Optimizing Analytical Thresholds for Low-Template DNA Analysis: Insights from Multi-Laboratory Negative Controls

Author:

Chen Dezhi1,Tan Mengyu1,Xue Jiaming1,Wu Mengna1,Song Jinlong1,Wu Qiushuo1,Liu Guihong1,Zheng Yazi1,Xiao Yuanyuan1,Lv Meili2,Liao Miao13,Qu Shengqiu3,Liang Weibo1ORCID

Affiliation:

1. Department of Forensic Genetics, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China

2. Department of Immunology, West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, Chengdu 610041, China

3. West China Forensics Center, Sichuan University, No. 16, Section 3, Renmin South Road, Wuhou District, Chengdu 610041, China

Abstract

When analyzing challenging samples, such as low-template DNA, analysts aim to maximize information while minimizing noise, often by adjusting the analytical threshold (AT) for optimal results. A potential approach involves calculating the AT based on the baseline signal distribution in electrophoresis results. This study investigates the impact of reagent kits, testing quarters, environmental conditions, and amplification cycles on baseline signals using historical records and experimental data on low-template DNA. Variations in these aspects contribute to differences in baseline signal patterns. Analysts should remain vigilant regarding routine instrument maintenance and reagent replacement, as these may affect baseline signals. Prompt analysis of baseline status and tailored adjustments to ATs under specific laboratory conditions are advised. A comparative analysis of published methods for calculating the optimal AT from a negative signal distribution highlighted the efficiency of utilizing baseline signals to enhance forensic genetic analysis, with the exception of extremely low-template samples and high-amplification cycles. Moreover, a user-friendly program for real-time analysis was developed, enabling prompt adjustments to ATs based on negative control profiles. In conclusion, this study provides insights into baseline signals, aiming to enhance genetic analysis accuracy across diverse laboratories. Practical recommendations are offered for optimizing ATs in forensic DNA analysis.

Funder

National Natural Science Foundation of China

Chengdu Science and Technology Program

Publisher

MDPI AG

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