A number of studies have looked at sample compositional stability associated with different conditions of storage. Carroll et al. [2] have provided the current benchmark for long-term ?80 °C storage, showing no significant changes in the faecal microbial community after 6 months of storage, with Bai et al. [3] reporting similar findings for the vaginal microbiota after a shorter 4 weeks of storage. For some studies, however, samples may need to be accrued over far longer periods, and the impact of longer term storage has not until now been systematically appraised. Here, we report that 2 years at ?80 °C leads to few significant changes in the microbial community of infant faecal samples, with a small reduction in the observed number OTUs and shifts in the abundance of some specific OTUs, although these were often not shared amongst similar lineages. The increases in abundance of the Lactobacillus and bacilli OTUs are a notable exception to this trend, and it may be important to consider the effects of long-term storage of samples where studies focus on these organisms. We also note that the results presented may not be representative of more complex mixtures and that the effects of long-term storage have only been described for the specific organisms featuring in our dataset.

Carroll et al. [2] and Cardona et al. [4] have investigated sample stability at room temperature. Both groups demonstrated compositional stability of faecal samples over a 24-h period. A lack of significant change in the relative proportions of different bacterial phyla has been reported over a 3-day period of storage at room temperature by Dominianni et al. [7]. Voigt et al. [1] and Flores et al. [6] have demonstrated stability out to 7 days when samples are preserved in the proprietary Stabilization Solution “RNAlater”. These findings have appeared to represent the limit. Cardona et al. [4] have shown that significant changes in the community are seen in samples analysed after 2 weeks at room temperature. Here, we have investigated the compositional stability of infant faecal samples held at room temperature for periods out to 2 weeks and confirm the findings of Carrol et al., Cardona et al. and Dominianni et al. [2, 4, 7] that by 1 week, significant skewing of the observed population has occurred. In our more complex community samples, this primarily affects the abundance of some elements of the bacterial community, with our analysis indicating significant shifts in the weighted measures of beta diversity by 48 h. The most extreme shifts appeared to be correlated with increasing relative abundance of Enterobacteriaceae (specifically an Enterobacter OTU). The less complex community samples were more strongly affected by loss and gain of low abundance OTUs, leading to significant increases in unweighted measures. Considerable differences in both bacterial OTUs and phyla were evident by 1 and 2 weeks of room temperature storage, and although no particular trends unified which groups are likely to be affected, increased proportional abundance of Bifidobacterium and decreased Veillonella were seen in both datasets.

We conclude accordingly that for preservation of bacterial community structure, faecal samples should be frozen within 2 days of collection. In the UK, this happily allows time for samples to be delivered to the laboratory by mail without loss of quality, provided that they are dispatched on the day of collection by first class mail and care is taken to avoid weekends. In settings where the mail service is unreliable, alternative means of sample delivery should be considered to ensure that quality is maintained.

By carrying out replicate DNA extractions (different researchers, different lots of reagents), we have concluded that this part of the overall protocol contributes negligibly to variation.

Reduction in faecal sample weight, even down to only an eighth of the generally applied ‘standard’ of 200 mg, also scarcely affected alpha diversity and the majority of beta diversity measures, noting that this related to our study of infant samples of inherently low diversity. The strongest effects were seen when considering weighted beta diversity measures, with OTU abundances suffering increased variation as lower amounts of faeces are sampled. However, despite variation being higher than expected, the actual magnitude of these changes appears to be relatively small, with samples still retaining close similarity. We consider that it would be prudent to establish on individual study basis a minimum sample size for DNA extraction to yield consistent results.