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

An alternative storage method for characterization of the intestinal microbiota through next generation sequencing. Rev Inst Med Trop Sao Paulo. 2018 Nov 29;60:e77 Authors: Ribeiro RM, Souza-Basqueira M, Oliveira LC, Salles FC, Pereira NB, Sabino EC 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....
Source: Revista do Instituto de Medicina Tropical de Sao Paulo - Category: Tropical Medicine Authors: Tags: Rev Inst Med Trop Sao Paulo Source Type: research