Sample storage conditions significantly influence faecal microbiome profiles

Abstract

Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to −80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants’ homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to −80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to −80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.

Comparison of Fecal Collection Methods for Microbiota Studies in Bangladesh

“…In general, our findings agree with those from many of the previous studies considering the impact of the collection method and short-term storage at room temperature in predominantly Western populations (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). In general, FOBT cards or Whatman FTA cards, which have feces smeared on the card itself that then dries, have been found to be relatively stable at room temperature and similar to immediately frozen samples without preservatives (6,10,15,21,22).…”

“…The impact of the type of fecal sample collection method on microbial data has been considered in a number of previous studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). However, the majority of these studies were conducted in North American or European populations near academic or clinical laboratories and typically included only a small number of methods.…”

Comparison of Collection Methods for Fecal Samples in Microbiome Studies

“…RNAlater has also been tested in a number of studies (10, 20-24, 26, 30, 32), but the results varied. The majority of studies concluded that RNAlater was an acceptable preservative for microbial analyses of fecal samples (10,22,23,26,30), although in some studies, researchers detected decreased alpha diversity (20,21,30), decreased DNA purity or yields (21,24), or lower stability at room temperature for longer periods (33) compared with immediately frozen fecal samples. Storage of fecal samples in 70% ethanol does not appear to be suitable for microbiome analyses (10), but 95% ethanol has been previously observed to adequately preserve fecal samples (23,33).…”

“…Investigators in a number of previous studies have assessed different collection methods for fecal samples and the impact of leaving a sample at room temperature on the fecal microbiome (10,(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). The FOBT card, or a similar Whatman FTA card (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania), has been tested in a few studies (10,21,26,32).…”

“…To our knowledge, this study has the largest sample of participants of all studies comparing fecal collection methods. Previously, the number of participants included ranged from 1 (20) to 28 participants (29). Because there is high interindividual variability in the gut microbiome (36) and variable microbial composition may be differentially affected by collection method, it is important to evaluate fecal collection methods in larger groups of people.…”