Optimization of microhaplotypes for advanced DNA mixture deconvolution

Detection of minor DNA components in biological mixtures has increased as molecular techniques have become more sensitive. Accordingly, mixture deconvolution has become a major concern and topic of debate in the forensic DNA community. Short tandem repeat (STR) profile data generated with capillary electrophoresis and massively parallel sequencing (MPS) are subject to inherent issues that complicate mixture deconvolution. Deconvolution may be improved by sequencing microhaplotypes as they are not subject to the amplification noise artifacts and stochastic effects that impact STRs. Before microhaplotypes can be implemented in casework, the following considerations should be addressed: definition of a consistent panel of microhaplotype loci; increased population studies to determine relevant haplotype allele frequencies; incorporation of advanced sequencing technologies into forensic laboratories; development of user-friendly bioinformatic analysis and mixture deconvolution methods; and a..., An AmpliSeq for Illumina assay was developed to target amplify and sequence 43 forensically relevant microhaplotype loci, specifically selected for their potential application to complex mixture deconvoution. Target loci were selected from previously published work compiled in the MicroHapDB database [https://microhapdb.readthedocs.io/en/latest/].
A set of 240 test samples was identified from a donor collection of nearly 500 individuals previously collected under IRB and housed at GWU. These donors represent eleven biogeographical populations. Additional donors were identified from purchased blood bank samples previously obtained for internal validation projects.
For sensitivity testing of the final assay, 2800M Control DNA (Promega Corp, Madison, WI), NA24385 (NIST RM8391), and two purchased blood samples were serial diluted to test inputs of 2 ng, 1 ng, 0.5 ng, 0.1 ng, 0.05 ng, and 0.025 ng. Each dilution was evaluated in triplicate libraries.
A set of 149 complex mixtures were constr..., # Optimization of microhaplotypes for advanced DNA mixture deconvolution

DOI: 10.5061/dryad.k98sf7mmt

This data set contains locus information, assay sensitivity metrics, and mixture deconvolution results for the evaluation of a target sequencing microhaplotype assay.
This project was supported by Award No. 15PNIJ-22-GG-04393-DNAX, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this presentation are those of the authors and do not necessarily reflect those of the Department of Justice.

## Description of the data and file structure

Tabular file 15pnij-22-gg-04393-dnax_LocusInfo.csv contains the chromosome locations for each of the 43 loci evaluated in this effort.

* MH Name: Locus name following general convention established in Kidd 2016 ([https://doi.org/10.1186/s40246-016-0078-y](https://doi.org/10.1186/s40246-016-0078-y))
* Chr#: Chromosome locations of ..., No personally identifiable data is provided in these data. Microhaplotype allele frequencies calculated within a population classification are disclosed; however, no genotype information of individuals will be provided. All samples were collected under IRB or purchased from a biorepository as anonymized donors. IRB consent allows for publishing of de-identified data in the public domain, so long as individual genotypes are withheld. Upon collection, all donors were provided a sample archiving code to de-identify the source donor and then given a secondary project code for further de-identification in this project. Two commercially available samples often used as control samples for genotyping studies were also purchased for use in this project: Promega 2800M and NIST RM8391/NA24385. Lastly, the NIST RGTM samples were obtained from NIST. These are also publicly accessible samples that have been de-identified and consented to disclose genotype information; however, no genotype information...

随着分子检测技术灵敏度的不断提升，生物混合样本中微量DNA成分的检出率逐步提高，因此混合图谱解析已成为法医DNA学界备受关注的核心议题与热议话题。通过毛细管电泳（Capillary Electrophoresis）与大规模并行测序（Massively Parallel Sequencing, MPS）获取的短串联重复序列（Short Tandem Repeat, STR）分型数据存在固有缺陷，会增加混合图谱解析的难度。而微单倍型（microhaplotype）测序则可有效提升混合图谱解析效果，因其不受影响STR分型的扩增噪声伪影与随机效应的干扰。在将微单倍型技术应用于实际案件检验前，需解决以下关键问题：建立统一的微单倍型位点组合；开展更多人群研究以确定相关单倍型等位基因频率；将先进测序技术融入法医实验室；开发易用的生物信息学分析与混合图谱解析方法；以及……；针对Illumina平台的AmpliSeq检测体系被开发用于靶向扩增并测序43个法医学相关的微单倍型位点，这些位点均经过筛选以适配复杂混合图谱解析的应用需求。目标位点选自此前发表的、整合于MicroHapDB数据库的研究成果[https://microhapdb.readthedocs.io/en/latest/]。

研究团队从近500名已采集的供体样本库中筛选出240份检测样本，该样本库此前已通过伦理审查委员会（Institutional Review Board, IRB）批准，并保存于乔治华盛顿大学（George Washington University, GWU）。这些供体来自11个生物地理人群。额外的供体样本则采购自此前用于内部验证项目的血库样本。

为对最终检测体系开展灵敏度测试，研究人员对2800M 对照DNA（普洛麦格公司，威斯康星州麦迪逊市）、NA24385（美国国家标准与技术研究院RM8391，National Institute of Standards and Technology, NIST RM8391）以及两份采购的血液样本进行梯度稀释，测试的DNA投入量分别为2 ng、1 ng、0.5 ng、0.1 ng、0.05 ng与0.025 ng。每个稀释梯度均设置三次重复文库构建与检测。

共构建149份复杂混合样本…… # 面向复杂DNA混合图谱解析的微单倍型优化研究

DOI：10.5061/dryad.k98sf7mmt

本数据集包含用于评估靶向测序微单倍型检测体系的位点信息、检测灵敏度指标及混合图谱解析结果。

本项目由美国司法部司法项目办公室国家司法研究所授予的编号为15PNIJ-22-GG-04393-DNAX的资助项目支持。本报告中表达的观点、发现、结论或建议仅代表作者本人，不一定代表司法部的官方立场。

## 数据与文件结构说明

表格文件15pnij-22-gg-04393-dnax_LocusInfo.csv 包含本研究中评估的全部43个位点的染色体位置信息。
* MH名称：遵循Kidd 2016年确立的通用命名规则的位点名称[https://doi.org/10.1186/s40246-016-0078-y]
* Chr#：染色体位置……

本数据集未包含任何可识别个人身份的信息。本研究公开了基于人群分类计算得到的微单倍型等位基因频率，但不会提供个体的基因型信息。所有样本均通过伦理审查委员会（Institutional Review Board, IRB）批准采集，或从生物样本库采购匿名供体样本。伦理审查委员会的知情同意书允许在公共领域发布去标识化数据，但需隐去个体基因型信息。样本采集时，所有供体均被赋予样本存档编码以实现供体来源的去标识化，随后再分配二级项目编码用于本项目中的进一步去标识化处理。本研究还采购了两种常用于基因分型研究的商业化对照样本：普洛麦格2800M对照DNA以及NIST RM8391/NA24385样本。此外，本研究还从NIST获取了NIST RGTM系列样本，此类样本同样为可公开获取的去标识化样本，已获得公开基因型信息的知情同意，但本数据集同样不会提供具体的基因型信息……