Forensics 2.0 comes to the Philippines

| Written by Andre DP Encarnacion

In 2016, National Geographic ran a story on the case of Sierra Bouzigard, a 19-year-old from Louisiana, USA who was found beaten to death seven years prior. Although in the fatal struggle Bouzigard managed to get some of her attacker’s tissue under her nails, traditional methods of matching DNA to suspect failed to yield any result. Policemen were stumped.

With the collected DNA their only lead, the case analyst decided to take a chance and send the evidence to Parabon Nanolabs, a company specializing in “DNA phenotyping.” Using so-called single nucleotide polymorphisms (SNPs, pronounced “snips”), which are variations in a single building block of DNA, Parabon promised to conjure a rough likeness of the sample’s owner that would include certain physical features and probable ethno-geographic ancestry.

While the analysis of these characteristics is in its relative infancy and not without controversy, its use in the Bouzigard case highlighted the question of how much more science could contribute in the pursuit of justice. If a broader variety of DNA markers could be simultaneously analyzed to build on what current methods can accomplish, how much more sophisticated could our power to discriminate and investigate become?

The DNA Analysis Laboratory's (DAL) Jazelyn Salvador. (Photo courtesy of the DAL)
The DNA Analysis Laboratory’s (DAL) Jazelyn Salvador. (Photo courtesy of the DAL)


That question is one of many that the DNA Analysis Laboratory, Natural Sciences Research Institute, UP Diliman (DNA Analysis Laboratory, NSRI-UPD) is currently trying to address. Using breakthrough technology called “Next Generation Sequencing” (NGS), University research associates Jazelyn M. Salvador and Dame Loveliness T. Apaga are now getting a first-hand glimpse of the answers.

The gold standard

The laboratory, headed overall by Dr. Maria Corazon A. De Ungria, is no stranger to breakthrough contributions. Its work was a major driver behind the approval by the Supreme Court of the 2007 Rules on DNA Evidence that set terms and guidelines for the conduct of DNA testing and its specific applications in Philippine courts.

The technology behind much of the team’s success to date is called capillary electrophoresis (CE). In creating a DNA profile for any individual using this method, members of the team look at what they call “short tandem repeats” (STRs). These are areas in the genome with sequences of nucleotides, made up of combinations of nitrogen bases: (G)uanine, (T)hymine, (A)denine and (C)ytosine. True to their name, STRs are sequences of these bases that repeat a certain number of times with successive repeats being located next to each other (i.e., TCGA-TCGA-TCGA…).

The laboratory typically examines a person’s DNA profile in 20 of these STR regions that are found across 22 so-called “autosomal” chromosomes, as well as in the X and Y, or human sex chromosomes. In a sample like blood or saliva, which has sufficient amounts of DNA, these pre-selected DNA regions or markers are amplified or “photocopied” via a process called the Polymerase Chain Reaction or PCR.


Researchers Jessalyn Parco and Jan Vincent Beltran at the PCARI Shared Genomics Core Laboratory. (Photo by El Bacani, UP MPRO)
Researchers Jessalyn Parco and Jan Vincent Beltran at the PCARI Shared Genomics Core Laboratory. (Photo by El Bacani, UP MPRO)


“After amplification,” Jazelyn says, “fragments of DNA are separated by length via CE, where they migrate along an electric field through a tube separating anode from cathode. Because of their size, smaller fragments can migrate from start to end much faster.

Fragments, distinguished by the number of repeats using a reference set, are then detected by a laser via fluorescent tags. The result of this process is a person’s DNA profile. As more STR regions are analyzed, the probability that two unrelated individuals would have the same DNA profile on each DNA marker becomes increasingly remote.

Dame also spoke about how CE continues to be the gold standard for human identification. “The system is stable and accurate, and is relatively easy to use for forensic applications.” The technique, however, has its limits. Because of the relative length of STRs, creating a suitable profile may not always be possible, especially with degraded DNA. The latter situation is unfortunately all too common in disaster areas and in many crime scenes.

