Evaluation of Automated Magnetic Bead–Based DNA Extraction for Detection of Short Tandem Repeat Expansions With Nanopore Sequencing

IF 2.6 4区医学 Q2 MEDICAL LABORATORY TECHNOLOGY

Journal of Clinical Laboratory Analysis Pub Date : 2024-03-20 DOI:10.1002/jcla.25029

Helene Faust, Patricia Duffek, Julia Hentschel, Denny Popp

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Abstract

Background

Long-read technologies such as nanopore sequencing provide new opportunities to detect short tandem repeat expansions. Therefore, a DNA extraction method is necessary that minimizes DNA fragmentation and hence allows the identification of large repeat expansions. In this study, an automated magnetic bead–based DNA extraction method and the required EDTA blood storage conditions as well as DNA and sequencing quality were evaluated for their suitability for repeat expansion detection with nanopore sequencing.

Methods

DNA was extracted from EDTA blood, and subsequently, its concentration, purity, and integrity were assessed. DNA was then subjected to nanopore sequencing, and quality metrics of the obtained sequencing data were evaluated.

Results

DNA extracted from fresh EDTA blood as well as from cooled or frozen EDTA blood revealed high DNA integrity whereas storage at room temperature over 7 days had detrimental effects. After nanopore sequencing, the read length N50 values of approximately 9 kb were obtained, and based on adaptive sampling of samples with a known repeat expansion, repeat expansions up to 10 kb could be detected.

Conclusion

The automated magnetic bead–based DNA extraction was sufficient to detect short tandem repeat expansions, omitting the need for high-molecular-weight DNA extraction methods. Therefore, DNA should be extracted either from fresh blood or from blood stored in cooled or frozen conditions. Consequently, this study may help other laboratories to evaluate their DNA extraction method regarding the suitability for detecting repeat expansions with nanopore sequencing.

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评估利用纳米孔测序检测短串联重复序列扩增的基于磁珠的自动 DNA 提取。

背景：纳米孔测序等长读技术为检测短串联重复扩增提供了新的机会。因此，有必要采用一种 DNA 提取方法，以最大限度地减少 DNA 断裂，从而鉴定大重复扩增。本研究评估了一种基于磁珠的自动 DNA 提取方法和所需的 EDTA 血液储存条件以及 DNA 和测序质量，以确定其是否适合用纳米孔测序检测重复扩增：从 EDTA 血液中提取 DNA，然后评估其浓度、纯度和完整性。然后对 DNA 进行纳米孔测序，并评估测序数据的质量指标：结果：从新鲜的乙二胺四乙酸（EDTA）血液以及冷却或冷冻的乙二胺四乙酸（EDTA）血液中提取的 DNA 显示出较高的 DNA 完整性，而在室温下保存 7 天以上则会产生不利影响。纳米孔测序后，读长 N50 值约为 9 kb，根据对已知重复扩增样本的自适应取样，可检测到重复扩增达 10 kb：结论：基于磁珠的自动 DNA 提取足以检测短串联重复扩增，无需使用高分子量 DNA 提取方法。因此，DNA 应从新鲜血液或在冷却或冷冻条件下储存的血液中提取。因此，本研究可帮助其他实验室评估其DNA提取方法是否适合用纳米孔测序法检测重复扩增。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Clinical Laboratory Analysis 医学-医学实验技术

CiteScore

5.60

自引率

7.40%

发文量

584

审稿时长

6-12 weeks

期刊介绍： Journal of Clinical Laboratory Analysis publishes original articles on newly developing modes of technology and laboratory assays, with emphasis on their application in current and future clinical laboratory testing. This includes reports from the following fields: immunochemistry and toxicology, hematology and hematopathology, immunopathology, molecular diagnostics, microbiology, genetic testing, immunohematology, and clinical chemistry.