In the realm of forensic science, advancements in DNA analysis play a pivotal role in resolving criminal investigations. Among the latest innovations is the use of next-generation sequencing (NGS) and the integration of the MiSeq FGx system with the ForenSeq DNA Signature Prep kit, representing a significant leap forward in genetic profiling. This article explores the practical application of this technology in a recent double homicide case, shedding light on its effectiveness in forensic analysis.
Central to this advancement is the ForenSeq DNA Signature Prep kit, designed to amplify comprehensive genetic information from DNA samples using next-generation sequencing (NGS) technology. By examining autosomal short tandem repeats (STRs), Y-chromosomal STRs (YSTRs), and identity single nucleotide polymorphisms (iSNPs), this kit provides detailed genetic profiles with high accuracy.
In a notable case in Kern County, California, the efficacy of this technology was put to the test during an admissibility hearing. Forensic experts presented the scientific basis of NGS technology, outlining its applications and limitations. Following this rigorous evaluation, the data derived from the MiSeq FGx system and ForenSeq DNA Signature Prep kit was deemed admissible in court.
During the trial, forensic scientists utilized this advanced technology to analyze evidence from the crime scene. Comparative analyses of autosomal STRs, YSTRs, and iSNPs provided valuable insights into the genetic composition of the samples, aiding in the identification of individuals involved.
Ultimately, the use of NGS technology proved instrumental in securing a conviction in the double homicide case. The comprehensive genetic profiles generated by the MiSeq FGx system and ForenSeq DNA Signature prep kit provided compelling evidence, leading to a successful outcome in court.
This case exemplifies the practical benefits of next-generation sequencing in forensic DNA analysis, highlighting its role in enhancing the accuracy and reliability of criminal investigations. As technology continues to advance, the potential for further improvements in forensic science remains promising.
Mandi Van Buren, former DNA Technical Lead at the Kern Regional Crime lab led her team through the validation and implementation process for NGS. She will share her experiences and insight into this process and how she prepared for the admissibility hearing and trial.
I began my career in forensics over 15 years ago. I started with the Kern Regional Crime Lab in 2016 and moved through the ranks until ultimately being promoted to the DNA Technical Lead in 2019. Currently I am working for DNA Labs International as a DNA analyst. I am looking forward to growing my DNA expertise with their company as they are pioneers in providing advanced technology and services to customers for DNA testing. I am excited to be a part of such an amazing team of field experts sharing in my same visions of how important this work is and giving everything we can to help provide quality scientific results to each case we work.
Mandi van Buren, DNA Analyst, DNA Labs International 1. Can you provide a summary of work your laboratory undertakes (types of cases, numbers of cases, for which police forces/customers)?
The Kern Regional Crime Laboratory is a full-service laboratory offering an expansive number of forensic services to the law enforcement community. The laboratory serves over 40 agencies across Kern County including local police agencies, numerous prisons, and the local school district. The DNA analysis unit specifically conducts serological testing for body fluid identification, DNA analysis, kinship for criminal paternity cases, familial searching, CODIS, and a complete local database for generation of investigative leads. On average, the DNA unit receives 700 requests for service annually and completes testing of over 2,500 DNA samples. Case submissions are approved for all types of cases including felonies and misdemeanors from homicides and sexual assaults to property crimes. In addition to current casework, the DNA unit received many requests for analysis of evidence related to cold case homicides and sexual assaults.
2. What were the major challenges your lab faced that led you to implement next generation sequencing for casework?
During the initial considerations of adopting NGS technology, the laboratory was operating with an out-of-date genetic analyzer, limited resources, and an increase of more complex DNA profiles for interpretation with the allowance for touch DNA samples to be submitted for testing. Additionally, the laboratory had Y-STR testing available but, due to the workflow being used, not all evidence items had enough sample available to test both autosomal STRs and Y-STRs, even if the sample was deemed suitable for Y-STR testing.
In 2018, the MiSeq FGx instrument and the ForenSeq DNA Signature Prep kit had been approved by NDIS for use on forensic samples. Since the DNA unit relies primarily on grant funds for equipment and reagents, this approval allowed the lab to use its grant funding to purchase the MiSeq FGx workflow with ForenSeq DNA Signature Prep. We believed the system would alleviate some of our challenges and streamline our current workflow by providing aSTR and Y-STR genetic information in a single amplification reducing analyst time in the lab. Additionally, we hoped for improved mixture deconvolution with the identification of isoalleles with NGS. We utilized probabilistic genotyping for all mixture interpretation for our CE workflow and often an analyst would struggle estimating the number of contributors in a complex mixture. The additional aSTR markers, combined with the availability of Y-STR data, SNP data, and identification of isoalleles would provide analysts with more information to accurately estimate the number of contributors during interpretation.
