Unidentified human remains often are associated with tragic events, such as fires, terrorist attacks, natural disasters, war conflicts, genocide, airline crashes, homicide, and human rights violations under oppressive totalitarian regimes. In these scenarios, bone may be the most viable sample type for DNA testing. Additionally, skeletal remains often are the only samples available in historical and archaeological cases.
Skeletal remains are among the most challenging sample types for DNA testing due to prolonged exposure to a variety of environmental insults, including the effects of soil acidity. Humic and fulvic acids in soil damage DNA and, if co-purified, inhibit PCR amplification.
Written by: Angie Ambers, UNTHSC
Since DNA recovered from bone often is degraded and in low quantities, autosomal STR typing sometimes fails or results in partial profiles which may not be sufficient for rendering an identification. In cases involving unidentified males, Y-chromosome lineage markers can serve to supplement autosomal STR results and anthropological metadata to increase statistical confidence in identification efforts.
Although the accuracy of identification of skeletal remains increases as the number of relatives typed increases, in some cases the number of reference samples available can be quite limited. One approach to improving the power of identification is to type additional markers. Lineage-based Y chromosome markers can provide additional data to support or refute putative familial relationships. In some cases, lineage markers may be the only informative markers for making associations between unidentified remains and living relatives.
Two early generation kits, PowerPlex® Y (Promega Corporation) and Yfiler™ (Thermo Fisher Scientific), contain reagents to simultaneously amplify 12 Y-STRs and 17 Y-STRs, respectively. Both of these kits contain the core Y-STR loci advocated by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the European minimal haplotype. Since the development of the two aforementioned kits, a considerable amount of effort has been dedicated to identifying additional Y-STR loci that may increase the discriminatory power of Y haplotype data, as well as be more effective for amplification of challenged samples. Both PowerPlex® Y23 (Promega) and Yfiler™ Plus (Thermo Fisher Scientific) are enhanced Y-STR multiplex kits that may increase the typing capability of Y-STRs in bone samples.
The Yfiler™ Plus kit includes the 17 Y-STR markers from the original Yfiler™ kit plus 10 additional highly polymorphic markers (DYS449, DYS460, DYS481, DYS518, DYS533, DYS570, DYS576, DYS627, and DYF387S1a/b). Seven of the ten additional loci included in the Yfiler™ Plus kit are rapidly mutating Y-STRs (DYS449, DYS518, DYS570, DYS576, DYS627, and DYF387S1a/b). Several validation and population genetic studies have been performed using the Yfiler™ Plus kit but none evaluating its ability to type DNA extracted from skeletal remains.
Our study evaluated the efficacy of the Yfiler™ Plus kit for typing degraded DNA from human skeletal remains from the American Civil War (1861-1865), the Black Hills Gold Rush in Deadwood, South Dakota USA (1874-1877), the Seven Years’ War (1756-1763), a 17th century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). Comparisons were made to the previous generation Yfiler™ kit.
Due to the particular challenges encountered with human skeletal remains and degraded DNA, data analyses focused on a few key areas that are crucial for identification: (1) total number of alleles detected, (2) performance with larger loci, and (3) sensitivity.
Regardless of the age, condition, or origin of the remains, 22 out of 24 bone samples tested yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. Although 1ng of input DNA is recommended for both Y-STR multiplexes, this target quantity was not available for the samples used in this study, a limitation commonly encountered with degraded skeletal remains. For this sample set, the range of input DNA was 0.100ng─0.827ng.
Furthermore, although a multiplex with a greater number of loci (e.g. Yfiler™ Plus) increases discriminatory power, another important consideration for degraded samples is amplicon size and potential inhibition. Skeletal remains often contain DNA of limited quantity and compromised quality, both of which contribute to reduction or loss of signal at larger loci. In these types of samples, incomplete genetic profiles due to allele and/or locus drop-out are well documented. Therefore, due to the degraded nature and/or quality of the bone samples used in this study, the performance of the seven largest loci that are common between the two kits (DYS385a/b, DYS389II, DYS392, DYS438, DYS448, DYS635) was compared. Out of 24 bone samples tested, higher success rates for the larger common loci were achieved with the Yfiler™ Plus kit compared to the Yfiler™ kit. These data suggest that the Yfiler™ Plus kit can overcome inhibition of the downstream assay better than the Yfiler™ kit.
