Charla Kristen Marshall is the Chief of the Emerging Technologies Section at AFMES-AFDIL. She received her B.A. from The Ohio State University, and M.A. and Ph.D. from Indiana University, Bloomington. Her background is in molecular anthropology and ancient DNA.
Dr. Marshall has a keen interest in new DNA sequencing methods. We asked her to comment on some of the more promising new technologies that are being added to laboratories around the world.
Which multiple parallel sequencing (MPS) applications are currently fully validated and implemented in labs?
I am more familiar with the status of MPS in the United States from my SWGDAM involvement on the Next Generation Sequencing (NGS) committee. There are a few laboratories that have MPS applications online right now.
For example, our lab, the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL) has two mitogenome sequencing applications in use. The first is a hybridization capture method for degraded samples, and the second is a PCR-based method for reference sample processing. Both methods use the MiSeq or MiSeq FGx for sequencing.
The California Department of Justice also uses MPS for its mtDNA sequencing in the missing persons unit. Their lab validated the Precision ID Identity Panel on the Ion Chef and Ion Torrent.
Then there are many other laboratories in various stages of the validation and implementation process. These include the FBI Laboratory and Ohio Bureau of Criminal Investigation that validated the PowerSeq CRM Nested System using the MiSeq. The DC Laboratory and the California Department of Justice validated the ForenSeq system for STR typing.
Additionally, Verogen led an inter-laboratory study of the ForenSeq system that involved AFMES-AFDIL, DC Laboratory and UNT, to demonstrate its utility for STR typing. UNT has certainly been playing a leading role in MPS validation efforts, and they will soon be implementing the Precision ID panel in their Center for Human Identification. Collectively, this progress has led to NDIS approval of three commercially available MPS kits (Precision ID Whole MtDNA Genome, PowerSeq Control Region MtDNA, and ForenSeq). I imagine that more US laboratories will be adopting MPS officially now that these kits have been given the green light.
The status of the international laboratories is changing rapidly. Some laboratories have MPS applications online already. These include, for example, the Laboratory of Scientific Police of Lyon in France, which is using the ForenSeq system. The Swedish National Board of Forensic Medicine uses the QIAseq Investigator SNP ID Panel and the MiSeq FGx for sequencing. The Institute for Legal Medicine at the Medical University of Innsbruck in Austria has implemented the Precision ID Whole MtDNA Genome Panel as well as and a primer extension capture method for mtDNA sequencing on the S5. The Netherlands Forensic Institute has an internally developed mtDNA control region assay that uses MPS.
Also based in the Netherlands, the International Commission on Missing Persons has developed a triallelic SNP panel for extended kinship analysis. They have been using the QIAseq chemistry with unique molecular indices (UMIs) and the GeneRead sequencing technology to validate their SNP panel for missing persons applications.
What are the hurdles to overcome for a lab hoping to bring MPS applications into practice?
It seems that the biggest hurdles to overcome for labs hoping to bring MPS applications into practice include the associated costs such as the initial investment in the sequencing system, the translational burden of validation and implementation, and the turnaround time since MPS is currently a longer process than CE.
There is also a significant gap in the implementation of MPS for STR typing, specifically, which is the lack of probabilistic genotyping software for MPS. Such software would be needed before many labs could even consider looking into MPS for their casework.
What benefits are offered by MPS compared to “traditional” STR methods?
There are several benefits that MPS offers over traditional CE methods for STR genotyping. These include enhanced discriminatory power because the sequence variation contained within the STR amplicon is revealed. This makes population frequency estimates of STR alleles more precise, in theory. Yet the STR sequence data must be produced from global populations, and practical aspects of STR sequencing have to be worked out, like nomenclature and allele calling guidelines. Many labs, such as NIST and UNT, are coming together to make STR sequence information of use to the forensic community.
Technologically speaking, the detection of STRs by sequencing offers several advantages to detection by size. First of all, size is no longer a limiting factor to the loci that can be multiplexed in a PCR reaction. In CE-based detection, only loci of disparate size can be assigned the same fluorophore for simultaneous electrophoretic separation, and most instruments allow for a maximum of five or six fluorescent signals. This means that a maximum of a few dozen STRs can be multiplexed for current CE technology. In MPS, almost any loci can be included in a multiplex, given compatible PCR primers. This means that autosomal, Y, and X STRs can be combined in the same multiplexed PCR reaction, which can be beneficial when DNA extracts have limited volume or low template DNA.
Another strength of MPS is locus-specific allele detection that is particularly beneficial for challenged samples with low signal intensity. The locus-specific detection of MPS reduces the background noise when compared to the size-based detection of CE, which is aggregated from all the amplicons in the same dye channel.
Do you expect to see MPS replace STRs at some future point? If so, when would you predict this would happen?
This is an interesting question. I do expect to see MPS replace STRs in the future. MPS is especially conducive to SNP genotyping, which has a number of benefits over STRs. I see the community adopting SNPs for forensic use in the near future, perhaps to be used in tandem with STRs for a defined or even a prolonged period. Then I envision the community phasing out STR typing as reference databases become antiquated due to changing population dynamics, or for criminal databases, due to generational turnover. Perhaps 30 years from now, we will only be looking at SNPs.
What are the most promising DNA sequencing methods on the horizon?
The most promising DNA sequencing methods on the horizon, in my opinion, involve hybridization capture and SNP genotyping. These are two methods that are widely used in clinical and medical genetics as well as ancient DNA. Therefore, these methods can be adapted to any sample type, from high quality reference samples to degraded and inhibited DNA. The flexibility of hybridization capture and SNP genotyping to accommodate the broadest range of DNA samples can revolutionize forensic casework. We will overcome the technical problem of having no results or having uninterpretable results, and we will be facing more of the ethical and fiscal problems of having to prioritize which samples to test - because they all will yield usable results.
What are the ethical and legal ramifications to be considered with new marker sets?
The ethical and legal ramifications to be considered with new marker sets are those that relate to genetic privacy and genetic discrimination. In this technological age in which the ascertainment of genetic information is an easy pursuit, it is important that forensic scientists consider what should be assayed. It is possible to perform whole genome sequencing (WGS) on DNA samples, and as sequencing costs decrease, this may be an affordable approach in the future. Yet WGS would reveal an individual’s genetic predisposition to disease, creating a genetic privacy issue. In my opinion, the basis for new marker sets should be scientifically driven and should take a targeted approach. Even though it may be more costly in the future to conduct targeted sequencing rather than WGS, we ought to limit ourselves to the analysis of the most critical genetic information that will allow us to solve our cases.
Genetic genealogy has become a powerful tool for solving cold cases. Do you see this expanding to more countries outside of the US?
Yes, genetic genealogy has become a powerful tool in the US and has seen some use in Europe as well. However in my opinion, the US will be unmatched in its use of genetic genealogy, and this tool will not hold the same value in other countries. The reasons are many-fold, ranging from the size of the direct-to-consumer DNA testing market outside of the US, the diversity in DNA testing laws worldwide, cultural views about genetic privacy, etc.
Charla, thank you for talking with us today. We look forward to seeing what the future holds for forensic DNA!
Chief, Emerging Technologies Section, AFMES-AFDIL
Charla Kristen Marshall is the Chief of the Emerging Technologies Section at AFMES-AFDIL. She received her B.A. from The Ohio State University, and M.A. and Ph.D. from Indiana University – Bloomington. Charla’s background is in molecular anthropology and ancient DNA. Before coming to AFDIL, she was a postdoctoral research associate at the University of Illinois. For fun, she enjoys spending time with her family and going to the beach.