Next Generation Sequencing

“That’s the advantage of Next Generation Sequencing (NGS), also known as Massively Parallel Sequencing,” Dame continues, referring to the newest technology being validated by the laboratory. “This technology enables a researcher to study and sequence several markers simultaneously, thereby significantly increasing the amount of information that can be mined from the sample.” In fact, NGS can be used to sequence an entire genome at a relatively shorter time compared to more traditional procedures.

The NGS project by the Laboratory funded by DOST-PCHRD has for the most part used Illumina’s MiSeq FGx Forensic Genomics System. Using this platform, billions of short, single-stranded templates of DNA are attached to a slide. Fluorescently-labeled nucleotides are added one by one to the templates, after which a photo is taken that captures light from color-coded bases. The process is repeated with these bases added one at a time until sequencing is complete.


UP Diliman researcher Jessalyn Parco using Illumina's MiSeq FGx TM Forensic Genomics System. (Photo by El Bacani, UP MPRO)
UP Diliman researcher Jessalyn Parco using Illumina’s MiSeq FGx TM Forensic Genomics System. (Photo by El Bacani, UP MPRO)


Jazelyn says this technique allowed the team to simultaneously analyze not only STRs, but also SNPs. In a paper published earlier this year, the team analyzed more than 200 markers from 143 unrelated Filipinos who volunteered to provide samples. These DNA markers included 28 autosomal STRs, 24 STRs from the Y chromosome and 7 from the X chromosome, providing supplementary information that can be vital in resolving complex kinship cases.

Moreover, they were also able to analyze 173 SNPs, including 22 phenotypic informative SNPs and 56 ancestry informative SNPs. “The purpose of ancestry SNPs is to determine the bio-geographic lineage of individuals,” explains Dame. “If you test an individual, these markers can predict whether one is likely to be Caucasian, East Asian, or from another group based on reference population datasets.”

“Phenotypic SNPs, on the other hand determine externally visible characteristics. These characteristics include eye color, skin color, and hair color,” Dame adds. Both SNPs, they say, might be extremely important in cases that involve people that have crossed national boundaries, as in the 2004 Madrid Bombing, and those who commit international crimes like human trafficking. In these situations, DNA may provide the lead to aid investigation in the absence of other clues.


The DNA Analysis Laboratory's (DAL) Jazelyn Salvador. (Photo by El Bacani, UP MPRO)
The DNA Analysis Laboratory’s (DAL) Jazelyn Salvador. (Photo by El Bacani, UP MPRO)


Asian-specific markers

Despite these findings, much work remains to be done before the technology can be used routinely in the Philippines. Since many of the markers used to create DNA panels originated from research that involved Europe and the US, the project aims to generate the “Southeast Asian reference population database” that will be more useful for local law enforcement agencies.

“We might find that many of the current DNA markers included in the panel are not useful for the Philippine population,” Jazelyn said. Citing the case of China, which manufactures DNA kits that better discriminate among the local population, she mentioned the possibility of creating kits that are both cheaper and better suited to the region. “What our population-based studies can do is maybe select the most useful markers and come up with something more applicable to the country.”


Dame Loveliness Apaga of the DNA Analysis Laboratory, UPD-NSRI. (Photo by El Bacani, UP MPRO)
Dame Loveliness Apaga of the DNA Analysis Laboratory, UPD-NSRI. (Photo by El Bacani, UP MPRO)


With the use of NGS, Jazelyn foresees an even larger role for DNA in forensic investigations. “Usually,” she said, “we use DNA to answer: Whom does this belong to? But with the discovery and existence of these SNPs, we can use Asian-specific ones to narrow the field, prior to pinpointing identities.”

Just a year after the Sierra Bouzigard story ran on National Geographic, officers arrested a suspect based on Parabon Nanolabs’ profile. As the DNA suggested (and contrary to police speculation), the man was not Hispanic, but Caucasian. He had fair skin and blue-green eyes. DNA later taken from an item he discarded finally matched the sample from under Bouzigard’s nails. Police believe they have their man.

Does this case represent the future of forensic analysis in the country? With a little more work, the answer seems to be “Yes.”