Dr. Bruce Budowle is the executive director of the Institute of Applied Genetics at the University of North Texas Health Science Center (UNTHSC). Since its establishment in 2009, the Institute has concentrated a large number of well-known scientists who are committed to improving genetics methods to enhance several research fields, including forensic DNA. Students enrolled in the UNTHSC forensic science project (only 14 in the United States) also participated in the Institute ’s forensic DNA project. The research team led by Dr. Budowle not only carried out case studies and human identity testing, but also participated in the research and Development, such as next-generation sequencing (NGS). Their goal is to determine how to best apply and optimize NGS and other methods, and develop operational procedures to simplify the application of these methods in forensic analysis.
Dr. Budowle talked to us about his thoughts on NGS and the potential impact of NGS on forensic genomics.
Q: What forensic DNA research and development work has been carried out by your team in the research institute?
Bruce Budowle (BB): We are developing methods to better recover DNA and remove inhibitors from the environment that may affect our analysis. We are also studying new genetic markers, allowing us to gain a deeper understanding of forensic and human genetics issues, and to identify new technologies that can expand our capabilities. We use a variety of new methods, from synchronous coefficient of drag alteration (SCODA) to the extraction, purification and concentration of DNA in challenging forensic samples, to NGS technology, which can analyze those samples and help us extract the most information.
Q: Do you think NGS will be transformative in forensics?
BB: I think it will become another tool of change that will greatly benefit forensic medicine. Over the years we have had many tools, from restriction enzyme digestion and Southern blotting to polymerase chain reaction (PCR), which have improved molecular biology. Today, we have new NGS tools that allow us to look at things more deeply and analyze more samples in parallel multiplex. NGS also gives us more flexibility to solve the forensic challenges we face.
Q: What are the limitations of the existing forensic process that NGS will intervene?
BB: The biggest gap in forensic medicine is to get high-quality results from the limited information we can get, which comes from a small number of poor quality DNA samples. NGS allows us to obtain higher quality results from a very small amount of DNA samples.
Another limitation is the limitation of our core tags. In many cases, more genetic markers will help us answer forensic questions. For example, when a blood spot is found on the floor, and we compare it with the suspect's sample (reference sample), we directly compare the genetic profile. We look for 1: 1 correspondence with reference samples. In cases of missing persons, we carry out kinship analysis, where we look at part of the genetic map of individuals, which may have the same parts as potential or so-called relatives. That means we don't have much information to compare the graphs. Relative immediate relatives may be simpler, such as parents, children, brothers, or siblings. Identifying half-brothers or cousins, or comparing grandparents to grandchildren can be much more complicated. With the ability to use NGS to create all genetic markers, we will be able to answer some kinship questions that were even unimaginable a few years ago.
Q: What do you think of the forensic laboratory using NGS?
BB: As I said before, one of the real benefits of NGS is that it allows us to analyze more tags. We can apply NGS to reference samples, which will bring us a wider space for typing forensic samples. So we will have an extended set of markers, including single nucleotide polymorphism (SNP), which gives us the flexibility to push the analysis in the direction that best answers the questions in our hands, whether it ’s kinship analysis, direct The analysis is something else, rather than pushing our analysis towards the existing core repeat marker [Standard Tandem Repeat (STR)].
STR is not wrong, but SNP performs better on degraded samples than repeat elements that require larger DNA fragments. If my laboratory wants to study extremely challenging samples, I will choose SNP.
In addition, NGS will bring us more marks, allowing us to cover the entire range of forensics, and allowing laboratories in different countries to communicate better. For example, if one laboratory uses 13 markers and another laboratory uses 20 different markers, the results cannot be easily compared or used to identify suspects. If we can type a group of markers of the reference sample at the same time, it includes the markers selected by each country, then we can communicate more efficiently.
Q: How does NGS compare with Sanger sequencing?
BB: When we carry out Sanger sequencing, the coverage is usually 1x or 2x. With NGS, our coverage can reach 1000x and see the differences that we could not see before. Mitochondrial DNA (mtDNA) may be an example. Due to the replication fidelity of the mitochondrial genome, our sequences will have some differences, resulting in individual heterogeneity or more than one mitochondrial species. Usually with Sanger, we can only see one or two categories. We can handle that level of variation because we build the entire system accordingly. With NGS, due to its depth of coverage, we will see 10-100 mtDNA differences. New problems arise, and we must draw the line of interpretation in another place, but this is not a bad thing. This is just the fact that NGS technology provides excellent depth of coverage.
Q: Illumina's system can sequence a large number of sites simultaneously and sequence multiple samples at once. What kind of information level will bring?
BB: We are currently limited to our analysis because we need a kit for each labeling system and a separate analysis run for each labeling group. The advantage of NGS is that all labels can be processed in the same way, using the same reagents to obtain results. Yes, you must design probes to identify your site, but the technique is the same, so we can use similar reagents to solve all the markers of interest, whether it is STR, SNP, Y-STR, or mtDNA. This will be a real benefit for forensic medicine because it means that an analysis run can provide a complete set of marked results.
