Li Ao, Sun Bingrui, Zhang Ying, Yu Ping, Qu Jicheng, Deng Hongkuan, Pang Qiuxiang, Yang Fengtang
School of Life Sciences and Medicine, Shandong University of Technology, Zibo, 255049, PR China.
BMC Genomics. 2025 May 26;26(1):532. doi: 10.1186/s12864-025-11731-6.
The isolation of intact, high-molecular-weight genomic DNA (HMW gDNA) is essential for achieving complete genome assemblies. However, extracting HMW gDNA from a single individual of Dugesia japonica remains a technical challenge using the standard protocol, probably due to the presence of abundant polysaccharides and nucleases.
In this study, we have developed a more robust protocol for preparing HMM gDNA, with high yields and quality, from a single D. japonica. The key step in our protocol involves the use of a Mg-dependent lysis buffer, rather than using metal cation chelation to block the activities of DNase I as in the standard protocol. Using this approach were able to achieve a yield of about 10-15 µg of HWM gDNA per worm. Our method showed species- and region-specific effectiveness, with optimal results observed at 20 mM Mg for our local D. japonica specimens. The extracted HMW gDNA is fully compatible with advanced long-read sequencing platforms such as PacBio HiFi and Oxford Nanopore. However, when applied to Schmidtea mediterranea and D. japonica specimens from Beijing, the method was ineffective and led to progressive gDNA degradation.
This protocol offers a simple and high-yield solution for isolating HMW gDNA from D. japonica. It also provides an alternative for organisms whose gDNA consistently exhibits unexplained degradation using established protocols.
分离完整的高分子量基因组DNA（HMW gDNA）对于实现完整的基因组组装至关重要。然而，使用标准方案从单个日本三角涡虫个体中提取HMW gDNA仍然是一项技术挑战，这可能是由于存在大量多糖和核酸酶。
在本研究中，我们开发了一种更强大的方案，用于从单个日本三角涡虫中制备高产高质量的HMM gDNA。我们方案的关键步骤是使用镁依赖性裂解缓冲液，而不是像标准方案那样使用金属阳离子螯合来阻断DNase I的活性。使用这种方法，我们能够实现每条虫约10 - 15微克HWM gDNA的产量。我们的方法显示出物种和区域特异性效果，对于我们当地的日本三角涡虫标本，在20 mM镁时观察到最佳结果。提取的HMW gDNA与PacBio HiFi和Oxford Nanopore等先进的长读长测序平台完全兼容。然而，当应用于来自北京的地中海涡虫和日本三角涡虫标本时，该方法无效并导致gDNA逐渐降解。
该方案为从日本三角涡虫中分离HMW gDNA提供了一种简单且高产的解决方案。它也为那些使用既定方案其gDNA一直出现无法解释的降解的生物体提供了一种替代方法。
