Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters.
Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters.
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DOI：
10.1093/nar/gkab199
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发表时间：
2021-07-09
影响因子：
14.9
通讯作者：
Chen IA
中科院分区：
文献类型：
--
作者：
Shen Y;Pressman A;Janzen E;Chen IA
表征生物分子（例如核酶）的基因型-表型关系需要准确的方法来测量大量分子的活性。使用高通量测序（例如 k-Seq）进行动力学测量是一种适用于各个领域的新兴检测方法，有可能将测量通量扩展到超过 106 个独特的核酸序列。然而，要最大化此类检测的回报，需要了解序列异质性和 DNA 测序带来的技术挑战。我们使用模拟数据集和来自先前识别的核酶构建的变体库的实验数据，从模型可识别性、测序误差的影响、准确性和精密度方面对 k-Seq 方法进行了表征。发现相对丰度、动力学系数和测量噪声会影响每个序列的测量。我们引入了引导法来稳健地量化估计模型参数的不确定性，并提出了可解释的指标来量化模型的可识别性。这些努力使得 k-Seq 实验中各个序列的数据质量能够得到严格的报告。在这里，我们提出详细的协议，定义关键的实验因素，并确定一般准则，以最大限度地提高 k-Seq 数据的序列数量及其测量准确性。类似的做法可用于提高其他基于测序的测定的严谨性。
Characterizing genotype-phenotype relationships of biomolecules (e.g. ribozymes) requires accurate ways to measure activity for a large set of molecules. Kinetic measurement using high-throughput sequencing (e.g. k-Seq) is an emerging assay applicable in various domains that potentially scales up measurement throughput to over 106 unique nucleic acid sequences. However, maximizing the return of such assays requires understanding the technical challenges introduced by sequence heterogeneity and DNA sequencing. We characterized the k-Seq method in terms of model identifiability, effects of sequencing error, accuracy and precision using simulated datasets and experimental data from a variant pool constructed from previously identified ribozymes. Relative abundance, kinetic coefficients, and measurement noise were found to affect the measurement of each sequence. We introduced bootstrapping to robustly quantify the uncertainty in estimating model parameters and proposed interpretable metrics to quantify model identifiability. These efforts enabled the rigorous reporting of data quality for individual sequences in k-Seq experiments. Here we present detailed protocols, define critical experimental factors, and identify general guidelines to maximize the number of sequences and their measurement accuracy from k-Seq data. Analogous practices could be applied to improve the rigor of other sequencing-based assays.
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