Knowing all the markers at once will benefit technologies such as family search. When the DNA map of the suspect cannot be directly matched, the forensic doctor uses this technique to search the evidence map in the convicted recidivism database. In order to find the potential source of evidence for the investigative clues, we then look to see if someone has a similar map to the evidence, which may be a relative of the true source of the evidence. Currently, there are limitations to that type of analysis. A special kit is also needed to view the Y chromosome and X chromosome markers. In the future, we hope to have a technology that everyone can rely on, and all tags are based on that technology. This is what NGS has.
Q: How do you view the value of obtaining NGS results to law enforcement agencies?
BB: Most crime laboratories provide law enforcement with investigative leads in order to refute or confirm a person ’s potential association with physical evidence. This is really important when you want to exclude someone. It helped those who were wrongly connected and allowed the case to move forward. Law enforcement now knows that this person is not a source of biological evidence and will investigate other people who may be involved.
NGS will also benefit cases of missing persons, identify people, and help families find relatives. Most cases of traced populations or unidentified human remains are not just people who just walked into the woods and died. The vast majority were murdered, and the discovery and identification of the corpse was the beginning of a murder investigation. Therefore, NGS technology will bring benefits in various ways. It will help innocent children, enable law enforcement to identify who may have committed crimes, and let the families of missing persons know exactly what happened. It also allows the police to focus their resources on cases that cannot be resolved or given guidance through DNA.
Q: What do you think of NGS 'new capabilities for forensic genomics?
BB: NGS will bring benefits to many areas – those areas that we think are beyond forensics today. One is what we are currently doing, combining pharmacogenomics with molecular autopsy. We know that many of us have a variety of genes, some of which are beneficial, some will cause problems, or will bring risks when faced with environmental stress or pressure.
There are many situations that may be important in viewing an individual ’s genetic makeup and can help determine the cause or manner of death. For example, a 13-day-old newborn died of morphine poisoning. Generally speaking, when this happens, the police will investigate, because this is an unusual death. They will first investigate the parents, or others who have the opportunity to approach the baby. They may also investigate whether the hospital is negligent. In this particular case, the mother's perineum was cut sideways during the birth process and then given codeine. Sequencing showed that she was a super-metabolic type of codeine, with twice the dose of specific genes. Because codeine is metabolized into morphine, infants get morphine from breast milk. This information takes the investigation into a completely different direction, because our strong evidence shows that death was an accident, not intentional murder. With NGS, we are able to sequence a large number of genes at once and treat all possible mutations objectively. This provides us with information that may help us determine the cause of death in challenging forensic cases.
Q: The Joint DNA Search System (CODIS) contains DNA maps submitted by the US federal, state, and local forensic laboratories. What do you think of CODIS's recent development?
BB: CODIS needs to start preparing its system to better accommodate NGS data. CODIS is currently focused on its core content, and everything else is not handled well in its database. With NGS, we think that 30, 40, 50 or 200 tags will be added in the near future, which will increase our ability to search for some tags in the reference data. Most people are driven by the ability to upload things to CODIS. Therefore, if the data from the NGS system cannot be uploaded to CODIS in the future, it will slow down the development of the database. Start thinking about how to accommodate NGS identifiable marks and include them in the next version, which is imperative for CODIS.
Q: How is NGS used for biological lineage?
BB: There are some markers in a group more frequently than others. If you find a variation, then you may infer something related to the biological lineage of the individual. For our missing people work, finding more marks is of great value to us. Currently, when law enforcement finds human remains and skull bones, we hand it over to an artist who can repair the skull bones and provide us with sketches of the dead. Sometimes the sketch is very similar to the real person, and sometimes it is far away.
In the field of phenotype, when we draw a picture of that person, we need to do a lot of work to determine the color – hair color, eye color, skin color. Now we want to focus on marks related to more specific features, such as the traits of the nose, ears, lips, and soft tissue, whether curly or straight. These characteristics may be enough to evoke someone's memories and help identify the repaired individuals, allowing us to enter the traditional DNA kinship analysis. I think this is the true value of NGS in the discovery of biological lineage markers.
Other areas of potential interest are microbiology forensics. Our microbial forensic history can be traced back to the days when anthrax letters attacked, and we continue to participate in biosecurity. I think the huge depth of coverage that NGS can provide will benefit metagenomic research, which will be carried out on the human microbiome, challenging samples, and potential threats. It is the ideal technology in this regard.
Q: What other benefits will NGS provide?
BB: NGS will allow us to automate more analysis and carry out through the software, instead of one person viewing the data and verifying, and the second person viewing and verifying. We will rely more on the ability of the semi-expert software system to solve what marks are there, and the quality value and other small amounts of information can make us more confident in the results. This will be done behind the scenes by the computer, so it can improve our throughput capabilities.
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