Kinetic sequencing (k-Seq) as a massively parallel assay for ribozyme kinetics: utility and critical parameters

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 metr...表征生物分子（如核酶（ribozyme））的基因型-表型关系，需要可精准测定大量分子活性的方法。采用高通量测序（如k-Seq）的动力学测定是一类新兴检测技术，可应用于诸多领域，有望将检测通量提升至10^6种以上独特核酸序列。然而，要最大化这类检测的应用价值，需明晰序列异质性与DNA测序所引入的技术挑战。本研究借助模拟数据集与由先前已鉴定核酶构建的变异体库的实验数据，从模型可辨识性、测序误差的影响、准确性与精密度等维度对k-Seq方法进行了表征。研究发现，相对丰度、动力学系数与测量噪声均会对各序列的测定造成影响。我们引入自助法（bootstrapping）以稳健量化模型参数估计中的不确定性，并提出了可解释的度量...