DNA methylation analysis using bisulfite conversion followed by pyrosequencing on the PyroMark Q48 has gained increasing interest in forensic applications, including age estimation and body fluid identification. While the Q48 platform offers enhanced automation, studies have reported technical challenges affecting signal performance. During our pre-trial evaluation, we observed lower signal intensity when identical samples were analysed on the Q48 compared to its predecessor, the Q24 system. Reduced signal strength may compromise signal-to-noise ratio and consequently affect the accuracy of methylation quantification and downstream age estimation. This study aimed to optimise the workflow parameters to improve signal performance within a forensic laboratory setting.

A series of experiments were conducted to evaluate the effects of PCR product input volume, sequencing primer concentration, PCR cycle number, and post-PCR concentration via vacuum centrifugation. Separately, bisulfite PCR chemistry was optimised by varying primer concentration, and assessing the inclusion of Q-solution, CoralLoad, and MgCl2 supplementation.

Our results demonstrated that post-PCR template concentration via vacuum centrifugation was the most substantial driver of signal enhancement, consistently increasing the peak height signal from baseline to higher levels. Increasing PCR product input volume yielded modest improvements, whereas increasing PCR cycle number and sequencing primer concentration showed limited or inconsistent effects. Within bisulfite PCR optimisation, CoralLoad supplementation notably improved signal, particularly when Q-Solution was omitted, indicating a reagent interaction effect. Primer concentration exhibited minimal standalone influence; however, 0.6 µM demonstrated marginally higher signal compared to 0.4 µM under optimised conditions. Additionally, MgCl2 did not improve performance.

Collectively, our findings suggest that signal intensity in pyrosequencing-based methylation assay is primarily limited by post-PCR template availability rather than amplification efficiency alone. A combined optimisation strategy involving post-PCR concentration and refined PCR chemistry is proposed to enhance assay signal.

This study provides a practical framework for improving analytical sensitivity and reproducibility of forensic DNA methylation workflows, supporting the Q48 implementation in operational casework.

Authors

• Zhonghui Thong (Health Sciences Authority Singapore, Singapore)
• Zhi Yi Goh (Health Sciences Authority Singapore, Singapore)
• Jolena Ying Ying Tan (Health Sciences Authority Singapore, Singapore)
• Afiqah Razanah Rosli (Health Sciences Authority Singapore, Singapore)
• Michelle Xue Ting Tung (Health Sciences Authority Singapore, Singapore)
• Christopher Kiu-Choong Syn (Health Sciences Authority Singapore, Singapore)