Next Generation DNA Sequencing

By Mass Communication Specialist 2nd Class Samantha Thorpe Armed Forces Medical Examiner System

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3/17/2016 - DOVER AIR FORCE BASE, Del. -- When American biologist James Watson and English physicist Francis Crick first discovered the double helix in 1953, they could not have imagined the huge strides DNA technology would take in just a few short years. From the 1977 development of the Sanger Sequencing technique by Frederick Sanger to the 1996 birth of Dolly the Sheep, the first ever cloned animal, to the 2003 completion of the Human Genome Project, which sequenced the human genome to 99.9 percent accuracy, scientists have continually improved upon their methods and techniques to lead us to the DNA Sequencing of today.

The DoD DNA Registry, Armed Forces DNA Identification Laboratory (AFDIL), which is part of the Armed Forces Medical Examiner System (AFMES) at Dover AFB, Del., is working with the newest innovation in Forensic DNA science, Mitochondrial DNA (mtDNA) Hybridization Capture and Next Generation DNA Sequencing (NGS).

Utilizing this new technology and process, scientists will be able to obtain mtDNA sequencing results from severely degraded DNA samples that previously failed with traditional sequencing methods.

"Over the past 15 years, AFDIL has been working with the Defense POW/MIA Accounting Agency (DPAA) to sequence mtDNA samples from 100 of 800 unknown Korean War service members interred, or buried, at the National Memorial Cemetery of the Pacific in Hawaii, also known as the Punchbowl," said Dr. Timothy McMahon, Deputy Director of Forensic Services, Contractor with the American Registry of Pathology Sciences LLC. "The problem is that the mtDNA within the cells has been damaged by the environment and preservation methods used in the 1950's."

McMahon explained that in 1953, as part of Operation Glory, North Korea turned over 4,167 deceased U.S. and NATO service member's remains, 849 of which were not identified. The remains were sent to Camp Kokura, Japan where they were prepared for burial using chemicals that were harmful to the mtDNA. Using new instrumentation, AFDIL was able to identify that the mtDNA obtained from these samples is far smaller than their current mtDNA sequencing testing capabilities. Once AFDIL was able to determine the size of the mtDNA, the scientists were able to develop a custom testing method using NGS technologies.

"Since 2000, one hundred of the unknown service members have been disinterred from the Punchbowl by DPAA," said McMahon. "Using current mtDNA sequencing methods, only one of the samples has been accurately sequenced. Thanks to NGS technologies, AFDIL will be the first forensic laboratory in the U.S. to utilize a laboratory developed mtDNA NGS sequencing method to conclusively sequence the smallest, most degraded forensic samples. Additionally, this method has the potential to allow for the identification of any unknown service member whose mtDNA has been chemically treated."

Before this most recent scientific breakthrough, Sanger Sequencing was the forensic standard for mtDNA sequencing, and had been for almost 40 years. The issue that occurred with this long-trusted technology was that it did not give AFDIL the capability to efficiently amplify small fragments, such as the remains from the Punchbowl, in order to sequence them using the Sanger method.

"These samples from the Punchbowl are unique because they were treated post-mortem and the chemicals used to preserve the remains severely damaged the mtDNA," said Supervisory DNA Analyst, Kerriann Meyers. "Our current technology uses primers to target mtDNA fragments that are 120 base pairs in length, but our fragments from the Punchbowl are under 100 base pairs. I think a lot of analysts used to shy away from working with the Punchbowl samples because only 0.1 percent of the samples would come back conclusive and so it was very discouraging."

In 2003, Senior Research Scientist, Dr. Odile Loreille, was hired to aid in the sequencing of the Punchbowl samples. Due to her background in ancient DNA, Loreille was able to understand the highly damaged mtDNA which then led her to begin work on a new sequencing method in 2010.

"Odile's specialty was working with ancient DNA, which is DNA from archeological and historical specimens," said Higginbotham. "That experience is what gave her the insight to begin work on the new protocol. From there Dr. Charla Marshall, Chief, Emerging Technologies Section, Kim Andreaggi, Research Scientist and I worked tirelessly to turn that idea into a forensic protocol, validate and then implement it."

The massive parallel sequencer used to create the new NGS protocol was originally created to sequence small fragments of DNA with fewer than 350 base pairs, but AFDIL took it even further when they began work on sequencing mtDNA that could be smaller than 120 base pairs.

