human gut metagenome Targeted loci environmental

The advent of next-generation sequencing has greatly supported investigations into the role of the gut microbiota in human health. Specifically, faecal samples are used as the non-invasive proxy for the intestinal microbiota. However, the need for bacterial DNA of high quality and integrity following appropriate faecal sample collection and preservation steps is crucial. In a study of dietary mineral balance in the context of type 2 diabetes (T2D), faecal samples were collected from healthy and T2D individuals throughout a 13-day residential trial. Despite being freeze-dried, the samples were collected in accordance with standard microbiological practice and remained at -20°C from the trial date in 2000/2001 until the current research in 2014. A total of 454 faecal samples were analysed for their microbial make up. The present study focuses on the assessment of sample antiquity and comparability to fresh human faecal samples given the relative antiquity of our data (~14 years). Molecular analysis involved the extraction of bacterial DNA using the MoBio PowerSoil® DNA Isolation Kit. Using gel electrophoresis, we observed that our DNA extracts were not prone to excessive shearing; likewise, spectrophotometric assessment of extracts revealed that they were of high quality and quantity. The bacterial 16S rRNA gene from a subset of samples was amplified and sequenced on the Illumina MiSeq platform. The sequencing results were compared with publicly available human faecal sequence data of similar methodology and cohort characteristics analysed by the American Gut Project (AGP). Notably, our microbial profiles were highly consistent with those from the AGP data. Our results suggest that when faecal specimens are stored appropriately, the microbial profiles are preserved and robust to extended storage periods.