In April 2017, a man set out from New York to hike the Appalachian Trail. For 15 months, he made his way south, stopping along the way to socialize with fellow hikers. Then, in July 2018, his dead body was found in Florida. Investigators determined that he had died of starvation, but they were unable to establish his identity.

His personal effects did not include any form of identification, and his interactions with fellow hikers offered few clues. He went by a trail name, “Mostly Harmless,” or an alias, Ben Bilemy, and he told fellow hikers that he had worked in the tech industry. He also shared that he was hiking the trail before he could be disabled by some unspecified illness.

Othram employees looking at a computer screen
Othram, a company specializing in the recovery, enrichment, and analysis of human DNA from trace amounts of degraded or contaminated materials, asserts that it is the first and only forensic laboratory to vertically integrate a genome-wide test and a human identification solution. In this image, Othram scientists review forensic genome sequencing data from a recent sequencing run.

Two years after Ben’s body was found, conventional investigative tools—fingerprints, witness statements, and missing persons reports—led nowhere. And Ben’s DNA samples, which prompted searches through the usual databases, failed to yield any hits. At this point, Florida authorities announced that they had partnered with a new company, Othram, a DNA laboratory in Texas that provides forensic genealogy services.

Forensic genealogy is just one of several new DNA-based techniques that are helping authorities continue investigations that defy conventional approaches, or even reopen investigations that were suspended long ago. Advances in the field of forensic DNA testing are helping to solve a broadening range of difficult cases, including unidentified persons, sexual assaults, and homicides.

DNA testing was first used to catch and convict a murderer in 1987. Since then, forensic science has become increasingly sophisticated, adopting new techniques in molecular biology and bioinformatics to analyze and identify samples once believed too scarce, too dirty, or too damaged to be useful.

A new generation of forensic analysis techniques

For decades, forensic DNA testing has been done using polymerase chain reaction technology and capillary electrophoresis (CE) fragment analysis to detect length variation in short tandem repeat (STR) markers. A set of 20 standard STR loci are included in the National DNA Index System (NDIS) and the Combined DNA Index System (CODIS), databases that are used by law enforcement.

Because the number of repeats for each of the markers is inherited, sequencing multiple markers can pin down identity matches and relationships with a high degree of accuracy. However, STR analysis has some limitations. Options beyond STR analysis include improved software and laboratory methods to examine mitochondrial DNA, single nucleotide polymorphisms (SNPs), and even whole genomes.

Powerful next-generation sequencing (NGS) technology is driving many of the breakthroughs in forensic DNA testing. Verogen provides the MiSeq FGx Forensic Genomics System. Thermo Fisher Scientific’s semiconductor-based mitochondrial DNA analysis platform is being used by the FBI to generate profiles for the CODIS database.

Promega technology for forensic DNA analysis
Promega technology for forensic DNA analysis is compatible with workflows for capillary electrophoresis (CE) and massively parallel sequencing (MPS). Note that these workflows are generally quite similar up the amplification step. Samples are preprocessed to optimize DNA extraction, and qPCR-based DNA quantification is performed on the extracted DNA. Total human and male DNA concentrations prior to amplification are determined, DNA integrity is assessed, and PCR inhibitors are detected. After the DNA quantification step, STR amplification is performed on a CE instrument, whereas target amplification and library preparation are performed on an MPS instrument.

On the reagent side, Promega recently launched new kits for massively parallel sequencing (MPS), the PowerSeq™ 46GY and PowerSeq™ CRM Nested Systems. The kits are designed to use familiar loci and offer streamlined workflows to allow laboratories to transition more easily to regular use of MPS in their casework. The PowerSeq CRM Nested System received NDIS approval earlier this year.

Older, proven methods are getting an upgrade, too. “Newer MPS-based genotyping assays can extract much more information from an individual sample but require increased time, cost, and interpretation for case reporting,” says Benjamin Krenke, senior global product manager, genetic identity, Promega. “Consequently, nearly every laboratory maintains CE as the ‘workhorse’ genotyping technology due to its balance of discrimination, cost, and time to answer.”

For CE, Promega aims to provide low-, medium-, and high-throughput instrumentation alternatives for the forensic community, which has historically had limited instrumentation options. Promega’s Spectrum Compact and Spectrum CE Systems provide an alternative solution for CE instrumentation, with the Spectrum CE System supporting up to eight-color chemistry, including existing five- and six-color STR kits. Its eight-color capability allows amplification and detection of more loci from challenging casework samples, resulting in more meaningful analyses.

Evidence-to-answer human identification

This wealth of new technologies has added options for once-hopeless cases like Ben Bilemy’s. With the usual options for identifying Ben exhausted, including a missing persons’ search and the CODIS database, Florida detectives partnered with Othram, which is dedicated to solving cold cases and unidentified persons’ cases from difficult samples using NGS and forensic genealogy.

matching genome haplotypes
Representation of matching genome haplotypes (indicated in color) for seven individuals when compared to the DNA of an unknown subject viewed using the DNASolves™ human identity toolkit. The segments are phased and clustered to indicate which groups of matches share relationships to the unknown subject via the same chromosome homolog, and thus via the same side of the family (that is, maternal vs. paternal relations).

Forensic genealogy hit the headlines in 2018 when police used GEDmatch, a database that builds family trees from commercial DNA testing profiles, to catch a serial killer that had been on the loose for over 40 years. The technology, which Othram is deploying in the Ben Bilemy case, could be used more widely by law enforcement, depending on developments in DNA privacy.

