An alternative storage method for characterization of the intestinal microbiota through next generation sequencing

Abstract

Gut microbiota has been the subject of various molecular studies mainly due to its importance and wide-ranging relationships with human hosts. However, the storage of fecal samples prior to DNA extraction is critical when characterizing the composition of intestinal microbiota. Therefore, we aimed to understand the effects of different fecal storage methods to characterize intestinal microbiota using Next Generation Sequencing (NGS) as well as to establish an alternative conservation method of bacterial genetic material in these samples using guanidine. Stool samples from 10 healthy volunteers were collected. Each sample was divided into five aliquots: one aliquot was extracted immediately after collection (fresh) and two aliquots were subjected to freezing at -20 °C or -80 °C and extracted after 48 h. The other two aliquots were stored in guanidine at room temperature or 4 °C and extracted after 48 h. The V4 hypervariable regions of the bacterial and archeal 16S rRNA gene were amplified by PCR and sequenced using an Ion Torrent PGM platform for NGS. The data were analyzed using QIIME software. Statistical significance was determined using a non-parametric Kruskal-Wallis test. A total of 11,494,688 reads with acceptable quality were obtained. Unweighted principal coordinate analysis (PCoA) revealed that the samples were clustered based on the host rather than by the storage group. At the phylum and genus levels, we observed statistically significant differences between two genera, Proteobacteria (p=0.013) and Suterella (p=0.004), comparing frozen samples with guanidine-stored samples. Our data suggest that the use of guanidine can preserve bacterial genetic materials as well as freezing, providing additional conveniences.

Kestose supplementation exerts bifidogenic effect within fecal microbiota and increases fecal butyrate concentration in dogs

“…Increased production of SCFA within large intestine is known to have various beneficial effect to the host [1,9,15,21,47,63]. The supplementation of prebiotic oligosaccharide has been previously shown to increase the fecal SCFA concentration in dogs [40,44]. Supplementation with Kestose in particular, has been shown to result in a ten-fold increase of the butyrate concertation in rat cecal content [59], and similar effect was suggested to have taken place in dogs in this study.…”

“…Sample storage conditions, in general, may have an impact on results of microbiota analysis, however, with regard to the differences between −20°C and −80°C, immediate freezing of fecal samples in either temperature for a short period of time resulted in similar relative abundances of bacterial taxon and alpha-diversity estimations [32,58]. It is also previously reported that immediate freezing at −20°C or below has been considered the gold standard for microbiome preservation [44]. As for the current result, the microbiota in week 12, which is after four-week washing period, showed a pattern returning or approaching to that of week 0, different from weeks 4 and 8 which was under the influence of Kestose ingestion.…”

Low-cost sample preservation methods for high-throughput processing of rumen microbiomes

“…This suggests the solutions, in addition to feed type, time off feed and the rumen fill, had varying impacts on DNA preserving and extraction and consequently on its yield. Studies have suggested that 6 M GuHCl/EDTA solution is able to preserve bacterial genetic materials as well as freezing, providing additional conveniences [35]. Methodologically, TNx2 and GHx2 were more comparable between each other, and handled similarly compared to EtOH ( Table 1 Table 2).…”

Comparing Published Gut Microbiome Taxonomic Data Across Multinational Studies

“…Of the 36 studies that passed the first round of review, five reported their unfiltered, complete taxonomy tables as supplementary materials and therefore were directly downloaded for this work (Bhute et al, 2016; Chávez-Carbajal et al, 2019; Hevia et al, 2016; Hoarau et al, 2016; Ribeiro et al, 2018). For the remaining 31 studies, e-mails were sent to the corresponding or first authors requesting the raw operational taxonomic unit (OTU) tables for their healthy control samples.…”