粪便样本不同保存方式对肠道菌群测序结果的影响

Effects of different preservation methods for fecal samples on the sequencing results of gut microbiota

摘要:

【目的】本文探究了3种室温保存剂和–80℃冷冻保存对粪便样本中菌群结构的影响，为大规模、标准化的采样提供参考。【方法】本研究采集了5名健康志愿者的新鲜粪便作为测试样本，采用4种不同的保存方式保存：DETs室温保存、GITC室温保存、RNAlater室温保存和–80℃冷冻保存，在保存0、1、3、7、14、28 d后，采用高通量测序技术测定样本中微生物16S rRNA基因V3-V4区序列，通过比较不同保存剂、不同保存时间后的样本与新鲜样本的差异，分析保存剂和保存时间对粪便样本菌群组成及丰度的影响。【结果】5组样本共得到489个操作分类单元（operational taxonomic units，OTUs），其中488个OTUs为5组所共有，且样本alpha多样性无统计学差异。从属水平上看，与fresh组（新鲜样本）相比，DETs组的拟杆菌属相对丰度显著升高，且部分OTUs在保存超过3 d后发生明显变化；其余保存方式无显著差异。综合分析微生物群落多样性和丰度，结果表明，在保存不同天数后，4种保存方式的样本与fresh组的菌群结构均无显著差异，其中-80℃保存的样本与fresh组样本菌群结构相似程度最高，但不同志愿者与各自新鲜样本的相似程度变化较大，且整体相似程度随着时间的推移与fresh组差异呈下降趋势；而使用DTEs保存剂和GITC保存剂保存的样本，志愿者个体间差异小，保存效果稳定，且随时间变化小。聚类分析结果表明，无论使用哪种保存方式，保存方式和时间带来的差异均小于志愿者个体间的差异。【结论】在满足低温冷冻保存条件时优先选择–80℃冷冻保存粪便样本，无法满足立即冷冻保存的条件时，可以选择添加保存剂室温保存，且GITC保存剂优于其他保存剂。

Abstract:

[Objective] In this study, we compared three room-temperature preservation methods with −80℃ preservation and investigated the effects of these methods on the sequencing results of microbiota in the fecal samples, thence providing guidelines for large-scale and standardized sampling. [Methods] Fresh fecal samples from five healthy volunteers were collected and subjected to four different preservation methods:room-temperature preservation methods (DETs, GITC, and RNAlater) and storage at −80℃ without stabilizers. High-throughput sequencing was deployed to sequence the V3−V4 region of the 16S rRNA genes in the samples being stored for 0, 1, 3, 7, 14 and 28 days. The effects of preservation stabilizers and time on the microbiota composition and species diversity were determined by comparison between different treatments.[Results] A total of 489 operational taxonomic units (OTUs) were obtained from the five groups of samples (fresh samples, three room-temperature samples, and −80℃ preserved samples), among which 488 OTUs were shared by these five groups. No significant difference was observed in the alpha diversity of microbiota amory preservation methods. The relative abundance of Bacteroides in the samples preserved with DETs was higher than that in fresh samples, and some other OTUs in the samples preserved with DETs changed considerably after 3-day storage. No significant difference in the relative abundance of microbial genera was observed amory other groups apart from the DETs group. The microbiota diversity and abundance showed no significant difference between the samples preserved with the four methods and the fresh samples after the storage for different days. The samples stored at −80℃ had the highest similarity to the fresh samples. However, the similarity of the samples from each volunteer varied, and such variation tended to increase with the prolonging of storage time. Nevertheless, DTEs and GITC methods exhibited outstanding stability, especially after prolonged storage time. Cluster analysis revealed that the variation associated with preservation methods and time was neglectable. [Conclusion] Subject to the availability, storage at −80℃ is considered the gold standard for fecal samples, and storage at room temperature with the addition of GITC shows comparable performance to low temperature preservation.