Fig. S1. Construction of M1-2 strain. Table S1. Nematodes studied. Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. “
“Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly
or following freeze storage of three homogenized human fecal samples using three different extraction methods. No consistent differences were observed in DNA yields between extractions on fresh and frozen samples; however, differences AZD6244 clinical trial were observed between extraction methods. Quantitative PCR analysis was subsequently performed on all DNA samples using six
different primer pairs targeting 16S rRNA genes of significant bacterial groups, and the community composition was evaluated by comparing specific ratios of the calculated abundances. In seven of nine cases, the Firmicutes to Bacteroidetes 16S rRNA gene ratio was significantly higher in fecal samples that had been frozen compared to identical samples that had not. This effect was further supported by qPCR analysis of bacterial groups within these two phyla. The results demonstrate that storage conditions of fecal samples may adversely affect the determined Firmicutes to Bacteroidetes ratio, which is a frequently used biomarker in gut microbiology. Investigating the composition of the human buy BMS-354825 microbiota and correlating findings to specific clinical or physiological states such as irritable bowel diseases and obesity has demonstrated the collective importance of the bacterial community present in the gut as a regulatory factor in health and disease (Young et al., 2011). Because of the large diversity and complexity of interactions between the resident bacteria,
various simplifications have been sought. An example of this is the use of the ratio between the two most clonidine predominant phyla, namely the Firmicutes and the Bacteriodetes, as a biomarker in relation to human physiology (Armougom & Raoult, 2008; Guo et al., 2008; Mariat et al., 2009). Efficient and nonbiased extraction of total genomic bacterial DNA from the complex fecal samples is a crucial first step for many molecular-based studies of the bacterial community within the gut environment. Downstream applications, such as quantitative PCR and metagenomic sequencing, ultimately require unbiased DNA input to produce accurate and creditable research results. Previous studies have assessed the effectiveness of various DNA extraction procedures based on, for example, DNA yield, extent of DNA shearing, and use as template for subsequent PCR and have often been related to detection of specific pathogens (McOrist et al., 2002; Tang et al., 2008; Ariefdjohan et al.