Written by Kelly M. Elkins & Cynthia B. Zeller
SINCE ITS INTRODUCTION in the late 1990s, next generation sequencing (NGS) has found numerous applications in cancer and disease research and clinical applications, microbiology, and crop biology as well as bioforensics, biosurveillance, and infectious disease diagnosis (Kircher and Kelso 2010, Yang et al. 2014, Budowle et al. 2014, Schmedes et al. 2016, Arenas et al. 2017, Minogue et al. 2019). Important lessons were learned from sequencing the human genome that led to sequencing an individual genome (Collins 2003). Upon commercialization of the sequencing instruments, clinical applications began and continue to increase.
NGS has altered genomics research in the past 15 years. Testing that was not affordable or technically feasible has been made possible by NGS (Patrick 2007, Mannhalter 2017). While many laboratories still use Sanger sequencing for forensic and clinical diagnostic applications, NGS is increasingly finding applications in these labs especially as the price for NGS has gone down to approximately $1,000 (US dollars), making it more feasible for routine applications and casework (Mannhalter 2017). Full genomes are mapped with decreased cost and published almost weekly (Børsting and Morling 2015).
NGS has demonstrated that it can overcome issues of efficiency, capacity, and allelic resolution presented by capillary electrophoresis (CE) and reduce the number of false positives in mixture analysis. While NGS is currently being used for the most challenging samples such as human remains samples recovered in cold cases, we expect it will be employed to analyze more routine samples in the future. Labs will decide how and where in their workflow NGS fits.
It has only been since the 2010s that NGS has begun to make an impact in forensic science (Minogue et al. 2019). NGS is currently being used for human identification, phenotyping, and ancestry applications using human blood, buccal, bone, or teeth samples (Jäger et al. 2017). The first NGS kits approved for collecting human genotyping data for Combined DNA Index System (CODIS) searches in the United States criminal justice system were only approved in 2019 (Verogen media release). In parallel to the progress in applying NGS to human genotyping applications, the tool has been found useful in characterizing species of microorganisms for forensic applications (Minogue et al. 2019).
While the research landscape in this area is only beginning to develop, there are several trends and opportunities that are observed. As de Knijff wrote in 2019 in his paper, “From next generation sequencing to now generation sequencing in forensics,” forensic use of CE also took time to be appreciated and adopted.
Ongoing Challenges of Adopting NGS for Forensic Investigations
After years of development, NGS has demonstrated great promise for forensic casework. Although researchers and analysts have developed, piloted, and validated methods and kits, NGS remains a new tool that is being used in forensic casework. Even with all of the possibilities and advantages that NGS offers, there are still very real challenges that must be overcome for NGS to be widely adopted for forensic use.
Issues that need to be resolved prior to adoption for casework include nomenclature, data storage, funding, training, statistics, genetic privacy concerns, contamination issues, reporting, sample tracking, accreditation, casework needs, and acceptance by court (Alonso et al. 2017). The nomenclature for NGS- based STR alleles needs to be standardized. There is no convention for reporting isoalleles or accepted procedure for performing statistical evaluations of the newly identified alleles. Statistics need to be developed and uniformly employed to analyze new NGS alleles. Just as when any new technology is introduced, standard operating procedures (SOPs) need to be developed and the instrument and method need to be subjected to internal validations.
Beginning at the crime scene, investigators need to know the power and limitations of NGS in order to collect the appropriate samples or decide which samples are most promising to send to the lab. Other issues include the potentially long and complex chain of custody and speed of analysis if NGS is used (Gilchrist et al. 2015) and defining the analytical threshold (AT) to avoid overinterpreting the data (Young et al. 2017). As an example, to further analyze raw NGS data and noise to define the AT, FASTQ files were analyzed using the STRait Razor software and Python scripts instead of the Verogen UAS software (Young et al. 2017).