In addition to increased discriminatory power and successful typing of larger Y-STR loci, an equal or improved level of sensitivity of detection is desirable for forensic casework. The low-signal (RFU) data encountered in analyses of bone samples is an important consideration in assay selection. If addition of more loci to a multiplex reduces overall signal and performance, it may not be desirable for use with challenging casework samples. Even minimal reduction in signal across loci presents a risk of potentially losing valuable genetic data that could have otherwise been detected using a smaller, less discriminatory assay.
For the majority of bone samples tested (20 out of 24), Yfiler™ Plus performed the same or comparably (within one allele) on the common alleles observed from standard Yfiler™ amplifications. Allele designations were concordant between the two kits for all samples. More importantly, for most of the samples (21 out of 24) undetected Yfiler™ alleles were recovered with Yfiler™ Plus amplifications as well as the ancillary benefit of detection of additional alleles with the expanded loci. These observations further support that the Yfiler™ Plus kit is a more robust assay both in terms of sensitivity and in overcoming inhibition.
A general pattern observed was that samples which performed well with Yfiler™ also performed well with Yfiler™ Plus. However, some samples yielding poor results with Yfiler™ were substantially improved with Yfiler™ Plus. For example, although two of the Deadwood bone samples yielded no typable data with Yfiler™, partial profiles of 9 alleles and 10 alleles, respectively, were obtained using Yfiler™ Plus. In another case, a complete 27-locus Y-STR haplotype was obtained using Yfiler™ Plus for a sample which yielded only 3 detectable alleles using the Yfiler™ kit.
For the ten additional loci included in the Yfiler™ Plus multiplex (compared to the earlier generation Yfiler™ kit), an average of 6 additional alleles were obtained across all bone samples (range 2−10 alleles). This data provides improved discriminatory power than could have been possible using the earlier generation Yfiler™ assay. The potential for increased data acquisition from the same quantity of DNA using Yfiler™ Plus warrants its consideration for use with challenged forensic samples types such as skeletal remains and bone fragments.
Value of rapidly mutating Y-STRs
Another important potential benefit of the Yfiler™ Plus kit relates to the assay’s inclusion of rapidly mutating Y-STRs. Some studies have shown that rapidly mutating Y-STRs can increase the power of discrimination between unrelated males as well as between males of the same patrilineage. In a study of 305 males from 127 separate familial pedigrees, the rapidly mutating Y-STR panel included in Yfiler™ Plus could distinguish between 1) 48.7% of fathers and sons, 2) 60% of brothers, and 3) 75% of male cousins. The discriminatory power of the previous generation Yfiler™ kit was considerably less with the same data set, at 7.7%, 8%, and 25%, respectively [https://doi.org/10.1016/j.fsigen.2011.04.017]. This increased ability to distinguish between related males could have considerable application for mass disaster scenarios in which multiple victims from the same family were traveling together, as well as for mass graves from past war conflicts (which may contain related male soldiers), and for mass graves containing multi-generational victims of oppressive regimes.
The Center for Human Identification (CHI) is fortunate to have developed partnerships and collaborations with prominent, respected forensic scientists both here in the U.S. and abroad. This provides invaluable opportunities to share best practices, develop new ideas, and collectively assist in the advancement of our field. These collaborations also give us access to a diverse set of forensic casework and historical samples from which to evaluate the efficacy of our current methods. This project was supported in part by Thermo Fisher Scientific, the Department of Forensic Medicine at the University of Helsinki, Forensic DNA Service (Prague, Czech Republic), and the Czech Science Foundation.
Dr. Angie Ambers has a Ph.D. in molecular biology (with emphasis in forensic genetics and human identification) as well as master’s degrees both in forensic genetics and in criminology. She is currently a full-time forensic geneticist for the UNT Center for Human Identification. She specializes in characterization and identification of contemporary, historical, and archaeological human skeletal remains. Her recent casework has involved DNA testing of an American Civil War guerrilla scout, several Finnish World War II soldiers, unidentified late-19th century skeletal remains discovered by a construction crew in Deadwood, South Dakota, unidentified skeletal remains of Special Operations soldiers killed during the 1974 Turkish invasion of Cyprus, skeletal remains exhumed from Prague Castle in the Czech Republic, skeletal remains of soldiers from the Seven Years’ War (1756-1763), and bone samples purported to belong to a member of Jesse James gang (killed during a bank robbery in 1876). In October 2015, she co-presented a workshop on “Advanced Methods for DNA-based Analysis of Skeletal Remains” at the 26th International Symposium on Human Identification, and was an invited speaker at an international bone workshop/conference in Prague, Czech Republic in December 2015. In 2017, she traveled twice to India to train scientists from various Indian states and the Maldives Police Service on the processing of bone samples in forensic DNA casework.
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