Integrity of the Human Faecal Microbiota following Long-Term Sample Storage.

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PloS one, ISSN: 1932-6203, Vol: 11, Issue: 10, Page: e0163666

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10.1371/journal.pone.0163666; 10.1371/journal.pone.0163666.g003; 10.1371/journal.pone.0163666.g002; 10.1371/journal.pone.0163666.g001
Elahe Kia; Brett Wagner Mackenzie; Danielle Middleton; Anna Lau; David W. Waite; Gillian Lewis; Yih-Kai Chan; Marta Silvestre; Garth J. S. Cooper; Sally D. Poppitt; Michael W. Taylor; Daniel Paredes-Sabja Show More Hide
Public Library of Science (PLoS); Public Library of Science; Figshare
Biochemistry, Genetics and Molecular Biology; Agricultural and Biological Sciences; Microbiology; Cell Biology; Genetics; Ecology; Cancer; Science Policy; 110309 Infectious Diseases; 59999 Environmental Sciences not elsewhere classified; 69999 Biological Sciences not elsewhere classified; 39999 Chemical Sciences not elsewhere classified; AGP; faecal sample collection; 16 S rRNA gene amplicons; extract; type 2 diabetes; Long-Term Sample Storage; T 2D faecal samples; DNA; American Gut Project; T 2D individuals; Human Faecal Microbiota; support amplicon-based studies; faecal samples; Biochemistry
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In studies of the human microbiome, faecal samples are frequently used as a non-invasive proxy for the study of the intestinal microbiota. To obtain reliable insights, the need for bacterial DNA of high quality and integrity following appropriate faecal sample collection and preservation steps is paramount. 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. These samples were freeze-dried, then stored mostly at -20°C from the trial date in 2000/2001 until the current research in 2014. Given the relative antiquity of these samples (~14 years), we sought to evaluate DNA quality and comparability to freshly collected human faecal samples. Following the extraction of bacterial DNA, gel electrophoresis indicated that our DNA extracts were more sheared than extracts made from freshly collected faecal samples, but still of sufficiently high molecular weight to support amplicon-based studies. Likewise, spectrophotometric assessment of extracts revealed that they were of high quality and quantity. A subset of bacterial 16S rRNA gene amplicons were sequenced using Illumina MiSeq and compared against publicly available sequence data representing a similar cohort analysed by the American Gut Project (AGP). Notably, our bacterial community 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.