Sally Wasef talks about using next-generation sequencing coupled with Whole Genome target enrichment of degraded DNA to support investigations into Unrecovered War Casualties. Sally is developing new methods to provide lineage and phenotypic markers to identify service men and women from the Australian Defence Force fallen in World War I and World War II.
The Young Investigator Community is a forum for young forensic researchers and graduate students. Meet one of these talented researchers in our new Investigator blog where they share aspirations and achievements with their peers, friends and colleagues. Read on to find out why our blogger was drawn to study and practice forensic science. Let them share with you the excitement of discovery that sustains a passionate commitment to their work.
This new Investigator blog introduces Sally Wasef. Sally is currently a Research Fellow in Forensic Genomics based in the Ancient DNA Facility, Genomics Research Centre, Queensland University of Technology.
1. Tell us about your background and how you became interested in forensic science?
I have extensive experience and expertise in ancient DNA retrieval, next-generation sequencing (NGS), target enrichment and bioinformatics analyses. In fact, I've recovered more ancient DNA sequences from ancient remains in Sahul (the ancient Pleistocene continental shelf of Australia and Papua New Guinea) than any other researcher in the past 15 years. I'm proud to be the only woman of color among Australia's small group of ancient DNA experts.
Currently, I'm working on the Defence Genomics project, where I'm responsible for conducting all the ancient DNA analyses of historical remains. While I'm relatively new to the forensic field, I'm eager to contribute my ancient DNA knowledge to the Defence Genomics group and expand my understanding of the rules and regulations surrounding forensic samples.
Despite my limited knowledge and experience with routine forensic DNA casework, I'm enthusiastic about learning more about dealing with highly degraded samples and implementing Standard Operating Procedures (SOPs) in the newly established ancient DNA laboratory at QUT. I'm also keen to gain practical experience in the forensic DNA field and establish connections and collaborations with national and international researchers who are leaders in identifying war remains worldwide.
My interest in the forensic DNA field is strong. I'm considering pursuing a future career in collaboration with forensic laboratories in Queensland to provide a nationwide service for profiling highly degraded remains. Overall, I'm excited about the potential for ancient DNA analysis in forensic science and am dedicated to contributing my expertise to advance the field.
2. Can you provide a summary of the project you are working on?
The project I am involved in, as an ancient DNA expert, aims to identify Australian service members who have not been accounted for from past conflicts, specifically World Wars I and II. Modern criminal cases' current forensic DNA methods are unsuitable for nearly century-old remains. To overcome this issue, our team, led by Prof. Lyn Griffiths and funded by the Department of Defence Innovation Hub, is utilising NGS coupled with Whole Genome target enrichment of ancient DNA to identify these fallen service members. This method will provide lineage and phenotypic markers to aid identification efforts. Our project has three main goals:
- Establishing a Family Reference DNA Database for the Australian Defence Force to identify historical military remains;
- Developing new genomic methods based on ancient DNA techniques to extract DNA profiles from highly degraded remains to support investigations for identifying Unrecovered War Casualties across the Australian Defence Force;
- Developing analysis techniques for predicting biogeographical ancestry and externally visible characteristics in highly degraded military remains.
3. Please describe your typical day in the lab.
My typical day in the lab involves working in two different Queensland University of Technology genomic laboratories located in separate buildings: the ancient DNA facility and Defence Innovation Hub genomic laboratory. The ancient DNA facility adheres to international standards for maintaining a highly clean and contamination-controlled environment. The facility comprises two rooms equipped with positive pressure and UV HEPA air filtration systems.
To work in the ancient DNA main lab, I first don the necessary personal protective equipment (PPE), which includes a full-body suit, hair net, face mask, face shield and double gloves. After suiting up, I proceed to the ancient DNA main lab to begin my work.
In the ancient DNA main lab, I begin by decontaminating ancient remains, weighing them, taking photos and subsampling them. I then proceed to extract and purify DNA. After DNA extraction, I prepare NGS libraries then tranfer these to the modern DNA lab for PCR amplification. Once amplification is complete, I perform enrichment steps for the entire human genome, including mitochondrial DNA and all other necessary SNPs. I sequence the DNA using the in-house Illumina NextSeq 2000 system when ready.
4. What do you find most interesting about your project? Have you seen any surprising results?
To me, the most interesting aspect of this project is the way in which advancements in ancient DNA research can provide solutions to a significant issue in forensics, namely the handling of severely degraded remains. Each sample in this project poses unique challenges, particularly the younger ones from World War II, which were poorly preserved due to harsh conditions in Papua New Guinea. This contrasts with the better-preserved World War I remains from Europe. I find it fascinating to explore how to approach each sample and overcome the challenges of modern contamination. Additionally, this project has a significant social impact on the families of missing service members, adding to its significance. As for surprising results, it's still too early to say, but the journey thus far has been intriguing.
