Effect of storage and DNA extraction method on 16S rRNA-profiled fecal microbiota in Japanese adults

Abstract

The effect of two factors, storage and the bacterial DNA extraction method, that potentially affect the 16S rRNA-based profiling of the microbiota in the feces of Japanese adults, were evaluated. Profiles of the microbiota in feces stored in DESS (DMSO-EDTA-salt solution) for 1, 2 and 3 weeks at room temperature, and for 3 weeks at 4°C were compared with those in fresh feces and feces stored in guanidine thiocyanate solution for 3 weeks at 4°C. None of the storage variables (preservation solution, temperature and duration) considerably affected α- and β-diversity of the fecal microbiota and OTU profiles. Regarding the bacterial DNA extraction methods, four were evaluated; A) silica membrane DNA purification combined with bead-beating bacterial disruption, B) magnetic bead DNA purification combined with bead-beating bacterial disruption, C) manual DNA purification using phenol-chloroform and ethanol precipitation combined with enzymatic bacterial lysis, and D) DNA extraction by a commercially available DNA stool kit. While methods A, B, and C did not markedly affect α- and β-diversity of the fecal microbiota and the OTU profiles, method D noticeably altered both α- and β-diversity. In addition, method D caused significant changes in the abundance of two predominant genera; Bacteroides and Bifidobacterium.