DNA Typing Strategies via Real-Time Nanopore Sequencing for Forensic Analysis

Advancements in Next-Generation Sequencing (NGS) have progressed to the stage of forensic validation and development of interpretation guidelines, however newer sequencing technologies are rapidly evolving that may better serve forensic needs. The problem faced by forensic laboratories is that they are already encumbered with a costly array of instrumentation needed to perform capillary electrophoresis-based STR typing and the adoption of current NGS platforms is equally expensive with much more involved workflows and steep learning curve. Most laboratories cannot justify or allocate the funding needed to concurrently maintain casework instrumentation while investing in MPS platforms. Polymerase chain reaction (PCR) is a standard in forensic DNA analysis to amplify target STR regions. Amplification is necessary on most sequencing platforms that rely upon secondary signal, such as fluorescence, to detect the nucleotides. However, amplification also introduces artifacts that become intermixed with the data. Oxford Nanopore Technologies (ONT) has developed an inexpensive, compact single-molecule sequencer that allows for direct interrogation of single DNA molecules as they pass through nanopores. Since the nanopore is not reading a secondary signal, the biggest challenge for DNA analysis relies upon having sufficient copies of target DNA regions to overcome competing strands of non-target DNA. In this project, we will determine the ideal number of PCR cycles to effectively enrich STR regions, provide sufficient coverage for genotype determination, and minimize stutter, ultimately creating a micro-PCR protocol for STR sequencing. We will utilize the ONT platform to empirically assess the development of stutter, the most common PCR artifact observed in forensic STR analysis, by directly monitoring the process through real-time sequencing observing both the parental DNA strand and its synthesized complement generated during PCR. Through this we can model the effect of stutter in STR analysis and gain better understanding of the mutation process that naturally occurs in STRs. We have assembled a research team between UNT Health Science Center, University of Texas Arlington and the Human Genome Research Center at Baylor College of Medicine to provide the robust skill set and expertise to tackle this project. We will develop streamlined informatics pipelines, interpretation guidelines and quality assurance measures to consider in validation to facilitate rapid sequence analysis of forensic loci, providing a functional system that permits crime laboratories to explore this platform. Software pipelines will be made open source, hosted on GitHub and provided in popular software containers that ease the use and installation on multiple platforms.

This project contains a research and/or development component, as defined in applicable law, and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14).

CA/NCF