Reliability of a participant-friendly fecal collection method for microbiome analyses: a step towards large sample size investigation.

BACKGROUND: The effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods. RESULTS: Fourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods. CONCLUSION: Our results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.

Prognostic relevance of urinary bladder cancer susceptibility loci.

In the last few years, susceptibility loci have been identified for urinary bladder cancer (UBC) through candidate-gene and genome-wide association studies. Prognostic relevance of most of these loci is yet unknown. In this study, we used data of the Nijmegen Bladder Cancer Study (NBCS) to perform a comprehensive evaluation of the prognostic relevance of all confirmed UBC susceptibility loci. Detailed clinical data concerning diagnosis, stage, treatment, and disease course of a population-based series of 1,602 UBC patients were collected retrospectively based on a medical file survey. Kaplan-Meier survival analyses and Cox proportional hazard regression were performed, and log-rank tests calculated, to evaluate the association between 12 confirmed UBC susceptibility variants and recurrence and progression in non-muscle invasive bladder cancer (NMIBC) patients. Among muscle-invasive or metastatic bladder cancer (MIBC) patients, association of these variants with overall survival was tested. Subgroup analyses by tumor aggressiveness and smoking status were performed in NMIBC patients. In the overall NMIBC group (n = 1,269), a statistically significant association between rs9642880 at 8q24 and risk of progression was observed (GT vs. TT: HR = 1.08 (95% CI: 0.76-1.54), GG vs. TT: HR = 1.81 (95% CI: 1.23-2.66), P for trend = 2.6 × 10(-3)). In subgroup analyses, several other variants showed suggestive, though non-significant, prognostic relevance for recurrence and progression in NMIBC and survival in MIBC. This study provides suggestive evidence that genetic loci involved in UBC etiology may influence disease prognosis. Elucidation of the causal variant(s) could further our understanding of the mechanism of disease, could point to new therapeutic targets, and might aid in improvement of prognostic tools.