Section for Food Microbiology, Gut Health and Fermentation, Department of Food Science, University of Copenhagen, Rolighedsvej 26, Frederiksberg C, 1958, Denmark.
Section of Microbial Ecology and Biotechnology, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
Microbiome. 2024 Oct 24;12(1):219. doi: 10.1186/s40168-024-01935-5.
The gut virome is an integral component of the gut microbiome, playing a crucial role in maintaining gut health. However, accurately depicting the entire gut virome is challenging due to the inherent diversity of genome types (dsDNA, ssDNA, dsRNA, and ssRNA) and topologies (linear, circular, or fragments), with subsequently biases associated with current sequencing library preparation methods. To overcome these problems and improve reproducibility and comparability across studies, universal or standardized virome sequencing library construction methods are highly needed in the gut virome study.
We repurposed the ligation-based single-stranded library (SSLR) preparation method for virome studies. We demonstrate that the SSLR method exhibits exceptional efficiency in quantifying viral DNA genomes (both dsDNA and ssDNA) and outperforms existing double-stranded (Nextera) and single-stranded (xGen, MDA + Nextera) library preparation approaches in terms of minimal amplification bias, evenness of coverage, and integrity of assembling viral genomes. The SSLR method can be utilized for the simultaneous library preparation of both DNA and RNA viral genomes. Furthermore, the SSLR method showed its ability to capture highly modified phage genomes, which were often lost using other library preparation approaches.
We introduce and improve a fast, simple, and efficient ligation-based single-stranded DNA library preparation for gut virome study. This method is compatible with Illumina sequencing platforms and only requires ligation reagents within 3-h library preparation, which is similar or even better than the advanced library preparation method (xGen). We hope this method can be further optimized, validated, and widely used to make gut virome study more comparable and reproducible. Video Abstract.
肠道病毒组是肠道微生物组的一个组成部分，在维持肠道健康方面起着至关重要的作用。然而，由于基因组类型（双链 DNA、单链 DNA、双链 RNA 和单链 RNA）和拓扑结构（线性、圆形或片段）的固有多样性，以及与当前测序文库制备方法相关的固有偏差，准确描述整个肠道病毒组具有挑战性。为了解决这些问题，并提高肠道病毒组研究的可重复性和可比性，非常需要通用或标准化的病毒组测序文库构建方法。
我们重新利用基于连接的单链文库（SSLR）制备方法进行病毒组研究。我们证明 SSLR 方法在定量病毒 DNA 基因组（双链 DNA 和单链 DNA）方面表现出优异的效率，并且在最小扩增偏差、覆盖均匀性和组装病毒基因组的完整性方面优于现有的双链（Nextera）和单链（xGen、MDA+Nextera）文库制备方法。SSLR 方法可用于同时制备 DNA 和 RNA 病毒基因组的文库。此外，该 SSLR 方法显示出捕获高度修饰噬菌体基因组的能力，而使用其他文库制备方法往往会丢失这些基因组。
我们提出并改进了一种快速、简单、高效的基于连接的单链 DNA 文库制备方法，用于肠道病毒组研究。该方法与 Illumina 测序平台兼容，文库制备仅需 3 小时内的连接试剂，与先进的文库制备方法（xGen）相似甚至更好。我们希望这种方法能够进一步优化、验证和广泛应用，使肠道病毒组研究更具可比性和可重复性。
