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Optimization and evaluation of viral metagenomic amplification and sequencing procedures toward a genome-level resolution of the human fecal DNA virome
Journal of Advanced Research ( IF 10.7 ) Pub Date : 2022-08-20 , DOI: 10.1016/j.jare.2022.08.011
Guangyang Wang 1 , Shenghui Li 2 , Qiulong Yan 1 , Ruochun Guo 3 , Yue Zhang 3 , Fang Chen 1 , Xiangge Tian 4 , Qingbo Lv 3 , Hao Jin 3 , Xiaochi Ma 5 , Yufang Ma 1
Affiliation  

Introduction

Viruses in the human gut have been linked to health and disease. Deciphering the gut virome is dependent on metagenomic sequencing of the virus-like particles (VLPs) purified from the fecal specimens. A major limitation of conventional viral metagenomic sequencing is the low recoverability of viral genomes from the metagenomic dataset.

Objectives

To develop an optimal method for viral amplification and metagenomic sequencing for maximizing the recovery of viral genomes.

Methods

We performed parallel virus enrichment and DNA extraction to generate ∼ 30 viral DNA samples from each of 5 fresh fecal specimens and conducted the experiments including 1) optimizing the cycle number for high-fidelity enzyme-based PCR amplification, 2) evaluating the reproducibility of the optimally whole viral metagenomic experimental process, 3) evaluating the reliability of multiple displacement amplification (MDA), 4) testing the capability of long-read sequencing for improving viral metagenomic assembly, and 5) comparing the differences between viral metagenomic and bulk metagenomic approaches.

Results

Our results revealed that the optimal cycle number for PCR amplification is 15. We verified the reliability of MDA and the effectiveness of long-read sequencing. Based on our optimized results, we generated 151 high-quality viruses using the dataset combined from short-read and long-read sequencing. Genomic analysis of these viruses found that most (60.3%) of them were previously unknown and showed a remarkable diversity of viral functions, especially the existence of 206 viral auxiliary metabolic genes. Finally, we uncovered significant differences in the efficiency and coverage of viral identification between viral metagenomic and bulk metagenomic approaches.

Conclusions

Our study demonstrates the potential of optimized experiment and sequencing strategies in uncovering viral genomes from fecal specimens, which will facilitate future research about the genome-level characterization of complex viral communities.



中文翻译:

病毒宏基因组扩增和测序程序的优化和评估,以实现人类粪便 DNA 病毒组的基因组水平分辨率

介绍

人类肠道中的病毒与健康和疾病有关。破译肠道病毒组依赖于从粪便标本中纯化的病毒样颗粒 (VLP) 的宏基因组测序。传统病毒宏基因组测序的一个主要局限是宏基因组数据集中病毒基因组的可恢复性低。

目标

开发病毒扩增和宏基因组测序的最佳方法,以最大限度地恢复病毒基因组。

方法

我们进行了平行病毒富集和 DNA 提取,从 5 个新鲜粪便标本中每一个生成约 30 个病毒 DNA 样本,并进行了实验,包括 1) 优化基于酶的高保真 PCR 扩增的循环数,2) 评估重复性优化整个病毒宏基因组实验过程,3) 评估多重位移扩增 (MDA) 的可靠性,4) 测试长读长测序改进病毒宏基因组组装的能力,以及 5) 比较病毒宏基因组和批量宏基因组方法之间的差异。

结果

我们的结果表明 PCR 扩增的最佳循环数是 15。我们验证了 MDA 的可靠性和长读长测序的有效性。基于我们的优化结果,我们使用短读长和长读长测序相结合的数据集生成了 151 种高质量病毒。对这些病毒进行基因组分析发现,其中大部分(60.3%)以前是未知的,表现出显着的病毒功能多样性,尤其是存在206个病毒辅助代谢基因。最后,我们发现了病毒宏基因组和大量宏基因组方法在病毒识别效率和覆盖范围方面的显着差异。

结论

我们的研究证明了优化实验和测序策略在从粪便标本中发现病毒基因组方面的潜力,这将促进未来对复杂病毒群落的基因组水平表征的研究。

更新日期:2022-08-20
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