Exploring the Value of Biogeographic Ancestry in the Absence of a DNA Database

There are many scenarios encountered in forensic casework in which bone may be the only viable sample type for DNA testing, including fires, terrorist attacks, natural disasters, war conflicts, airline crashes, homicides, human trafficking, and mass graves. In these cases, the condition of recovered human remains can range from relatively intact to highly degraded and/or fragmented. In some homicides, perpetrators dismember the bodies of their victims and hide or disperse the individual parts across a wide geographic region in order to impede discovery of the remains, as well as to hinder subsequent identification efforts. Additionally, in terms of forensic investigation, unidentified remains in an advanced state of decomposition often essentially become ‘skeletal’ cases because the soft tissue is too decomposed to obtain usable data for positive identification. Decomposition of soft tissue prevents visualization of distinguishing tattoos or scars on the skin, and can nullify the possibility of a fingerprint match or facial feature recognition. In these cases, bone is the most viable sample for DNA testing and therefore may be the only avenue for identifying a decedent.


Written by: Dr. Angie Ambers, University of New Haven



In the U.S., thousands of children and adults vanish without a trace and/or under suspicious circumstances every year. The latest statistics available indicate that the National Crime Information Center (NCIC) contains 88,089 active missing persons records and a registry of 8,634 unidentified (skeletonized) remains. However, the actual number of unidentified skeletal remains that exists likely is substantially higher, given that many victims remain undiscovered to date.


Fortunately, the U.S. has a well-established database containing DNA profiles of missing persons and/or their relatives, for comparison when skeletal remains are recovered and to assist in identification efforts. However, although forensic DNA typing is widely conducted around the world, many countries still have not developed and/or populated reference DNA databases to assist in cases involving unidentified human remains (UHR).


In 2017, I traveled twice to the Gurgaon/Gurugram region of Delhi, India to train scientists from various Indian states and the Maldives Police Service on the processing of bone samples in forensic DNA casework. During the workshop training, I had the opportunity to work on an unidentified skeletal remains case with Dr. Vivek Sahajpal and Dr. Arun Sharma, two exceptional forensic DNA scientists from the State Forensic Science Laboratory in Himachal Pradesh.


Himachal Pradesh is a northern state of India, situated in the western Himalayas. Most of the state lies in the foothills of the Dhauladhar Range, with a scenic landscape of snow-clad mountains, deep gorges, densely forested valleys, large lakes, and cascading streams. Due to the majesty of the region, tourism is a major contributor to the state’s economy and growth. Popularly referred to as “The Land of Wanderlust,” it is a well-known trekking and climbing destination for adventure- and spiritual seekers. Additionally, Himachal Pradesh is the home of the Dalai Lama and the Tibetan government-in-exile. The strong Tibetan presence is reflected in the state’s Buddhist temples and monasteries, as well as during its vibrant festivals and celebrations.


In 2016, human skeletal remains were discovered along Indrahar Pass, a mountain pass in the Dhauladhar range of the Himalayas, located at an altitude of 4,342 meters (14,245 ft) above sea level. Indrahar Pass is near the town of Dharamshala in Himachal Pradesh and attracts substantial tourist traffic during the trekking season (April – October). In the last two decades, approximately 30 foreign tourists have gone missing in Himachal Pradesh, notably near the tourist hotspot of Dharamshala. The majority of these tourists were of European descent, including trekkers from the United States, Australia, France, England, Russia, Italy, Sweden, Ireland, Yugoslavia, and the Netherlands. When unknown skeletal remains were discovered in 2016, Indian authorities understandably assumed that the remains belonged to one of these missing tourists, and implored forensic scientists to attempt to recover DNA in an effort to identify the decedent.


Although the decedent’s endogenous STR profile had been determined, a DNA database was not available for kinship analysis and/or comparison to exemplars. At this stage, the only feasible option for identifying this individual involved reaching out to government officials in the “home” countries of each of the 30+ missing tourists and requesting that family reference samples be collected for comparison to the profile obtained from the skeletal remains. Aside from logistical challenges in undertaking this extensive effort, there are sensitivity issues that must be considered with such an approach. The families of these 30+ missing persons had already experienced a tremendous amount of trauma and grief due to unanswered questions associated with the disappearance of their loved one. Although discovery of a loved one’s remains and the ability to provide a proper burial often can provide some measure of “closure,” the reality in this case is that we had no reasonable level of confidence in assuming that the remains belonged to one of these families.


Since STR genotyping revealed that the remains were male – and Y haplogroups can be used to infer biogeographic ancestry – a decision was made to perform additional testing with markers that are specific to the Y chromosome (using the 27-locus Yfiler™ Plus multiplex by Thermo Fisher Scientific). With the alleles in the decedent’s Y-STR haplotype, Y haplogroup prediction was performed using the ancestry feature and metapopulation tool of the Y-STR Haplotype Reference Database (YHRD). The YHRD-based prediction was cross-checked with HaploGrep software. Analysis of the Y-STR alleles in the profile obtained from the skeletal remains revealed that the decedent was of Asian descent (not European).


In addition to traditional capillary electrophoresis (CE)-based methods for Y-chromosome typing and the utility of Y-STR markers for inferring biogeographic ancestry, the advent of massively parallel sequencing (MPS) technology has greatly expanded the tools available to scientists for predicting the ancestral origin of unidentified skeletal remains. In this case, samples from the decedent were further tested using the ForenSeq™ DNA Signature Prep Kit (Verogen, San Diego, California USA) and the MiSeq™ MPS platform.


Ultimately, although the identity of this individual remains unknown to date, the possibility of causing further emotional trauma to families of 30+ missing European and American tourists was circumvented due in large part to recent advances in forensic DNA technology. Although massively parallel sequencing (MPS) is also known as next-generation sequencing (NGS), the reality is that it is no longer “next generation.” Research laboratories have been using the technology for years, and many of the accompanying assays/kits have been through the validation process in preparation for implementation into forensic DNA casework. The changing face of forensic genetics now offers the capability to obtain biogeographic ancestry and phenotype information from unknown samples, which could provide important investigative leads in cases that otherwise may have progressed in the wrong direction. The capability to obtain far more information from an evidentiary sample than was previously possible in the traditional casework landscape likely will alter the way forensic genetic investigations are approached in the future, particularly with unidentified human remains (UHR).


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