APC Microbiome Institute, University College Cork, Cork, Ireland.
Department of Food Biosciences, Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland.
Microbiome. 2018 Apr 10;6(1):68. doi: 10.1186/s40168-018-0446-z.
Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing.
Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 °C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples.
The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.
最近的研究表明，人类肠道中存在着复杂的、高度个体化和稳定的病毒群落，其中大多数是噬菌体。虽然在 IBD、艾滋病和急性营养不良中观察到肠道噬菌体组的疾病特异性改变，但人类肠道噬菌体组仍未得到充分描述。在人类肠道噬菌体组的宏基因组研究中，一个重要的障碍是不同研究小组获得的结果之间存在高度差异。这通常是由于用于富集病毒样颗粒、核酸纯化和测序的不同方案造成的。本研究的目的是开发一种相对简单、可重复且具有成本效益的方法，从人类粪便样本中提取病毒核酸，适用于高通量研究。我们还分析了某些潜在混杂因素（如储存条件、反复冻融循环和操作人员偏差）对所得噬菌体组谱的影响。此外，还通过向粪便样本中添加外源性噬菌体标准来定量分析宏基因组测序后的噬菌体组。在 16S rRNA 扩增子测序后，还对噬菌体组谱与细菌组谱进行了比较分析。
与细菌组谱相比，粪便噬菌体组谱总体上具有更高的个体特异性。当储存在+4°C 或室温下时，噬菌体组和细菌组都表现出中度变化。噬菌体组谱受多次冻融循环的影响小于细菌组谱，但在噬菌体组处理过程中，操作人员的影响更大。成功地向粪便样本中添加外源性噬菌体表明，个体样本之间的总病毒载量存在很大差异。
本研究中开发的粪便噬菌体组测序方案为人类肠道微生物群提供了有价值的补充视角，与 16S 扩增子测序和/或粪便总 DNA 的宏基因组测序互补。该方案针对处理粪便样本时遇到的几种混杂因素进行了优化，以减少研究人类肠道噬菌体组的不同研究小组之间观察到的差异。建议快速储存、限制冻融循环和向粪便样本中添加外源性噬菌体标准，以获得最佳结果。
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