Home-Made Cost Effective Preservation Buffer Is a Better Alternative to Commercial Preservation Methods for Microbiome Research

Abstract

The investigation of wildlife gastrointestinal microbiomes by next-generation sequencing approaches is a growing field in microbial ecology and conservation. Such studies often face difficulties in sample preservation if neither freezing facilities nor liquid nitrogen (LQN) are readily available. Thus, in order to prevent microbial community changes because of bacterial growth after sampling, preservation buffers need to be applied to samples. However, the amount of microbial community variation attributable to the different preservation treatments and potentially affecting biological interpretation is hardly known. Here, we sampled feces of 11 sheep (Ovis aries sp.) by using swabs and analyzed the effect of air-drying, an inexpensive self-made nucleic acid preservation buffer (NAP), DNA/RNA Shield™, and RNAlater®, each together with freezing (for 10 days) or storing at room temperature (for 10 days) prior to 16S rRNA gene high-throughput sequencing to determine bacterial communities. Results revealed that the proportions of operational taxonomic units (OTUs) belonging to a bacterial phylum were affected by the preservation treatments, and that alpha diversities [observed OTUs, Shannon index, and phylogenetic diversity (PD)] were lower in all preservation treatments than in samples taken by forensic swabs and immediately frozen which is considered as the favored preservation treatment in the absence of any logistic constraints. Overall, NAP had better preservation qualities than RNAlater® and DNA/RNA Shield™ making this self-made buffer a valuable solution in wildlife microbiome studies.

Methodological Insight Into Mosquito Microbiome Studies

“…We thus encourage future attempts on the development of efficient laboratory-made buffers that would overcome this issue and facilitate their massive usage, being an easy to prepare and more economic alternative to commercial buffers. Almost all preservation treatments produced a slight increase in the OTU richness found in the samples compared to the control (i.e., specimens freshly extracted upon collection), with the exception of All Protect combined with freezing at −20 • C. These results are contrary to those obtained by Menke et al (2017), yet the differences found in our study are not significant. In addition, the control treatment they used involved sample freezing.…”

“…The finding of absolute ethanol as an efficient RNA preservative may seem surprising, but it has previously been shown for insect larvae and nymphs (Astrid et al, 2016). In general, our results agree with previous studies where NAP buffer was found as an efficient nucleic acid preservative for various samples (e.g., rat tissues: Camacho-Sanchez et al, 2018;and frog tissues Montero-Mendieta et al, 2017), including those for microbiome studies (e.g., fecal samples: Menke et al, 2017). Nevertheless, we faced difficulties fully submerging the mosquito specimens in NAP buffer.…”

“…The storage temperature was more important in determining the significant differences found than the preservation buffer used. Menke et al (2017) found a similar effect of their different preservation treatments, with a strong effect of storage temperature irrespective of the preservation buffer used. Nevertheless, none of the preservation buffers combined with storage at 4 • C or freezing at −20 • C significantly differed from the freshly extracted controls in our beta-diversity analyses.…”

Peer Review #3 of "Impact of inter- and intra-individual variation, sample storage and sampling fraction on human stool microbial community profiles (v0.1)"

“…Biological samples are usually chilled or mixed with preservative media to suppress opportunistic microorganisms from flourishing and dominating community profiles (Vandeputte et al, 2017), although this problem may be somewhat alleviated by setting a maximum allowed transit duration. A few studies have compared the use of preservation buffers in maintaining microbial community profiles and generally recommend its use especially when samples cannot be immediately frozen due to logistical constraints (Menke et al, 2017;Flores et al, 2015;Voight et al, 2015). Our results, however, show that preservation media may not be necessary for human stool samples for short transit durations of up to 24 hours as the use of a medium was associated with larger shifts in microbial community composition compared to samples stored at ambient and lowered temperatures (Fig.…”

High Throughput Sequencing for Detection of Foodborne Pathogens

“…The extraction and purification of DNA from the samples is of major importance as the presence of inhibitors may hamper the analysis further down the line (Ceuppens et al, 2014 ; Moore et al, 2015 ). The sample storage conditions and preparation steps can introduce taxonomic biases linked to recovery (Ceuppens et al, 2015 ; Menke et al, 2017 ). Similar or even stronger effects on the microbiome and RNA population of samples are predictable, as also the intraspecific diversity and gene expression can be affected.…”