While Sanger sequencing is still the optimal method in terms of time and cost for sequencing short targets and pyrosequencing is ideal for probing DNA methylation variants, NGS technologies have several advantages when many loci need to be sequenced for each sample or for sequencing complete genomes or chromosomes.
An issue that needs to be addressed with regard to NGS is cost. More competitors are needed in the industry to drive down the costs of forensic DNA analysis. Labs need to invest in and implement more automation to reduce preparation time and inter-operator variability. Centralized labs may help counties, states, territories, and countries achieve the economies of scale needed to make NGS cost-effective rather than cost-prohibitive. While Sanger sequencing is still the optimal method in terms of time and cost for sequencing short targets and pyrosequencing is ideal for probing DNA methylation variants, NGS technologies have several advantages when many loci need to be sequenced for each sample or for sequencing complete genomes or chromosomes.
Funding must be made available in the form of grants or included in allocated annual budgets to facilitate access to NGS, either in the form of new a local capability or for sample out- processing. The US DNA Capacity Enhancement and Backlog Reduction Program which funds grants totaling $82 million a year has been approved for the purchase of laboratory equipment and reagents, as well as training for systems that are approved for use with NDIS; grants can help reduce direct costs to labs seeking to implement new technology such as NGS (Verogen media release).
Even if funding is granted through a special program or allocated by states in the annual budget (Funding information for U.S. Forensic Laboratories), labs still must decide which NGS platform to adopt and validate the new kits and instruments at their labs. Labs need to develop and validate SOPs and workflows to process samples and store the high resolution and large sequence datasets (Gilchrist et al. 2015, Aly et al. 2015, Clarke et al. 2017, Mannhalter 2017). As some of the kits are sequencer-specific, the lab will need to decide upon the kit and sequencing instrument. The commercial NGS kits that have been introduced and are approved for input into CODIS are reliable and robust. The Promega PowerSeq 46GY system is sequenced on a MiSeq and the Verogen ForenSeq Signature Prep kit amplicons are sequenced using the MiSeq FGx. The Applied Biosystems Precision ID system amplicons can be sequenced using a ThermoFisher Ion series instrument. Qiagen’s kits are compatible with the MiSeq.
After sequencing is complete, the data analysis begins. Verogen sells its Universal Analysis Software (UAS) for analyzing STR and SNP data and a different version for analyzing mitochondrial DNA typing data. ThermoFisher’s Torrent Suite and Converge software can be used to analyze data from its Ion series instruments. Qiagen’s CLC Genomics Workbench can analyze and visualize data from all major NGS platforms. Versions are available for Windows, Mac OS X, and Linux platforms. Methods still in development will have to demonstrate that they are also sensitive, specific, reliable, and robust through developmental validation.
Many of the commercial software packages developed for NGS data analysis are hosted on the cloud which could be susceptible to outages and cyberattacks. Verogen’s UAS and Illumina’s BaseSpace applications are cloud-based software that can be accessed from any computer using a virtual private network (VPN) client and a lab can make unlimited accounts for its users. ThermoFisher’s Torrent Suite Software can be accessed via the local area network.
Another challenge is the quantity of data produced. NGS generates a huge quantity of data. The data output from the MiSeq is approximately 1 GB per sequencing run. Labs must consider data storage options including external hard drives, cloud storage, and internal server storage when adopting NGS technology. Whereas forensic labs routinely maintain paper backup files of CE and quantitative data with NGS, it is no longer feasible to print hard copies of all of the data. While the actual sequence data files are not large in storage size, the raw digital photographs recorded after each base is added are cumulatively substantial in size. For example, the server shipped with the Verogen UAS can store approximately one hundred sequencing runs. A lab could choose to save only the original raw data and final analyses as intermediate data interpretation files can be reconstructed, as needed, using the software. Labs will need to establish which data to save and back up, and whether off-site services will be acceptable. Adoption of cloud storage is an option for storing a copy of all of the data that will be collected or in-house servers can be purchased and maintained to house the data. All of these options require additional infrastructure and funding, and supporting an in-house server may require additional IT support. Labs will also face the question of which sequencing output files need to be stored indefinitely.
