Exploring nanopore direct sequencing performance of forensic STRs, SNPs, InDels, and DNA methylation markers in a single assay

IF 3.2 2区医学 Q2 GENETICS & HEREDITY

Forensic Science International-Genetics Pub Date : 2024-10-12 DOI:10.1016/j.fsigen.2024.103154

Desiree D.S.H. de Bruin , Martin A. Haagmans , Kristiaan J. van der Gaag , Jerry Hoogenboom , Natalie E.C. Weiler , Niccoló Tesi , Alex Salazar , Yaran Zhang , Henne Holstege , Marcel Reinders , Amade Aouatef M’charek , Titia Sijen , Peter Henneman

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引用次数: 0

Abstract

Introduction

The field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow.

Methods and results

In our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90–92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson’s correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath’s clock. Additional age-associated markers exhibited Pearson’s correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21–72, showing 100 % concordance with reference data.

Discussion

Our exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases.

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在单次测定中探索法医 STR、SNP、InDels 和 DNA 甲基化标记的纳米孔直接测序性能。

导言：近几十年来，法医 DNA 分析领域取得了突飞猛进的发展。大规模并行测序（MPS）的整合显著扩展了法医工具包，从身份匹配扩展到预测表型特征和生物地理祖先。在传统 DNA 分析无法确定单一嫌疑人的情况下，这种转变具有特别重要的意义。用估计生物年龄来补充法证分析可能很有价值，但涉及复杂耗时的 DNA 甲基化分析。本研究利用牛津纳米孔技术公司（ONT）的自适应直接测序方法，探索并验证了综合性法医第三代测序分析的性能。我们纳入了最广泛使用的法医标记，即 STR、SNP、InDels、线粒体 DNA（mtDNA）和两个基于甲基化的时钟分类器，从而将法医遗传学和表观遗传学分析结合在一个工作流程中：在我们的调查中，使用 ONT 标准自适应直接测序方法对六个匿名个体的 DNA 进行了测序，每个样本的平均目标读数百分比从 6.6% 到 7.7% 不等。ONT 数据与标准 MPS 数据和 Illumina EPIC DNA 甲基化图谱进行了比较。采用推荐的 ONT 软件包进行基线调用。TREAT 用于基于 ONT 的常染色体和 Y 染色体 STR 分析，根据等位基因读取深度阈值的不同，正确调用率为 90-92%。由于读取深度不够，对两个质量较低的样本进行 InDel 分析具有挑战性，而其余四个样本对观察到的标记百分比（60.9%）和正确调用率（97.8%）做出了重大贡献。SNP 分析的调用率达到 98%，只有两个错配和两个等位基因漏检。ONT 生成的 DNA 甲基化数据与 EPIC 数据的皮尔逊相关系数从 0.67 到 0.97 不等。其他与年龄相关的标记物在读取深度为 0.14（ELOVL2）到 0.96（FHL2）之间显示出与年代年龄的皮尔逊相关系数：我们使用 ONT 自适应测序法对传统法医和年龄相关 DNA 甲基化标记进行的探索性研究显示，大量标记的测序准确率很高，表明 ONT 是一种很有前途的（外）遗传法医方法。未来的研究必须解决三个关键问题：确定明确的数量和质量衡量标准以及准确性的检测阈值；优化输入 DNA 的数量以满足法医办案的期望；解决与表型和祖先分析相关的伦理问题以防止种族偏见。

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来源期刊

Forensic Science International-Genetics 生物-医学：法

CiteScore

7.50

自引率

32.30%

发文量

132

审稿时长

11.3 weeks

期刊介绍： Forensic Science International: Genetics is the premier journal in the field of Forensic Genetics. This branch of Forensic Science can be defined as the application of genetics to human and non-human material (in the sense of a science with the purpose of studying inherited characteristics for the analysis of inter- and intra-specific variations in populations) for the resolution of legal conflicts. The scope of the journal includes: Forensic applications of human polymorphism. Testing of paternity and other family relationships, immigration cases, typing of biological stains and tissues from criminal casework, identification of human remains by DNA testing methodologies. Description of human polymorphisms of forensic interest, with special interest in DNA polymorphisms. Autosomal DNA polymorphisms, mini- and microsatellites (or short tandem repeats, STRs), single nucleotide polymorphisms (SNPs), X and Y chromosome polymorphisms, mtDNA polymorphisms, and any other type of DNA variation with potential forensic applications. Non-human DNA polymorphisms for crime scene investigation. Population genetics of human polymorphisms of forensic interest. Population data, especially from DNA polymorphisms of interest for the solution of forensic problems. DNA typing methodologies and strategies. Biostatistical methods in forensic genetics. Evaluation of DNA evidence in forensic problems (such as paternity or immigration cases, criminal casework, identification), classical and new statistical approaches. Standards in forensic genetics. Recommendations of regulatory bodies concerning methods, markers, interpretation or strategies or proposals for procedural or technical standards. Quality control. Quality control and quality assurance strategies, proficiency testing for DNA typing methodologies. Criminal DNA databases. Technical, legal and statistical issues. General ethical and legal issues related to forensic genetics.