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

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.