Battelle and the Ohio Bureau of Criminal Investigation (Ohio BCI) have made significant progress in optimizing and validating methods, equipment and workflows for Massively Parallel Sequencing (MPS) in an Ohio BCI lab. Three new studies, to be presented at the International Symposium for Human Identification (ISHI) this month, highlight some of the latest results.
Battelle is working with the Ohio BCI to implement MPS (sometimes referred to as Next-Generation Sequencing, or NGS) in their forensic laboratories in London, Ohio. MPS, while widely used in academic labs, is still a relatively new technology for forensic laboratories. DNA typing using MPS requires additional laboratory steps compared to more familiar CE-based technologies. Implementing MPS for forensics requires optimization and validation of technologies, workflows and methods specific to forensic applications. The joint Battelle/Ohio BCI project is breaking new ground for the industry and defining methods that will soon be able to be applied by other forensic labs across the country.
The three Battelle studies examine specific aspects of the project.
- Optimization of the Promega PowerSeq Auti/Y System Protocol to Effectively Integrate into a Forensic Laboratory: In this study, the Promega PowerSeq™ Auto/Y System was optimized with modifications designed to improve sample processing in a forensics laboratory. The system includes Promega’s PowerSeq Auto/Y prototype amplification kit, Illumina’s TruSeq® HT DNA PCR-Free library prep kits, and the Illumina MiSeq®FGx System sequencer in Research-Use-Only mode with MiSeq v2 300-cycle sequencing kits. Experiments assessed streamlining some quality control steps, adjusting sample purification procedures, incorporating sample-processing automation, and reducing the amount of hands-on manipulation of amplified products. The results indicated that the protocol enhancements had no detrimental impact on data quality, and were streamlined improvements in sample processing. The study establishes a precedence to more easily integrate this MPS procedure into a current forensics laboratory.
- Implementation and Validation of an Autosomal and Y Short Tandem Repeat Workflow for Massively Parallel Sequencing: In this study, both developmental and internal validation are being performed for MPS workflows in the Ohio BCI lab. The studies were conducted using Promega’s PowerSeq™ Auto/Y prototype amplification kit along with Illumina’s TruSeq® DNA PCR-Free library preparation kits for sequencing on the Illumina MiSeq FGx™. Battelle ExactID®, an innovative MPS software program for forensic analysis developed by Battelle, is being used to process and evaluate the large amount of data produced. As this workflow is new to the forensic DNA community, validation guidelines published by SWGDAM were followed as closely as practicable for the MPS study. The data from the studies performed thus far confirmed the typing accuracy of the workflow across many variables, including concordance with CE results. The studies show that the PowerSeq System workflow is suitable for integration at a forensic DNA laboratory
- MPS Data Analysis – Methods to Identify Locus-Specific Analytical Thresholds And Stutter Percentages: In order to perform effective validation of MPS technologies and workflows, it is necessary to set an analytical threshold and determine applicable stutter percentages. Sequencing data from a dilution series of a NIST Standard, a set of buccal swabs from five different individuals, and a set of mock casework-type samples were analyzed to determine a method of setting a potential analytical threshold and calculating stutter percentages. The data was analyzed using Battelle ExactID® software and a custom analysis program written specifically for this application. Results showed that calculated analytical thresholds were consistent across both sample types and concentrations, while varying across loci. This suggests that a dynamic method for setting an analytical threshold may be desirable. Stutter analysis suggests that a per-locus stutter percentage may be applied by the software when interpreting sequencing data in a forensic context.
Results of all three studies will be presented by Battelle researchers and Ohio BCI personnel during poster presentations at ISHI. Battelle and Ohio BCI will build on this work as they complete the MPS technology transfer project over the next several months. Lessons learned will be used to develop guidelines for other forensic laboratories planning to implement MPS.