Genomic GC-content affects the accuracy of 16S rRNA gene sequencing bsed microbial profiling due to PCR bias

Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5–73.7%) for all 20 included strains in the mock community across five separate sequencing runs, with underrepresented strains generally showing the largest degree of variation. In terms of accuracy, mock community species belonging to Proteobacteria were underestimated, whereas those belonging to Firmicutes were mostly overestimated. This could be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification from 30 to 120 s resulted in an increased average relative abundance of the three mock community members with the highest genomic GC%, but did not significantly change the overall evenness of the community distribution. Therefore, efforts should be made to optimize the PCR conditions prior to sequencing in order to maximize accuracy

Microbiota composition of simultaneously colonized mice housed under either a gnotobiotic isolator or individually ventilated cage regime

Germ-free rodents colonized with microbiotas of interest are used for host-microbiota investigations and for testing microbiota-targeted therapeutic candidates. Traditionally, isolators are used for housing such gnotobiotic rodents due to optimal protection from the environment, but research groups focused on the microbiome are increasingly combining or substituting isolator housing with individually ventilated cage (IVC) systems. We compared the effect of housing systems on the gut microbiota composition of germ-free mice colonized with a complex microbiota and housed in either multiple IVC cages in an IVC facility or in multiple open-top cages in an isolator during three generations and five months. No increase in bacterial diversity as assessed by 16S rRNA gene sequencing was observed in the IVC cages, despite not applying completely aseptic cage changes. The donor bacterial community was equally represented in both housing systems. Time-dependent clustering between generations was observed in both systems, but was strongest in the IVC cages. Different relative abundance of a Rikenellaceae genus contributed to separate clustering of the isolator and IVC communities. Our data suggest that complex microbiotas are protected in IVC systems, but challenges related to temporal dynamics should be addressed

Dietary xylo-oligosaccharide stimulates intestinal bifidobacteria and lactobacilli but has limited effect on intestinal integrity in rats.

BACKGROUND: Consumption of prebiotics may modulate gut microbiota, subsequently affecting the bacterial composition, metabolite profile, and human health. Previous studies indicate that also changes in intestinal integrity may occur. In order to explore this further we have investigated the effect of the putative prebiotic xylo-oligosaccharides (XOS) on the gut microbiota and intestinal integrity in male Wistar rats. As changes in intestinal integrity may be related to the expected bifidogenic effect of XOS, we additionally addressed effects of supplementation with a commensal Bifidobacterium pseudolongum (BIF) isolated from the same breed of laboratory rats. RESULTS: Changes in faecal and caecal bacterial composition determined by 16S rRNA gene sequencing and quantitative PCR for selected bacterial groups revealed that the overall bacterial composition did not differ markedly between the control (CON), XOS, and BIF groups, when correcting for multiple comparisons. However as hypothesised, the relative abundance of Bifidobacterium spp. was increased in XOS-fed rats as compared to CON in faecal samples after the intervention. Also Lactobacillus spp. was increased in both the XOS and BIF groups in caecum content compared to CON. Intestinal permeability determined in vivo by FITC-dextran permeability and in vitro using extracted caecum water in trans-epithelial resistance (TER) assay showed no effect on intestinal integrity in either the XOS or the BIF groups. However, the expression of occludin, which is part of the tight junction complex, was increased in the XOS group compared to the CON group. CONCLUSIONS: Supplementation with XOS or a commensal Bifidobacterium pseudolongum had very limited effects on intestinal integrity in rats as only significant change in expression of a single tight junction protein gene was found for the XOS group