3. Did you have any concerns about bringing on NGS?
As with any new technology or instrument, there are always going to be concerns. The decision to implement NGS, I think, came with even more concerns at that time because no other labs had successfully implemented this instrument and testing kit in a forensic crime lab for evidence samples yet. In addition, we were bringing this technology online with the plan to eventually replace and phase out CE technology in our lab completely. I was a newly appointed DNA Technical lead and was handed this project at a time when the unit was understaffed and dealing with a backlog of cases. I knew my team would not be welcoming this change with open arms, as all change slows down the process, adds additional training obligations, and requires more from everyone when our plates were already full. There were additional concerns about what support we would have from all involved stakeholders including lab management, law enforcement customers, the DA office, and technical support from vendors. As the project continued, other concerns arose including data storage and cost, training, and implementation strategy.
4. Which NGS products did you validate/implement and why? Why did you decide on the MiSeq FGx?
We validated the MiSeq FGx system with the ForenSeq DNA Signature Prep kit. Our validation focused on both primer sets A and B. We had researched different NGS instruments and kits but we felt this combination was best suited for our needs, could be easily integrated into our current workflow, and would work with our existing Hamilton liquid handling robots. At the time this project started, this was the only NDIS approved kit and instrument which we needed in order to utilize grant funds to fund the project.
5. What types of cases do you/do you intend to apply NGS to?
NGS was initially validated to be used on all casework samples, including evidence and known reference items. During implementation, the plan shifted to samples from violent crime cases only. The workflow would be to evaluate samples at quantification and then decide if a whether to use primer set A or B. Some considerations for this decision included how much sample did we have, did we consume the item, is there any potential subjects or are they unknown.
The first 10 cases utilizing NGS were violent crime cases and focused on samples originating from biological fluids. Our interpretation protocol states that all mixtures must be interpreted with probabilistic genotyping software, so our NGS testing was limited until the completion of additional validation projects. Once NGS and probabilistic genotyping validations are fully implemented, all samples of any case type would be acceptable for NGS.
6. Your lab was a part of the first accepted admissibility hearing for NGS. Can you tell us more about that?
The case was a double homicide in Dec 2023. We selected this case because it had additional evidence available from biological fluids (blood) that we suspected would be single source and therefore interpretable with NGS. We tested samples first with CE and then an additional set of samples with NGS and presented the aSTRs, YSTRs and iSNPS with statistics in court. Using the iSNPs was a big deal because this was the first time SNP to SNP comparisons were presented for identification in a criminal court case.
We had a team of experts present at the admissibility hearing; Bruce Budowle laid the foundation of the science and the evolution of DNA testing; Meredith Turnbough covered the details of the MiSeq FGx and ForenSeq DNA Signature Prep Kit and what happens at a molecular level; I covered the validation data and procedures developed in the lab; and Brooke Ramirez presented the data and results from the case evidence. The admissibility was successful and the accused was convicted of two counts of murder.
7. What advice or recommendation would you give other labs?
a. Develop a core team early in the project that includes analysts, stakeholders, vendors, and outside experts as needed
b. Gain buy in early from your team and management. This project takes time and time away from cases so everyone needs to be understanding.
c. Know the benefits up front and use those to sell the project to your leadership and team members
d. Look at ALL the applications. So many people are only focusing on FIGG but this can be used in your everyday casework as well. This is especially important if you want to use grant funding to fund the project.
e. Do the validation yourself or choose a hybrid method with the vendor, I would not recommend having someone else do the whole validation. Though time consuming and challenging, this is where your understanding of the system, kit, and process will grow deeper to help you mentor and train others and help you present this in court.
f. Reach out to others that have completed this. Do not reinvent the wheel or get stuck and think there is no one to help- there is!
8. What would you say to other labs considering validating and implementing NGS for HID casework?
Do it now and stop waiting! There is a strong support system in this field with experts on hand waiting and wanting to help. I believe this technology improves our current testing capabilities and can provide more investigative information to solve more cases faster. I believe all crime labs can benefit from implementing NGS in some fashion. There are numerous applications and ways to integrate it into a lab’s current workflow. The process will still be overwhelming at times and you will still face challenges, but there is light at the end of the tunnel and now a clear path to follow from beginning to end.