Othram CEO David Mittelman, PhD, asserts that the company offers in-house “evidence to answer” analysis using advanced DNA testing techniques: “We evaluate nearly any kind of evidence; we’ll pull the DNA from it and clean it up. Then we get it onto a sequencer and measure anywhere from tens of thousands to hundreds of thousands of markers. We can use that information to reconstruct a representation of the genome for that forensic evidence.”

Othram scientist loads a flow cell into the Illumina NovaSeq 6000
An Othram scientist loads a flow cell into the Illumina NovaSeq 6000, a sequencer that provides exceptionally high throughput. Othram Forensic-Grade Genome Sequencing leverages this platform for maximum
flexibility and scale.

For years, a common limitation of DNA forensic analysis has been the inability to get a whole genome from a poor-quality sample. That could be something like a hair without a root, or DNA that is degraded or burned. Othram has a portfolio of techniques to enhance these evidence types and get usable sequence information from them.

Mittelman says that forensics, unlike other areas in human genomics, is a relatively unexplored frontier. He is helping to start a new scientific journal, Forensic Genomics, as editor-in-chief. Mittelman adds that he expects the technology to be deployed globally: “I’m interested in how the next ten thousand cases are going to be solved and how this technology can be deployed broadly to law enforcement, here in the United States and abroad, to clear as many cold cases as possible.”

By solving Ben’s case and many others that the company is working on, Othram hopes to fulfill a basic need of every human, the need to be connected to others, even if the need can be fulfilled only after death.

More than just software

Software and database management are also important areas of innovation in forensic DNA analysis. Sexual assault cases require a high standard for data management and record keeping.

The status and location of a rape kit after the initial exam, however, is not always available to survivors. Some jurisdictions have lost, misplaced, or failed to process rape kits, leading to major scandals. In one high-profile example, 11,000 untested rape kits were found in an abandoned evidence warehouse in Wayne County, MI, in 2009.

Traditionally, the only way rape survivors can get information about the status of their evidence in the system is directly from the detective on their case. Because of this lack of transparency, background of institutional neglect, and the potential to feel “lost in the system,” lawmakers are giving survivors more access to information about their rape kits.

Tim Stacy, a sales director for STACS DNA, says there’s no reason survivors shouldn’t be able to keep tabs on their kits. “We’re tracking packages with Amazon and FedEx,” he points out. “Why not apply that to sexual assault kits?”

Stacy is slated to give an update on STACS DNA’s tracking system at Bode’s 19th Annual Forensic DNA Conference, which is scheduled to be held virtually on October 26 and 27. Survivors can access STACS DNA’s Track-Kit system with a password and track the kit as it moves through the system. The information available varies between states that have implemented the system. Michigan is currently the only state that will post results through Track-Kit. Other states will just show whether the case is completed.

“A lot of survivors talk about losing their power,” observes Stacy. “What we’re simply doing is giving them some control back.” Having a tracking system is also an important prerequisite if there are going to be requirements for how quickly a kit will be analyzed.

Stacy says that because states implement a tracking system in response to legislation, STACS DNA’s solutions are custom tailored for each state. Rollout generally starts with one city’s law enforcement agencies and hospitals, then expands outward to the rest of the state, but it is always focused on the survivor’s need for updates and privacy. Stacy concludes, “I’ve talked with a number of survivors to get a sense of what they struggle with. For a lot of people, it’s just a software program. For us, it’s a lot more than that.”

GEDmatch gets a makeover

Genealogical DNA matching services are expanding and upgrading to meet law enforcement requirements. GEDmatch originally was founded in 2010 to support genealogical research using results from commercial DNA testing companies. In 2019, Verogen, an NGS company, acquired GEDmatch. Verogen is scheduled to present at the Bode conference about plans to develop an assay that will run on the Illumina MiSeq Forensic Genomics System and integrate GEDmatch for kinship reporting.

Verogen’s strategy recognizes the gap between conventional NGS approaches designed to find medically relevant DNA variations and the methods used for kinship and genealogy analysis. Verogen’s sequence-based approach to SNP genotyping uses short amplicons to overcome the problem of degraded DNA.

“Current methods, including whole genome sequencing and genotyping by consumer-oriented arrays, were not designed for the challenges inherent in forensic samples,” asserts Julian Husbands, Verogen’s vice president of marketing. “As a result, the yield of useful data for genealogy analysis can be somewhat limited.

“Moreover, most labs are not able to process and analyze these samples internally. When they are analyzed, a vast majority of the data generated, such as medically relevant DNA variations, is not relevant to kinship and genealogy analysis.”

Husbands states Verogen’s goal is to orchestrate a seamless solution for its users while maintaining the existing GEDmatch site, and to improve the solution’s underlying security features. The company also plans to create a separate portal for law enforcement matching for users who have opted-in to those searches.

“Cold cases continue to accumulate, leaving many lives disrupted for many years,” informs Husbands. “This includes the lives of family members directly impacted by different types of crime and the law enforcement individuals who search desperately for leads and resolution. We’re excited to be a part of a solution that improves resolution and closure for many.”

The transfer of advances from the life sciences and medical fields to the field of forensic DNA analysis has led to a burst of innovation. Sophisticated genetic analysis techniques are now being used to unlock difficult samples, coupled with software and database tools capable of accurately and securely connecting the information with those who need it.

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