NGS reporting and implementation guidelines have been released and continue to be rolled out. A new version of SWGDAM was released on January 12, 2017, that included NGS in the Internal Validation Guidelines for the first time (SWGDAM). On April 23, 2019, an NGS Addendum to the SWGDAM Autosomal Interpretation Guidelines included background information, core comparison of references and statistical weight (SWGDAM). The application of NGS is included in the FBI Quality Assurance Guidelines and Standards that became effective July 1, 2020 (FBI).
NGS is extremely sensitive. This is a great strength of NGS but also can lead to mixture profiles from samples contaminated with trace contaminating DNA.
NGS is extremely sensitive. This is a great strength of NGS but also can lead to mixture profiles from samples contaminated with trace contaminating DNA. Since NGS is much more sensitive than previous kits and methods, the quality assurance program needs to ensure that all products the lab uses from tips to tubes and other consumables are DNA- free otherwise a plant worker’s non- relevant DNA could be typed. Scientists must be able to differentiate between mutation and error, especially in mixture samples. Error reads typically occur only once. The minimum number of reads under various conditions (e. g., mixtures) needs to be established (e.g., 10 or 50 or 100 reads for low-level contributor) for each locus and sample. Labs need to allocate more analysis time for mixtures.
NGS poses challenges in implementation. Implementing a technology such as NGS requires training of existing staff and validation of the new technology, reagents, kits, and writing new or amended SOPs (Budowle et al. 2014). While the DNA extraction and quantitation instrumentation are largely transferable, staff will need to be trained in NGS technology. While most graduates of Forensic Science Education Programs Accreditation Commission (FEPAC)-accredited programs are well-versed in STR DNA typing methods using CE, as of early 2021, they likely have not had training in NGS. Skilled and experienced analysts will need advanced training courses. They should be reassured that the commercial library preparation kits and sequencing manuals for forensic applications are easy to follow and that their skills are easily transferrable to performing the new protocols. Verogen and ThermoFisher offer training to labs who purchase their instruments and adopt their kits. Colleges and universities are conducting NGS research and adding NGS courses. FEPAC-accredited institutions including Pennsylvania State University, Sam Houston State University, and Towson University are training new scientists in NGS applications for forensic science and developing new NGS-related forensic biology methods. In 2019, Towson University added undergraduate and Masters-level courses in forensic science (FRSC 422 and FRSC 622, respectively) focused on NGS for both autosomal and mitochondrial DNA analysis (Elkins and Zeller 2020). Other institutions offer online NGS courses.
On May 2, 2019, the US FBI approved profiles generated using Verogen’s MiSeq FGx Forensic Genomics System for upload to the National DNA Index System (NDIS). With support from a contractor, Ohio participated in a pilot study of the ForenSeq kit. The Washington, DC and California forensic labs have adopted ForenSeq in their labs. To introduce NGS to the Washington, DC Department of Forensic Sciences, Dr. Jenifer Smith utilized a contractor for implementation support to mitigate the burden on her staff. The Baltimore City Police Department laboratory obtained an Illumina MiSeq instrument with grant support and is validating ForenSeq for casework.
While processing and preparing samples for NGS requires a similar amount of time as CE, the library preparation steps are more time-consuming and the sequencing step takes much longer. Whereas STR fragment analysis on CE takes approximately twenty minutes a sample, and several capillaries are routinely run in parallel, an NGS run with a MiSeq FGx instrument must run to completion to obtain the data for the samples so while 96 samples take a similar amount of time as CE, the time required is standard, so for one sample it is prohibitive. Furthermore, remote labs are challenged with continuous power for long NGS sequencing runs (Minogue et al. 2019). Microbial community profiling of human body fluids, human body site and geographic locations, and evidence items needs to be accepted by courts. Technical and biological validation of the various NGS applications will be required before it can be adopted as a standard tool for use in casework and acceptance into courts (Kuiper 2016).
