Gut Microbes and Health Research Programme, Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UQ, UK.
Norwich Medical School, University of East Anglia, Norwich NR4 7TJ, UK.
Viruses. 2021 Oct 18;13(10):2093. doi: 10.3390/v13102093.
The human intestinal microbiota is abundant in viruses, comprising mainly bacteriophages, occasionally outnumbering bacteria 10:1 and is termed the virome. Due to their high genetic diversity and the lack of suitable tools and reference databases, the virome remains poorly characterised and is often referred to as "viral dark matter". However, the choice of sequencing platforms, read lengths and library preparation make study design challenging with respect to the virome. Here we have compared the use of PCR and PCR-free methods for sequence-library construction on the Illumina sequencing platform for characterising the human faecal virome. Viral DNA was extracted from faecal samples of three healthy donors and sequenced. Our analysis shows that most variation was reflecting the individually specific faecal virome. However, we observed differences between PCR and PCR-free library preparation that affected the recovery of low-abundance viral genomes. Using three faecal samples in this study, the PCR library preparation samples led to a loss of lower-abundance vOTUs evident in their PCR-free pairs (vOTUs 128, 6202 and 8364) and decreased the alpha-diversity indices (Chao1 -value = 0.045 and Simpson -value = 0.044). Thus, differences between PCR and PCR-free methods are important to consider when investigating "rare" members of the gut virome, with these biases likely negligible when investigating moderately and highly abundant viruses.
人类肠道微生物群中富含病毒，主要由噬菌体组成，偶尔数量是细菌的 10 倍，被称为病毒组。由于其高度的遗传多样性以及缺乏合适的工具和参考数据库，病毒组的特征仍然很差，通常被称为“病毒暗物质”。然而，测序平台、读长和文库制备的选择使得病毒组的研究设计具有挑战性。在这里，我们比较了使用 PCR 和无 PCR 方法在 Illumina 测序平台上构建序列文库，以描述人类粪便病毒组。从三个健康供体的粪便样本中提取病毒 DNA 并进行测序。我们的分析表明，大多数变异反映了个体特异性的粪便病毒组。然而，我们观察到 PCR 和无 PCR 文库制备之间的差异会影响低丰度病毒基因组的回收。在这项研究中使用三个粪便样本，PCR 文库制备样本导致 PCR 自由对中明显的低丰度 vOTU 丢失（vOTU 128、6202 和 8364），并降低了 alpha 多样性指数（Chao1 值= 0.045 和 Simpson 值= 0.044）。因此，当研究肠道病毒组中的“稀有”成员时，PCR 和无 PCR 方法之间的差异是需要考虑的重要因素，而当研究中度和高度丰富的病毒时，这些偏差可能可以忽略不计。
