Center for Systems and Therapeutics, Gladstone Institutes, 1650 Owens Street, San Francisco, CA, 94158, USA.
Center for Systems and Therapeutics, Gladstone Institutes, 1650 Owens Street, San Francisco, CA, 94158, USA; Departments of Neurology and Physiology, University of California, San Francisco, San Francisco, CA, 94158, USA.
Redox Biol. 2023 Jun;62:102680. doi: 10.1016/j.redox.2023.102680. Epub 2023 Mar 24.
DNA damage is a common cellular feature seen in cancer and neurodegenerative disease, but fast and accurate methods for quantifying DNA damage are lacking. Comet assays are a biochemical tool to measure DNA damage based on the migration of broken DNA strands towards a positive electrode, which creates a quantifiable 'tail' behind the cell. However, a major limitation of this approach is the time needed for analysis of comets in the images with available open-source algorithms. The requirement for manual curation and the laborious pre- and post-processing steps can take hours to days. To overcome these limitations, we developed AutoComet, a new open-source algorithm for comet analysis that utilizes automated comet segmentation and quantification of tail parameters. AutoComet first segments and filters comets based on size and intensity and then filters out comets without a well-connected head and tail, which significantly increases segmentation accuracy. Because AutoComet is fully automated, it minimizes curator bias and is scalable, decreasing analysis time over ten-fold, to less than 3 s per comet. AutoComet successfully detected statistically significant differences in tail parameters between cells with and without induced DNA damage, and was more comparable to the results of manual curation than other open-source software analysis programs. We conclude that the AutoComet algorithm provides a fast, unbiased and accurate method to quantify DNA damage that avoids the inherent limitations of manual curation and will significantly improve the ability to detect DNA damage.
DNA 损伤是癌症和神经退行性疾病中常见的细胞特征，但缺乏快速准确的方法来定量 DNA 损伤。彗星实验是一种基于断裂 DNA 链向正极迁移来测量 DNA 损伤的生化工具，这会在细胞后面产生可量化的“尾巴”。然而，这种方法的一个主要限制是，使用现有的开源算法分析图像中的彗星需要时间。手动注释和繁琐的预处理和后处理步骤可能需要数小时到数天。为了克服这些限制，我们开发了 AutoComet，这是一种用于彗星分析的新的开源算法，它利用自动彗星分割和尾部参数的定量。AutoComet 首先根据大小和强度对彗星进行分割和过滤，然后过滤掉没有连接良好的头部和尾部的彗星，这显著提高了分割准确性。由于 AutoComet 是完全自动化的，它可以最小化注释者的偏见，并具有可扩展性，将分析时间减少了十倍以上，每个彗星的分析时间不到 3 秒。AutoComet 成功地检测到诱导 DNA 损伤前后细胞尾部参数的统计学差异，并且与手动注释相比，与其他开源软件分析程序的结果更可比。我们得出结论，AutoComet 算法提供了一种快速、无偏和准确的定量 DNA 损伤的方法，避免了手动注释的固有限制，并将大大提高检测 DNA 损伤的能力。