Microbial NGS methods especially lack standardization of targets and analysis approaches including databases for statistical analysis, especially when unknown and rare taxa are encountered for interpretation using limited published study data. Further development of bioinformatics tools and processed and referred databases are needed (Minogue et al. 2019). The bioinformatics methods need to be able to map and identify sequence variants by critically evaluating raw sequence data (Gilchrist et al. 2015). The data output needs to be presented in an actionable format (Gilchrist et al. 2015). Other NGS- centric issues include depth of sequencing, higher error rates than Sanger sequencing, reproducibility, sensitivity, AT-sequence bias, and large number of targets and markers (Gilchrist et al. 2015, Aly et al. 2015). More studies of robustness of the method are needed (Gilchrist et al. 2015, Aly et al. 2015).
Familial DNA searching has begun in jurisdictions that allow it but this poses privacy concerns. Similarly, there are ethical considerations to consider. The ForenSeq NGS panel contains loci that codes for unique traits, as opposed to solely “junk” DNA. GEDmatch was a database founded to help users use their genetic profiles to locate family members based upon similarities in the genetic makeup. Individuals can upload their DNA profiles from one of several personal genealogy services. Initially, GEDmatch gave users the option of opting out of other uses including investigations. Verogen recently purchased GEDmatch for use in cold case and other criminal investigations. Now users and potential family member matches must opt into investigative use; otherwise, their samples are protected from these searches and their privacy is maintained. There were concerns when users previously had to opt out that GEDmatch was turning all users into suspects. Still, database security breaches are a concern.
Figure 1 summarizes many of the issues that need to be resolved including allele naming, data storage, statistics, and acceptance by the courts.
In spite of the concerns and challenges, countries around the world are working to bring NGS to the forensics workflow due to its advantages. NGS has been used in casework and missing persons investigations.
Early Successes of NGS in Forensic Cases
Genetic genealogy has been employed in investigations. DNA profiles of non-offenders from commercial companies including 23andMe, Ancestry DNA, and My Heritage DNA tests are being used in searches to support law enforcement. NGS data and websites such as GEDmatch and Family Tree databases as well as traditional history research methods using the United States Federal Census, state birth indexes, Newspapers.com Obituary Index, US City Directories, US Obituary Collection, US Social Security records, and church membership and baptismal records have proved to be valuable in their approach. As a recent case example, GEDmatch was used by law enforcement to solve the decades-long cases of the Golden State Killer (Selk 2018).
The ThermoFisher HID-Ion AmpliSeq Ancestry Panel was used in a forensic case involving a carbonized corpse (Hollard et al. 2017). The autosomal STR profile did not lead to a profile, so NGS was used to determine the eye color and biogeographical origin of the deceased. The team also conducted Y typing and mitotyping. NGS did lead to more information but lack of a sufficient database for interpretation was a drawback. Xiao (2019) recently described the design and implementation of a large-scale, high-throughput automated DNA database construction using NGS.
The first case of NGS was on trial in Dutch Courts in January 2019 (de Knijff). The case included a sample from a complex sexual assault with a minor contributor at less than 10% that of the major contributor in the STR profile. The STR repeats were reported, but the traditional CE method does not permit the determination of underlying sequencing information. The DNA sample was analyzed using traditional PCR and CE methods and generated a hit in their convicted criminal database. The case resulted in an acquittal because many of the minor contributor’s alleles were in the stutter position of the major contributor’s alleles. Upon appeal by the prosecution and reanalysis of the samples using the MiSeq FGx, the minor contributor was distinguished from stutter, and likelihood ratio statistics were performed on the results. Upon hearing the new data and analysis, the judge ruled that the defendant was guilty of sexual assault (de Knijff 2020).
