Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling
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DOI：
10.1128/msystems.00271-18
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发表时间：
2019-01-01
期刊：
影响因子：
6.4
通讯作者：
Chan, Paul K. S.
中科院分区：
文献类型：
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作者：
Chen, Zigui;Hui, Pak Chun;Chan, Paul K. S.
适当保存粪便样本，以最大程度地减少微生物群落变化并灭活传染原，对于在冷链运输不可行时需要运送到实验室的自采集标本非常重要。在本研究中，我们评估了六种保存解决方案（Norgen、OMNI、RNAlater、CURNA、HEMA 和 Shield）在这些方面的性能。将含有这些防腐剂的人类粪便样本在室温下保存 7 天后，对细菌 16S rRNA 基因的三个高变区（V1-V2、V3-V4 和 V4）进行了扩增子测序。我们发现，与其他储存条件相比，在 Norgen 和 OMNI 两种防腐剂中收集的样品相对于 -80 摄氏度标准的群落组成变化最小，并且都有效抑制需氧和厌氧细菌的生长。 RNAlater 并没有阻止细菌活动，并且表现出相对较大的群落变化。尽管与受试者间变异相比，保存溶液的影响很小，但观察到微生物群组成的显着变化，这可能会在下游数据分析中产生偏差。当比较从不同 16S rRNA 基因高变区推断的群落概况时，我们发现引物组在识别整体微生物群落和某些细菌类群方面的敏感性不同。例如，V4 引物对生成的读数显示肠道微生物群落具有更高的 α 多样性。简并 27f-YM 引物未能检测到大多数双歧杆菌。我们的数据表明，保存溶液和 16S rRNA 基因引物对的选择是影响肠道微生物群分析的关键决定因素。 重要性 大规模人类微生物群研究需要从多个地点和/或时间点收集样本，以最大限度地检测微生物与宿主相互作用中的微小影响。然而，由实验方案（例如样本收集、大规模并行测序和生物信息学分析）引起的批次偏差仍然很重要，应尽量减少。这项工作评估了保存液和细菌 16S rRNA 基因引物对在揭示人类肠道微生物群组成方面的效果。由于在选择防腐剂和引物对时观察到检测细菌组成和丰度的显着变化，因此一致的方法对于最大限度地减少其影响以促进数据集之间的比较至关重要。
Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to -80 degrees C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling.IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.