Evaluation of the ForenSeq™ MainstAY workflow for forensic DNA samples

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• The ForenSeq™ MainstAY Kit on the MiSeq FGx® Sequencing System is approved for National DNA Index System (NDIS) use.
• Evaluating its performance is crucial for forensic laboratories.

Purpose of the Study:

• To comprehensively evaluate the performance and limitations of the ForenSeq™ MainstAY chemistry.
• To provide practical guidance for forensic laboratories adopting this kit.

Main Methods:

• Baseline performance, sensitivity, and reproducibility studies were conducted.
• Challenging casework samples were analyzed, comparing different library preparation methods, PCR buffers, and flow cell types.
• Comparisons were made with capillary electrophoresis (CE).

Main Results:

• The kit demonstrated high reliability and consistent performance across users, with minimal locus dropout until 62 pg.
• It outperformed CE for low-input and degraded samples.
• Limitations were observed at higher plexities, with increased sample numbers reducing read counts in degraded samples.

Conclusions:

• The ForenSeq™ MainstAY Kit is reliable for forensic DNA analysis, especially for challenging samples.
• Strategic adjustments in workflow, such as batching degraded samples or lowering plexity, can mitigate performance limitations.
• Automated library preparation showed comparable or improved results without cross-contamination.