NGS has also been demonstrated for use in paternity cases. In a study, DNA isolated from sperm cells of monozygotic twins and blood from one of the twins’ children was typed using ultra-deep NGS. The researchers used VarScan 2 to analyze the sequence data for somatic mutations. Individualizing SNPs were identified in samples originating from the child’s father that were not found in the father’s twin (Weber-Lehmann et al., 2014).
The France National Police implemented a decision tree for deciding whether to analyze samples with NGS or traditional CE. In summary, if a sample is limited or degraded or if a Y-STR profile is needed, NGS using ForenSeq library preparation is supported (Alvarez-Cubero et al. 2017). If the DNA profile is urgent (<72 hours), PCR followed by CE is recommended. Their workflows include mini-STR typing, Y-STR typing, autosomal STR typing, and phenotypic SNP typing using CE and mitotyping using Sanger sequencing. They suggest NGS use in complex kinship cases, to identify a very minor contributor, to obtain a genetic profile from highly degraded DNA, and to deconvolute a mixture using possible isomutations. The French National Police used NGS in 2018 to analyze a 2011 cold case. The first analysis was performed using Identifiler and the two DNA extracts showed a mixture of the victim’s DNA and that of a very minor male contributor. Using NGS, the profile at D2S1338 was determined to contain two different seventeen-repeat alleles (isoalleles) which led to assignment of the minor profile.
To date, only a few cases processed using NGS have been presented in court; each jurisdiction will have to assess allowing the introduction of data produced with the new methods in accordance with the law. Forensic laboratories may utilize NGS for serious crimes and cold cases in the future, although caseloads may preclude using DNA typing for all cases.
In summary, John Butler wrote in 2005 that “the future is always challenging due to unforeseen innovation.” While NGS continues to challenge scientists and labs, NGS is here and providing new opportunities for analysis to solve crimes. The developmental validation of NGS for forensic applications has been published in peer-reviewed journals. NGS is being applied successfully to criminal cases, mass disaster, and missing persons forensic casework.
About the Authors
Kelly M. Elkins, PhD, is an Associate Professor of Chemistry at Towson University and a founding editor-in-chief of the Journal of Forensic Science Education. She has authored the books Forensic DNA Biology: A Laboratory Manual and Introduction to Forensic Chemistry, in addition to ten invited book chapters and more than thirty-five journal papers on her research in journals, including the Journal of Forensic Sciences, Analytical Biochemistry, Drug Testing and Analysis, and Medicine, Science and the Law. She has taught courses in forensic biology and forensic chemistry under various course numbers at four colleges and universities since 2006. She is an active member of the American Chemical Society and a Fellow of the American Academy of Forensic Sciences. She is a member of the Council of Forensic Science Educators and served as its President in 2012. She is a member of the ACS Ethics Committee and co- wrote the 2017 ACS Exams Institute Diagnostic of Undergraduate Chemical Knowledge (DUCK) exam. Her research has been funded by the Forensic Sciences Foundation, NSF, NIH, Maryland TEDCO, and ACS. She enjoys communicating science in the classroom, via outreach activities, in interviews, and on television. She is the editor for two books in production.
Cynthia B. Zeller, PhD, is an Associate Professor of Chemistry at Towson University. She has taught several forensic biology and DNA typing courses at Frederick Community College and Towson University for over fifteen years. After completing postdoctoral appointments in the School of Medicine at Johns Hopkins University for six years, she served as a Serologist and DNA Analyst at the Maryland State Police Forensic Science Division for six years. She is a member of the Midatlantic Association of Forensic Scientists. She has published ten scientific publications and has delivered more than hundred conference and seminar presentations. Her work has been published in the Journal of Forensic Sciences, Fibrogenesis Tissue Repair, and The American Journal of Physiology, and it has been funded by the National Institutes of Justice. This is her first book.
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