Moving Forward: Applying Massively Parallel Sequencing to Achieve Quality Mitochondrial Sequencing Results

While capillary electrophoresis (CE) instrumentation is the current standard for genetic profiling of forensic samples, there are advantages to using massively parallel sequencing (MPS) in challenging casework. With lower limits of detection and higher multiplexing capabilities, MPS instrumentation has potential applications with challenging samples including mixtures, degraded, and low template samples. While these capabilities will greatly improve short tandem repeat (STR) analysis, mitochondrial sequencing can also benefit through increased sensitivity, high sample throughput, and more streamlined reporting. Unlike CE instrumentation, MPS platforms can be used to analyze STRs and mitochondrial sequencing results without the need to change consumables on the platform, lowering the barrier for entry to mitochondrial sequencing. By adopting and validating MPS platforms for use with typical STR analysis with forensic casework, laboratories can also increase their available services to include mitochondrial sequencing allowing for a more versatile approach to analyzing forensic samples.

In addition to the inherent advantages of MPS platforms, the recent availability of commercial amplification and library prep chemistries such as the PowerSeq™ CRM Nested System and the ForenSeq™ mtDNA Control Region Kit allows for simplified approaches to generating and reporting data. The resulting data from these sequencing chemistries can then be analyzed using bioinformatics tools that are easily learned and implemented.

In this research challenging samples were prepared for mitochondrial analysis on a MiSeq FGx® using both the PowerSeq™ CRM Nested System and ForenSeq™ mtDNA Control Region Kit. These results were confirmed using Sanger sequencing as a control method. The results between the two MPS chemistries were compared in order to confirm the concordance between the two novel sample preparation methods. Samples were extracted with a variety of methods and sample types included donor buccal swabs and hair as well as cadaver bones. These chemistries were confirmed to be sensitive and reliable at a variety of input DNA amounts and able to recover profiles from samples identified as low input and degraded during real-time quantification.