Nakamae Kazuki, Ide Saya, Ohnuki Nagaki, Nakagawa Yoshiko, Okuhara Keisuke, Bono Hidemasa
Genome Editing Innovation Center, Hiroshima University, 3-10-23 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-0046, Japan.
PtBio Inc., Hiroshima, 739-0046, Japan.
BMC Bioinformatics. 2025 Apr 29;26(1):114. doi: 10.1186/s12859-025-06139-8.
Tracking of Insertions and DEletions (TIDE) analysis, which computationally deconvolves capillary sequencing data derived from the DNA of bulk or clonal cell populations to estimate the efficiency of targeted mutagenesis by programmable nucleases, has played a significant role in the field of genome editing. However, the detection range covered by conventional TIDE analysis is limited. Range extension for deconvolution is required to detect larger deletions and insertions (indels) derived from genome editing in TIDE analysis. However, extending the deconvolution range introduces uncertainty into the deconvolution process. Moreover, the accuracy and sensitivity of TIDE analysis tools for large deletions (> 50 bp) remain poorly understood.
In this study, we introduced a new software called PtWAVE that can detect a wide range of indel sizes, up to 200 bp. PtWAVE also offers options for variable selection and fitting algorithms to prevent uncertainties in the model. We evaluated the performance of PtWAVE by using in vitro capillary sequencing data that mimicked DNA sequencing, including large deletions. Furthermore, we confirmed that PtWAVE can stably analyze trace sequencing data derived from actual genome-edited samples.
PtWAVE demonstrated superior accuracy and sensitivity compared to the existing TIDE analysis tools for DNA samples, including large deletions. PtWAVE can accelerate genome editing applications in organisms and cell types in which large deletions often occur when programmable nucleases are applied.
插入和缺失追踪（TIDE）分析通过对来自大量或克隆细胞群体DNA的毛细管测序数据进行计算反卷积，以估计可编程核酸酶靶向诱变的效率，在基因组编辑领域发挥了重要作用。然而，传统TIDE分析的检测范围有限。在TIDE分析中，需要扩展反卷积范围以检测源自基因组编辑的更大的缺失和插入（indel）。然而，扩展反卷积范围会给反卷积过程带来不确定性。此外，对于大缺失（>50 bp）的TIDE分析工具的准确性和灵敏度仍知之甚少。
在本研究中，我们引入了一种名为PtWAVE的新软件，它可以检测范围广泛的indel大小，最大可达200 bp。PtWAVE还提供了变量选择和拟合算法选项，以防止模型中的不确定性。我们通过使用模拟DNA测序的体外毛细管测序数据（包括大缺失）评估了PtWAVE的性能。此外，我们证实PtWAVE可以稳定地分析源自实际基因组编辑样本的微量测序数据。
与现有的用于DNA样本（包括大缺失）的TIDE分析工具相比，PtWAVE表现出更高的准确性和灵敏度。PtWAVE可以加速在应用可编程核酸酶时经常出现大缺失的生物体和细胞类型中的基因组编辑应用。
