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

PLoS One. 2016 Oct 4;11(10):e0163666. doi: 10.1371/journal.pone.0163666. eCollection 2016.

Abstract

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.

MeSH terms

  • Biodiversity
  • DNA, Bacterial
  • Feces / microbiology*
  • Humans
  • Metagenome
  • Metagenomics
  • Microbial Viability*
  • Microbiota*
  • Preservation, Biological*
  • RNA, Ribosomal, 16S
  • Time Factors

Substances

  • DNA, Bacterial
  • RNA, Ribosomal, 16S

Grants and funding

We gratefully acknowledge funding support from the University of Auckland Faculty Research Development Fund grant 9841/3706738 (to MWT and SDP), and the Biomedical and Applied Discretionary Funding round within the School of Biological Sciences. This work was also supported by the Endocore Research Trust.