massively parallel sequencing (MPS) [5]. Some aDNA studies have been conducted by the research facilities of forensic labs [6], [7], but the majority of aDNA work has been carried out in the academic environment.
Ancient DNA presents researchers with a number of problems that are at least partially similar to the challenges faced by forensic DNA scientists. Therefore, standard molecular biology techniques have been widely modified by aDNA researchers to address problems of DNA degradation, contemporary contamination [8], [9] and the presence of various inhibitors [10], [11]. The addition of BSA (bovine serum albumin) to PCR and other enzymatic assays to combat the effects of copurified inhibitors [12] can serve as an excellent example of what is likely the first knowledge transfer from aDNA researchers [13], [14] to forensic laboratories [15], [16] and subsequently to manufacturers of commercial forensic typing kits. The properties of aDNA have been described previously [3], but we describe them here because of their importance in working with aDNA and other types of DNA of similar quantity and quality.
First and foremost, aDNA is present in relatively low quantities [17], at least when compared to modern samples. Therefore, it is of the utmost importance that every step in the process of obtaining and processing DNA from a sample is optimized to maximize DNA yield and minimize DNA loss. This starts with the choice of sample and continues through DNA extraction, PCR or construction of MPS libraries (and potentially includes hybridization capture and sequencing in the case of MPS) and finally bioinformatic analyses. In this context, it is important to keep a second characteristic of ancient DNA in mind, namely, its short fragment length. Although this has long been known [18], [19] (especially since the introduction of MPS methods, which allow harvesting of information from molecules as short as 30 bp), aDNA extraction methods have focused on fragments below 50 bp, which form the majority of aDNA molecules [20], [21]. Thus, from DNA extraction to bioinformatic analyses, the methodologies used in aDNA research must be optimized not only for small amounts of DNA but for small amounts of short fragments of DNA. Third, due to their extended (tens to hundreds of thousands of years) postmortem interval, samples from which aDNA is obtained often contain inhibitors, such as humic acids, that hinder enzymatic reactions used in aDNA processing steps. This creates a conflict since methods that maximize DNA yields often result in relatively high concentrations of coextracted inhibitors. Thus, aDNA extraction methods must be tailored to optimize DNA yield while simultaneously minimizing coextraction of inhibitors. Fourth, in addition to its short length, aDNA often contains additional types of DNA damage that prevents enzymatic manipulation, such as abasic sites and blocking lesions. Interestingly, very little is known about the types and frequencies of these lesions, but the few studies performed [18], [22], [23] suggest that they may be common in at least some samples. Finally, aDNA contains miscoding lesions in the form of deaminated cytosines, which result in C to T substitutions. In contrast to the previously mentioned lesions, the frequency, spatial distribution and genesis of cytosine deamination are well studied and understood.
In this review, we discuss the similarities and differences between ancient and forensic DNA as well as the implications these have on working with forensic DNA from skeletal remains. We do so in the context that aDNA extracted from human skeletal samples can be used to address a variety of questions, including but not limited to determination of familial relationships [24], [25], [26], sex [27], [28], geographical origin [29], phenotype [30], presence of pathogens [31] or even past migrations [32]. Ancient DNA analysis of animal samples can aid in species determination [33], [34], [35], wildlife seizures [36], domestication [37], [38], biogeography [39], and phylogenetics [40], [41], [42]. Ancient DNA analysis is also capable of addressing diverse questions surrounding the origin, distribution, susceptibility, or evolutionary changes in a pathogen and the disease it may cause [43].
We will show that aDNA know-how can aid standard forensic laboratories dealing with challenging samples or working on cases in which a multidisciplinary approach is required. Our analyses suggest that there is room for substantial improvement in forensic protocols regarding skeletal and, more generally, samples with heavily degraded DNA. However, we do not want to imply that sharing and communication of knowledge between the aDNA and forensic community do not currently occur as the two communities have indeed exchanged knowledge for a long time.
