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确定高通量测序的精度及其对Aptamer选择的影响

Juan Canoura¹, Obtin Alkhamis¹, Caleb Byrd¹

¹Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Raleigh, North Carolina 27695, United States.

Analytical chemistry

|October 25, 2024

概括

高通量测序 (HTS) 对于体外选择至关重要,但其在0.1%以下测量序列丰度的精度不可靠. 准确的HTS数据对于识别功能性核酸和胺体至关重要.

科学领域:

分子生物学分子生物学
生物技术是生物技术.
基因组学就是基因组学.

背景情况:

在体外选择从随机图书馆中识别出功能性核酸.
高通量测序 (HTS) 在选择过程中监测寡核酸池的演变.
对于aptamer选择的HTS的精度尚不清楚.

研究的目的:

在实验室选择中实证地确定HTS用于测量序列丰度的精度.
评估HTS精度对体发现的影响.
为在核酸选择中使用HTS的研究人员提供实用见解.

主要方法:

在实验室中对选定的池进行了多次独立的基于Illumina的测序试验.
经验地确定了序列丰度测量的精度.
分析了跨不同序列丰度值的HTS数据可靠性.

主要成果:

对于丰度≥0.1%的序列的HTS测量通常是可靠的 (RSD<25%).
低于0.1%的丰度值的测量是高度不可重现的.
HTS数据精度直接影响丰富度指标用于预测功能性质的实用性.

结论:

HTS数据的可靠性对于成功的体外选择和aptamer发现至关重要.
实践者必须考虑HTS精度限制,特别是对于低丰度序列.
这些发现有助于优化基于HTS的策略,以隔离和表征功能性寡核酸.

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