5. What are the benefits of your project?
The project has several benefits, including being the first human identification project for post-mass fatality events using ancient genomics and NGS. It will provide a tool to recover ancestral and phenotypic data from highly degraded samples. It will help identify World War I and World War II human remains with greater certainty and build a family reference dataset. The project has significant reconciliation benefits, showing a commitment to resolving the complex issue facing human identification today. The developed kit will be best suited for highly degraded, tiny samples from skeletal remains found in harsh environmental conditions where human identification using standard forensic techniques has been impossible.
Improving the quality and quantity of the recovered DNA from skeletal material will contribute to the profiling and identification of human remains, increasing the capabilities of military investigations in live theatres and providing law enforcement with faster initiation of skeletal identification. Moreover, improving DNA recovery from human remains may provide adequate amounts of DNA for biogeographical ancestry and externally visible characteristics profiling, giving visual traits to faceless skeletons and providing intelligence.
The outcome of this research will apply to various kinds of cases in forensic science where skeletal materials are the predominant biological remains for DNA typing, including cold cases, aged, buried, burnt, submerged, desiccated and mummified skeletal human remains, missing person cases, unrecovered war casualties, natural disasters, mass graves (including pandemic graves), war crimes (e.g., the Balkans), and crimes against humanity (e.g., East Timor and the Balkans).
6. What are the major challenges faced while working on your project and how do you overcome them?
The major challenge faced while working on this project is to recover variants of Short Tandem Repeats (STRs) that facilitate robust forensic identification. Y-STRs are especially helpful for forensic genetic genealogy but the recovery of long amplicon sequences, such as Y-STRs, remains challenging. Extracts with high endogenous DNA fractions are sometimes not possible due to age or preservation conditions of the specimen. This is despite advances in NGS and extraction techniques.
I aim to develop and improve a methodology to recover Y-STRs, mini-Y-STRs, whole mitochondrial genome and autosomal SNPs from a minimal amount of the highly degraded remains and challenging historical samples thereby maximising NGS on target sequences.
Verogen already has an established series of NGS forensic kits to retrieve Y-STRs, among other markers, but they are not designed to accommodate the nature of highly degraded, historical DNA samples. Historical remains from hot and arid environments have highly fragmented DNA. In areas like Australia and Papua New Guinea, fragments have an average length of between 30 and 150 bp. So, I hope to optimise sampling strategies by targeting tissues with reduced levels of contamination, particularly the petrous bone and the dense outer layer of long bones. Then I will introduce changes to the Verogen kit to recover Y-STRs, mini-Y-STR, mitochondrial, ancestral and phenotypic SNPs from ancient samples.
7. Which QIAGEN products do you use and what do you like about the products?
I use several QIAGEN products for my modified ancient DNA extraction protocol such as the DNeasy Blood and Tissue Kit, the MiniElute PCR Purification Kit and Buffer PB that binds to shorter DNA fragments of less than 30bp.
I am running a pilot study with QIAGEN, and my focus is on developing a new version of the Verogen ForenSeq MainstAY product line for highly degraded World War I and World War II remains. The changes will involve designing a unique primer mix and modifying the Verogen NGS protocol to prepare double-stranded genomic libraries optimized for ancient DNA with partial Uracil DNA Glycosylase (UDG) and Endonuclease VIII enzyme treatment. Finally, the Verogen MiSeq FGx Sequencing System will be used to sequence the degraded samples, and an integrated workflow will be developed to analyze the data in Verogen ForenSeq Universal Analysis Software (UAS).
8. Outside of forensic science, what are your hobbies?
I indulge in some of my hobbies when I'm not busy with my academic pursuits or engaged in forensic science. One of my favorite pastimes is crafting, and I love exploring different crafting techniques and materials. One of the crafts that I enjoy is creating gift baskets. I find that it's a great way to personalise a gift and make it extra special.
Another crafting activity that I find enjoyable is creating dried floral arrangements. I love working with natural materials and experimenting with different colour combinations and textures. There's something very calming and meditative about creating a beautiful floral arrangement that can be enjoyed for weeks or even months. I also enjoy working with balloons and creating event decorations. Whether for a birthday party, a wedding, or any other event, I love the challenge of creating unique and eye-catching balloon decorations that will make the occasion extra special.
Engaging in these creative activities helps me unwind, destress and recharge my batteries. They are a great way to explore my creative side and tap into my imagination, and I always look forward to spending time on these hobbies whenever I get the chance.