"Other laboratories are also making progress on [forensically] validating NGS, however none of them are working with samples as degraded as the ones we see coming from the Punchbowl," said Higginbotham. "What we are doing with these samples is unique thanks to our mission, and I don't think other laboratories realize how damaged our samples really are."

In order to begin the process of sequencing the extremely damaged mtDNA, the analysts treat the samples, which they receive from DPAA, similar to how the samples would be treated using the current method.

"The bones are sanded and ground down to a powder, the [DNA] extraction is carried out using a demineralization process and it is then allowed to incubate overnight," said Higginbotham. "The extract then contains the isolated mtDNA which is run through a bio-analyzer instrument to ensure there are enough quality fragments to get an authentic result."

Next, the samples are treated with a Uracil Specific Excision Reagent (USER) kit to remove damaged bases.

"While we call this step a repair, we are actually damaging the DNA more by leaving open sites and overhanging ends where the damaged bases were removed and leaving the fragments shorter," said Research DNA Analyst, Jennifer Higginbotham. "After this, we begin our library preparation end repair step."

During this step, a negative control is added to the sample to monitor for any possible contamination throughout the process and a positive control is added to ensure the final reaction functions properly. Next, the open sites and overhanging ends are filled with complementary bases. This forms blunt ends on the samples to which adapters are ligated, or attached.

"We then place a unique barcode on each end of the sample fragments," said Higginbotham. "Once the barcodes are added we can pool, or combine, all the samples in the sequencer at one time. The unique barcodes allows the computer software to later separate and group matching samples."

When the library preparation is completed, the samples are screened by the bio-analyzer to assess the quality of the libraries. If the quality is poor, the library preparation is repeated.

"At this point samples using traditional NGS methods could be sequenced, however, the Punchbowl samples have extremely high amounts of non-human DNA," said Higginbotham. "If we were to sequence them at this time we would have around one percent of the reads mapping as human. So what we do is enrich for the human DNA using a process that the ancient DNA community calls hybridization capture."

Hybridization capture consists of baits, or probes, which are made up of 75-base-pair sequences from the human genome DNA reference sequence (rCRS). These baits are created to be complimentary to and target specific sequences in the mtDNA genome. The baits are added to the sample and incubated for about 24 to 36 hours. Magnetic beads, which have a strong affinity to the baits, are then introduced. When removed, the magnets pull the baits out and with the baits come the sections of the mtDNA fragment they were attached to. This process, which is also called target capture, allows the non-human DNA in the sample to be removed from the human mtDNA being targeted.

Next, a Polymerase Chain Reaction (PCR) copies the target mtDNA in order to increase its yield, giving the sequencer more fragments to work with and a better chance at sequencing them.

"The last step is to combine our samples in equal volume to create a pool which is loaded on the sequencing instrument," said Higginbotham. "Included in the pool is the reagent blank from the extraction process, a negative control which we added in library preparation, three samples and a positive control which is used to ensure the reaction, in whole, is successful."

The pool is then placed in the sequencer and takes approximately 24 hours to complete sequencing. Once this is accomplished, a profile is made for the service member's mtDNA in order for it to be later compared to possible familial matches already on file.

"The whole NGS process from beginning to end takes approximately two weeks to complete and is extremely labor intensive with very low through-put compared to AFDIL's current past accounting processing methods," said Lt. Col. Alice Briones, Deputy Chief Medical Examiner and Director of the DoD DNA Registry. "Although it has taken 10 years of research to develop a method for getting mtDNA forensic results from chemically modified samples, I am extremely proud and honored to be a Medical Examiner and the current director of the DoD DNA Registry as we bring new hope to numerous families of our nation's fallen who thought their loved ones would never be identified."

Over the years, technology and science has continued to evolve and with this growth comes the great opportunity to not only discover new possibilities but also make a difference in many people's lives.

"When I was asked to be part of the NGS team I immediately said 'yes, absolutely' because after all the inconclusive results I have had to report, it will be amazing to get the opportunity to report positive results from the Punchbowl mtDNA samples," said Meyers. "I also think the families of these fallen service members have been waiting a very long time for technology to catch up to their unique situations, so I think that it's huge for the families in helping them get answers and maybe some closure. To be part of this process has been just amazing."

As technologies in mtDNA sequencing continue to rapidly grow and evolve, who knows where we will be another 40 years from now. What we do know now, is that the families of our fallen service members from the Korean War are this much closer to getting the answers they have been searching for, for so long.
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