法医遗传标记二代测序深度阈值设定方法研究——以微单倍型为例
Research on the method of setting depth thresholds for second generation sequencing of forensic genetic markers-Taking microhaplotypes as an example
贺琳 1康克莱 2冯耀森 2赵杰 2张驰 2马温华 2潘坤鹏 1陈璠 1季安全 1王乐1
作者信息
- 1. 昆明医科大学法医学院,云南 昆明 650500;法医遗传学公安部重点实验室,公安部鉴定中心,北京 100038
- 2. 法医遗传学公安部重点实验室,公安部鉴定中心,北京 100038
- 折叠
摘要
目的 测序深度是二代测序检验的关键参数,与测序结果的准确性紧密相关.法医物证检验对于准确性要求极高,科学合理地设定法医学二代测序检验的测序深度分析阈值至关重要.方法 本研究采用 50 份已知分型样本的二代测序微单倍型靶向测序数据,通过计算各阈值条件下各基因座的准确率、精确率、召回率和F1 分数,细致研究了基于固定读段数和基于固定序列占比两类分析阈值设定方法.结果 当设定分析阈值为 50'、100'时,均观察到假阳性;当设定分析阈值为200'时,观察到假阴性;当设定分析阈值为 1.5%、3.0%、4.5%时,均观察到假阳性.本研究进一步提出第三类分析阈值设定方法——基于序列占比散点图,基于该方法的分析结果未观察到假阳性或假阴性情况.通过讨论导致法医学二代测序深度差异显著的四个因素,类比毛细管电泳技术的分析阈值设定方法,讨论了分析阈值高低与混合DNA区分能力的相关性.结论 采用序列占比散点图方法设定分析阈值在法医遗传标记二代测序检验中具有重要应用价值.
Abstract
Objective Sequencing depth is a key parameter in next generation sequencing,which is closely related to the accuracy of sequencing results.Forensic biological evidence examination requires extremely high accuracy.It is crucial to scientifically and reasonably set the sequencing depth analysis threshold for forensic next generation sequencing testing.Methods This study used targeted sequencing data of microhaplotypes from 50 samples with known genotypes.By calculating the accuracy,precision,recall,and F1 score of each locus under various threshold conditions,two types of analysis threshold setting methods,which were based on fixed read count and fixed sequencing depth ratio,were studied extensively.Results The results showed that false positives were observed when the analysis threshold was set at 50×or 100×.When the analysis threshold was set at 200×,false negatives were observed.When the analysis threshold was set at 1.5%,3.0%,or 4.5%,false positives were observed.This study further proposed a third type of analysis threshold setting method,which was based on sequencing depth ratio scatter plots.With this method,no false positive or false negative was observed in the results.This article then explored four factors that lead to significant differences in the sequencing depth of forensic next generation sequencing experiments,compared with the analysis threshold setting method for capillary electrophoresis technology,and discussed the correlation between analysis thresholds and the ability to distinguish mixed DNA.Conclusion Employing the sequencing depth ratio scatter plot method to set analysis threshold has significant application value in next generation sequencing-based forensic genetic marker genotyping.
关键词
法医物证学/二代测序/测序深度/分析阈值Key words
Forensic biological evidence/Next generation sequencing/Sequencing depth/Analysis threshold引用本文复制引用
基金项目
国家自然科学基金(81971797)
公安部科技强警基础工作专项(2022JC09)
出版年
2024
NETL
NSTL
万方数据