PO.CL01.10 · 临床研究
基于磁珠的低起始量靶向DNA富集cfDNA提取的分析性能
Analytical performance of magnetic bead-based cfDNA extraction with a low input targeted DNA enrichment
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：cfDNA提取化学与下游靶向DNA检测之间的分析一致性对于可靠的分子计数至关重要，尤其是在低血浆起始量的情况下。本研究评估了两种基于磁珠的cfDNA提取方法在低起始量靶向DNA富集工作流程中是否产生等效或可区分的性能。
方法：实验I（人血浆，n = 8）：每位供体的16份3 mL血浆等分样本采用两种磁珠法试剂盒——Revolution cfDNA Max 20 Kit和MagMAX——并行提取，从相同起始样本产生配对洗脱液。质控包括电泳法cfDNA定量（50-700 bp）、核小体图谱评估以及高分子量（HMW）残留检测。实验II（人工加标）：含有0.2%等位基因频率cfDNA加标物的血浆基质，采用相同的提取试剂盒从1 mL起始量中一式两份进行提取。主要终点为按照Agilent的Avida低起始量靶向DNA富集工作流程处理后的文库可用cfDNA浓度和分子回收率。次要终点包括VAF一致性、靶向比例、背景噪声和文库复杂度。
结果：在两项实验中，两种磁珠法提取的cfDNA均达到最低质控阈值；然而，Revolution cfDNA Max 20 Kit始终表现出更优的分析性能，包括：更高的文库可用cfDNA浓度回收率（50-700 bp）、更清晰的单核小体/双核小体峰结构、更低的HMW残留频率，以及更强的低频（0.2%）变异检测质控适用性。加标实验表明，该方法产生的洗脱液更可靠地满足低起始量靶向DNA富集的要求，支持对下游分子计数更高的置信度。配对洗脱液的测序正在进行中，将量化分子回收率、VAF精度和背景抑制方面的差异。
结论：两种磁珠法均产生了适用于低起始量靶向测序的cfDNA；然而，Revolution cfDNA Max 20 Kit提供了更优的cfDNA产量、片段质量指标和整体分析前性能。待测序结果将进一步明确分析优势的幅度，并支持对低起始量液体活检检测分析前工作流程的优化建议。
AI声明：本摘要的部分内容使用AI辅助工具生成，随后由作者审阅和编辑。
查看英文原文 English abstract
Introduction: Analytical alignment between cfDNA extraction chemistry and downstream targeted-DNA assays is essential for reliable molecular counting, especially at low plasma input. This study evaluated whether two magnetic bead-based cfDNA extraction methods generate equivalent or distinguishable performance within a low-input targeted DNA enrichment workflow.
Methods: Experiment I (Human plasma, n = 8): Sixteen 3-mL plasma aliquots per donor were extracted in parallel using two bead-based kits-the Revolution cfDNA Max 20 Kit and MagMAX-producing paired eluates from identical input samples. QC included electrophoretic cfDNA quantification (50-700 bp), nucleosomal-profile assessment, and detection of high-molecular-weight (HMW) carryover. Experiment II (Contrived spike-in): Plasma matrices containing a 0.2% allele-frequency cfDNA spike-in were extracted in duplicate from 1-mL inputs using the same extraction kits.The primary endpoints were the library-eligible cfDNA concentration and molecule recovery following Agilent's Avida low-input targeted DNA enrichment workflow. Secondary endpoints included VAF concordance, on-target fraction, background noise, and library complexity.
Results: Across both experiments, cfDNA from the two bead-based extractions met the minimal QC thresholds; however, the Revolution cfDNA Max 20 Kit consistently demonstrated superior analytical performance, including: higher recovery of library-eligible cfDNA concentration (50-700 bp), more defined mono-/di-nucleosomal peak structure, less frequent HMW carryover, and stronger QC suitability for low-frequency (0.2%) variant interrogation.Spike-in experiments showed that eluates generated by this method more reliably met the requirements for low-input targeted DNA enrichment, supporting higher confidence in downstream molecular counting. Sequencing of paired eluates is ongoing and will quantify differences in molecule recovery, VAF precision, and background suppression.
Conclusions: Both magnetic bead-based methods generated cfDNA suitable for low-input targeted sequencing; however, the Revolution cfDNA Max 20 Kit delivered superior cfDNA yield, fragment-quality metrics, and overall pre-analytical performance. Pending sequencing results will further define the magnitude of analytical advantage and support optimized recommendations for pre-analytical workflows in low-input liquid biopsy assays.
AI Disclosure: Portions of this abstract were generated using an AI-assisted tool and subsequently reviewed and edited by the authors.
利益披露 Disclosure
M. Saidian,
nRichDX Employment.
Y. Wang,
Agilent Technologies, Inc. Employment.
H. Wang,
Agilent Technologies, Inc. Employment.
Y. Bao,
Agilent Technologies, Inc. Employment.
J. Saenz,
nRichDX Employment.
C. Hernandez,
nRichDX Employment.
C. Van Dieren,
nRichDX Employment.
D. Cedeno,
nRichDX Employment.