High-molecular-weight DNA extraction for long-read sequencing of plant genomes: An optimization of standard methods

IF 4.6 Q2 MATERIALS SCIENCE, BIOMATERIALS ACS Applied Bio Materials Pub Date : 2023-06-13 DOI:10.1002/aps3.11528

Myoungbo Kang, Andre Chanderbali, Seungyeon Lee, Douglas E. Soltis, Pamela S. Soltis, Sangtae Kim

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引用次数: 5

Abstract

Premise

Developing an effective and easy-to-use high-molecular-weight (HMW) DNA extraction method is essential for genomic research, especially in the era of third-generation sequencing. To efficiently use technologies capable of generating long-read sequences, it is important to maximize both the length and purity of the extracted DNA; however, this is frequently difficult to achieve with plant samples.

Methods and Results

We present a HMW DNA extraction method that combines (1) a nuclei extraction method followed by (2) a traditional cetyltrimethylammonium bromide (CTAB) DNA extraction method for plants with optimized extraction conditions that influence HMW DNA recovery. Our protocol produced DNA fragments (percentage of fragments >20 kbp) that were, on average, ca. five times longer than those obtained using a commercial kit, and contaminants were removed more effectively.

Conclusions

This effective HMW DNA extraction protocol can be used as a standard protocol for a diverse array of taxa, which will enhance plant genomic research.

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用于植物基因组长读测序的高分子量DNA提取:标准方法的优化

开发一种有效且易于使用的高分子量(HMW) DNA提取方法对于基因组研究至关重要，特别是在第三代测序时代。为了有效地利用能够产生长读序列的技术，最大限度地提高提取DNA的长度和纯度是很重要的;然而，这通常很难在植物样本中实现。方法与结果提出了一种HMW DNA提取方法，该方法结合了(1)核提取法和(2)传统的十六烷基三甲基溴化铵(CTAB)提取植物DNA的方法，并优化了影响HMW DNA回收率的提取条件。我们的方案产生的DNA片段(片段百分比> 20kbp)平均比使用商业试剂盒获得的DNA片段长约5倍，并且更有效地去除污染物。结论该提取方案可作为植物基因组研究的标准方案，为进一步开展植物基因组研究奠定基础。

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