Section snippets
Lessons learned
A key characteristic of scientific progress is that it rarely proceeds in a systematic manner. Rather, scientific breakthroughs have ramifications for related fields as well as steps that lie before and after the step of the technology in which the breakthrough was achieved. Analyses of DNA sequences are no exception. Here, the introduction of MPS technologies, beginning in 2005 with the release of the first 454 sequencer by Roche [44], clearly had a transformative effect on all research fields
Sample selection
The first step in DNA analysis is always the selection of a sample for extraction. For a long time, sampling in aDNA research was basically opportunistic, i.e., researchers took the part of the skeleton that was available or where sampling was deemed acceptable by the respective curator. However, in recent years, there have been important insights regarding the part of a skeleton that is ideal for sampling. In contrast, in forensics, there have long been recommendations regarding which part of
DNA extraction
DNA extraction is the next analytical step after sample selection. Researchers working with fresh DNA samples traditionally pay little attention to this step, except when high molecular weight DNA is the aim of the extraction, mainly because the amount of DNA that can be obtained from fresh samples is rarely an issue. This is strikingly different in both forensic and aDNA research because endogenous DNA, i.e., the DNA originating from the species to which the respective sample belongs, is
Repair of DNA modification in crude aDNA
The next step in ancient and forensic DNA analyses can be performed in several ways; the only common denominator is the use of enzymes. The classical use of ancient and forensic DNA extracts was to perform PCR amplification. However, since 2005, there has been an alternative to this approach, namely, construction of an MPS library. This library can then be sequenced directly, or alternatively, certain regions of the genome can be enriched via various types of hybridization capture. Finally,
MPS and associated bioinformatics
As noted previously, there are currently three main approaches for short-read MPS: Illumina, Ion Torrent and the recently available BGISEQ platform. Of course, library preparation procedures as well as the principles of sequencing and base calling differ between the platforms to a greater or lesser degree. Therefore, each platform has a slightly different sequencing error rate, different rates for different types of errors and different biases. These issues have been discussed in both the
Examples for transformation of forensic DNA work
An excellent example of the utilization of aDNA approaches by the forensic community is DNA-based forensic identification of victims from mass graves, which is a forensic niche discipline that utilizes aDNA know-how to deliver scientifically sound results [185], [186], [187], [188], [189], [190], [191], [192], [193], as shown in examples from Guatemala, Former Yugoslavia, World War I, Vietnam War and Spanish Civil Wars I & II. Research on skeletal remains of historical persons, such as the
Potential pitfalls and some notes of caution
While the advantages of MPS for forensic DNA analyses are in our view overwhelming, and we think we have backed up this claim with ample evidence, as with any technology, MPS has its pitfalls. Obviously, as in any DNA analysis technology, a key issue is to avoid contamination, be it from investigators, other samples or previous experiments. This problem was vividly illustrated by one of the earliest aDNA studies using MPS [253] at a time when the field was still rather naïve with regard to the
An outline for the way forward
In this section, we want to make suggestions regarding how the progress made in aDNA research could be incorporated into the more formalized process of forensic DNA work. Of course, there are many means to this end, and the path we suggest is only one of many possible. Since testing all parameters in every possible combination is simply not doable, we suggest a stepwise process that aims at minimizing work while maximizing insight. Additionally, given the endogenous content of many forensic
Concluding remarks
The aDNA typing field began to influence and stimulate forensic DNA identification immediately after pioneering studies in the late eighties [13], [257] demonstrated that aDNA is amplifiable, exemplified by the identification of the infamous Nazi doctor Josef Mengele [258] or the DNA-based identification of murder victim skeletal remains [259]. This process has continued into the present day, mostly in a rather random manner; however, more recently, a technological gap has opened between
Acknowledgments
The work was partially supported by the Charles University Grant Agency (No. 852119).
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2023, Forensic Science International: GeneticsCitation Excerpt :However, it is clear from the 100′s of records evaluated in this review, there has not been a systematic approach to this type of research in the forensic community, and instead consists of many “trial and error” studies of small sample sizes with little to no statistical strength, thus this further supports the merging of samples via conducting SLRs. The lack of a structured approach to tackling this research topic is staggering considering the first wide-spread systematic evaluation in the ancient DNA community took place in 2007, as detailed in [54]. Significantly, in this latter review the authors explain how demineralisation buffers are often discarded in forensic laboratories, yet they have been found to contain large amounts of DNA, which are regularly pooled with “pure” extracts in ancient DNA labs.
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2023, Forensic Science International: GeneticsCitation Excerpt :Failure of this method for genotyping these bone samples may partially be attributed to the requirement for bone chips vs bone powder, due to the latter clogging the microfluidic channels within the RHID sample cartridge. With bone chips, a smaller surface area is exposed to the DNA extraction reagents than when bone powder is used, resulting in lower DNA yields [38,41,42]. In addition, due to the lack of a pre-treatment step to digest bone material, and the short on-instrument bone digestion time during RHID Rapid DNA processing, less than optimal amounts of DNA may be released from the bone matrix.