Mitochondrial DNA analysis is an alternative to conventional STR typing for challenging samples because of its characteristic high copy number. Traditionally, Sanger-based sequencing methods have been used for whole mitochondrial genome sequencing, but labor-intensive workflows and limited multiplexing ability present significant limitations. The evolution of massively parallel sequencing (MPS) provides a method for higher sample throughput and multiplexing to cover the whole 16,569 base pair mitochondrial genome with increased sample coverage. Along with the emergence of MPS, commercially available whole mitogenome kits including the PowerSeq® Whole Mito System have been developed.
ISHI Student Ambassador, Ashley Welk’s, project aims to sequence the entire mitogenome of a subset of samples, previously collected with informed consent from a Lebanese population, using the PowerSeq® Whole Mito System. The study assessed concordance with existing data generated via long-range PCR and liquid handling robots and evaluated the performance of the PowerSeq® Whole Mito System in combination with the MiSeq FGxTM instrument.
We chatted with Ashley to learn a little more, and if you’ll be at ISHI this September, be sure to stop by during the poster sessions to learn more!
Briefly describe your work/area of interest.
Upon entering graduate school, I was very interested in many facets of forensic biology and DNA analysis, but I was unsure exactly what avenue I wanted to explore. After starting coursework and a graduate assistant position at the Center for Forensic Science Research and Education (CFSRE) I was exposed to the hands-on process of massively parallel sequencing, and it sparked an interest in emerging technologies in forensic science. I was fortunate that the CFSRE was in the initial phase of work with a kit manufactured for whole mitochondrial genome sequencing, leaving an opening for me to take over the project for my master’s thesis.
More specifically, I am working on evaluating the Promega PowerSeq® Whole Mito System in conjunction with the MiSeq FGxTM on a subset of Lebanese saliva samples previously collected with informed consent. The goal of the research is to determine concordance between whole mitochondrial genome sequencing data generated with the Promega PowerSeq® Whole Mito System, and data generated by the Armed Forces DNA Identification Laboratory (AFDIL) using their in-house methodology. Concordance between mitochondrial haplotypes and variants will shed light on the effectiveness of the PowerSeq® Whole Mito System.
My key collaborators for this project are the members of the biology team at the Center for Forensic Science Research and Education including Dr. Mirna Ghemrawi, Lindsay Kotchey, and Leah Nangeroni, as well as AFDIL team members who kindly provided their sample data for the concordance study. Finally, the Promega Corporation has supported the research by supplying the kits tested throughout this project. I am truly grateful for all the support and guidance collaborators have provided me throughout this process.
The pipeline of processing starts with a ten-fold dilution of the original extracts, followed by an initial quantification using the Promega PowerQuant® kit. Once quantification values are obtained, a serial dilution is performed to bring samples to a concentration of 0.05 ng/µL for input into amplification. Amplification is then performed using two primer sets and product is then purified. Next, libraries are prepared and purified, and library quantification is completed using the PowerSeqTM Quant MS Kit. Once library concentrations are obtained, libraries are normalized, pooled, diluted, denatured, and loaded into a reagent cartridge for sequencing on the MiSeq FGxTM. Data analysis is initiated after sequencing and includes recording overall run metrics, input of FASTQ files into the GeneMarker® HTS software, analyzing major and minor variants, input of variants into EMPOP to determine haplotype, and finally comparison to AFDIL data to evaluate concordance.
To date, I have completed whole mitochondrial genome sequencing for 144 of 195 Lebanese samples and am hopeful to have all sample sets represented in my results for ISHI. So far, I have seen 100% concordance in haplotypes between data generated with the PowerSeq® Whole Mito System, and data generated using AFDIL’s in-house method. Interestingly, I have observed slight variation in individual variants in samples within the two data sets, but I am still in the process of interpreting these variants in more detail to determine if the differences are significant or due to cluster density.
How did you become interested in this work? Why did this particular project appeal?
I got interested in this work through communication with my biology team advisors and professors at the CFSRE. Each team member has unique experience in the field, and hearing from team members with enthusiasm for whole mitochondrial genome sequencing inspired me to further research in this area of forensic biology. The opportunity to utilize the MiSeq FGxTM for massively parallel sequencing was another draw to the project. As the field of forensics moves forward, the prospect of having experience with next generation sequencing (NGS) technology was extremely appealing.
What has been the most rewarding part of working on this project?
The most rewarding part of working on this project has been troubleshooting. I have been so fortunate to learn techniques used for NGS throughout my research, but it is not without roadblocks. When I have faced a problem or setback during my research whether it be a quantification experiment that isn’t working as expected or an instrument acting up, being able to workshop the problem, reset, and then get a successful result is extremely satisfying and rewarding.
Are there any further areas of research you think are needed based on your findings?
After I conclude my initial concordance study, I would like to see my research continued with an internal validation at the CFSRE, along with further testing on case-type and degraded samples. Additionally, the kit could be evaluated on other populations that are underrepresented in databases. The possibilities for further testing and application of the PowerSeq® Whole Mito System are endless.
How do you hope your research will be applied in real-world scenarios?
I hope to see my research being used to increase knowledge about the performance of the PowerSeq® Whole Mito System to provide a basis for its potential implementation into casework. When degraded samples are encountered that may be incompatible with nuclear DNA analysis, a reliable method of mitochondrial DNA analysis can be used to provide leads to law enforcement, identify victims of a mass disaster, and trace lineage. My research with the PowerSeq® Whole Mito System certainly needs further investigation but could lead to implementation as a streamlined and systematic method of mitochondrial genome processing for casework and research.
