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使用超低起始DNA输入的细菌的短读和长读PCR无序测序

Thania Sbaghdi^1,2, Florence Jagorel¹, Marc Monot¹

¹Institut Pasteur Universite Paris Cite Plate-forme Technologique Biomics.

Journal of biomolecular techniques : JBT

|May 7, 2025

概括

现在可以对菌体DNA进行测序,输入量比以前想象的要低得多. 这项研究表明,在Illumina和纳米孔测序技术中,使用1000倍少的DNA来获得高质量的结果.

科学领域:

微生物学微生物学
基因组学就是基因组学.
分子生物学分子生物学

背景情况:

菌体DNA用于测序的提取通常受到低产量的限制,特别是如果没有敏感的细菌宿主.
基于标记的测序,同时避免PCR,可以导致菌体基因组末端的损失和偏差覆盖.
无PCR测序是无偏差菌体基因组表征的理想选择,但需要高DNA输入,这对低产量样本构成挑战.

研究的目的:

评估使用非常低的起始材料测序菌体基因组DNA的可行性.
用Illumina和纳米孔技术确定减少DNA输入对测序质量的影响.
为可靠的菌体基因组测序建立新的最小输入DNA要求.

主要方法:

从不同低度的菌体基因组DNA中评估了测序质量和基因组组.
使用了Illumina短读和Nanopore长读测序平台.
对比结果与制造商推的每个技术的DNA输入水平.

主要成果:

高质量的菌体DNA测序可以通过1000倍低于推的DNA输入来实现.
成功地测序了菌体DNA,仅使用Illumina的1 ng和纳米孔测序的0.4 ng.
证明了PCR无序测序的可行性,即使使用最小的DNA数量.

结论:

确定低DNA输入水平 (短读低至1ng,长读低至0.4ng) 足以进行高质量的菌体基因组测序.
克服了低DNA输入对PCR无序测序的挑战,使无偏向的菌体基因组表征成为可能.
提供了对菌体研究的实用解决方案,以有限的样本材料促进基因组测序,功能研究和治疗开发.

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