Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 8;18(1):171.
doi: 10.1186/s12876-018-0896-6.

Evaluating gut microbiota profiles from archived fecal samples

Trine B Rounge  1 , Roger Meisal  2 , Jan Inge Nordby  3 , Ole Herman Ambur  2   4 , Thomas de Lange  5   6 , Geir Hoff  5   7
Affiliations

Evaluating gut microbiota profiles from archived fecal samples

Trine B Rounge et al. BMC Gastroenterol. .

Abstract

Background: Associations between colorectal cancer and microbiota have been identified. Archived fecal samples might be valuable sample sources for investigating causality in carcinogenesis and biomarkers discovery due to the potential of performing longitudinal studies. However, the quality, quantity and stability of the gut microbiota in these fecal samples must be assessed prior to such studies. We evaluated i) cross-contamination during analysis for fecal blood and ii) evaporation in stored perforated fecal immunochemical tests (iFOBT) samples, iii) temperature stability as well as iv) comparison of the gut microbiota diversity and composition in archived, iFOBT and fresh fecal samples in order to assess feasibility of large scale microbiota studies.

Methods: The microbiota profiles were obtained by sequencing the V3-V4 region of 16S rDNA gene.

Results: The iFOBT does not introduce any cross-sample contamination detectable by qPCR. Neither could we detect evaporation during freeze-thaw cycle of perforated iFOBT samples. Our results confirm room temperature stability of the gut microbiome. Diverse microbial profiles were achieved in 100% of fresh, 81% of long-term archived and 96% of iFOBT samples. Microbial diversity and composition were comparable between fresh and iFOBT samples, however, diversity differed significantly between long-term archived, fresh and iFOBT samples.

Conclusion: Our data showed that it is feasible to exploit archived fecal sample sets originally collected for testing of fecal blood. The advantages of using these sample sets for microbial biomarker discovery and longitudinal observational studies are the availability of high-quality diagnostic and follow-up data. However, care must be taken when microbiota are profiled in long-term archived fecal samples.

Keywords: Archived samples; Diversity; Fecal immunochemical tests; Fecal samples; Microbiota; Storage.

PubMed Disclaimer

Conflict of interest statement

Ethics approval and consent to participate

The collection of iFOBT and archived samples was approved by the Regional Committees for Medical and Health Research Ethics in South-Eastern Norway (2011/1272 and 2010/3087 A, respectively). An additional ethical approval for the feasibility study was not required since all samples were anonymized and the purpose of the study was method related, as stated by the Norwegian regional ethics committee (Regional Committees for Medical and Health Research Ethics in South-Eastern Norway, ref. 2015/9).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests, with the exception of GH received payment from Amgen Norway for giving a lecture at a medical conference in 2017.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Alpha and beta diversity in iFOBT, fresh and archived samples. a Shows a boxplot of the number of observed OTUs in each sample group. b and c Shows boxplots of the Inverse Simpson index (b) and Shannon (c) index in fecal immunochemical tests (iFOBT samples, fresh fecal samples and fecal samples archived for approximately 16 years. The indexes are based on rarefied OTU data to minimize the impacts of uneven sampling. The Bray-Curtis dissimilarity index for comparisons of groups are shown (d)
Fig. 2
Fig. 2
Principal component plot of the community composition of iFOBT, archived and fresh sample. The first and second principal components of the community composition of iFOBT samples from a screening trial in Norway (BCSN), archived samples from the NORCCAP cohort stored for about 16 years and fresh samples
Fig. 3
Fig. 3
Clustering of archived, fresh and fecal immunochemical tests (iFOBT) fecal samples. Heatmap of the log transformed OTU table for all samples produced by hierarchical clustering and Euclidean distance. Only OTUs with a log sum > 20 were illustrated. iFOBT samples are marked in green, archived fecal samples stored for 14 to 16 years at − 30 °C are marked in blue and fresh samples are marked in pink. All samples are from presumably healthy individuals
Fig. 4
Fig. 4
Microbiota profiles in fresh samples frozen directly and frozen after 48 h in room temperature. a Boxplot of Inverse Simpson alpha-diversity index in samples frozen directly and frozen after 48 h. b Bray-Curtis dissimilarities between samples from the same individuals with different storage conditions, and between different individuals regardless of room temperature storage. c Heatmap of the log transformed OTU table from paired fecal samples from 8 presumably healthy individuals (serial number 01 to 10). One part of the samples was directly frozen (0 h, marked with green text) and the other part of the samples was frozen after 48 h in room temperature (48 h, marked with blue text). Hierarchical clustering and Euclidean distance produced the clustering showing higher inter-person variability than intra-person variability. Only OTUs with a log sum > 2 were illustrated
See this image and copyright information in PMC

References

    1. Schreuders Eline H, Ruco Arlinda, Rabeneck Linda, Schoen Robert E, Sung Joseph J Y, Young Graeme P, Kuipers Ernst J. Colorectal cancer screening: a global overview of existing programmes. Gut. 2015;64(10):1637–1649. doi: 10.1136/gutjnl-2014-309086. - DOI - PubMed
    1. Colorectal cancer estimated incidence, mortality and prevalence worldwide in 2012. [http://globocan.iarc.fr/old/FactSheets/cancers/colorectal-new.asp].
    1. Imperiale TF, Ransohoff DF, Itzkowitz SH, Levin TR, Lavin P, Lidgard GP, Ahlquist DA, Berger BM. Multitarget stool DNA testing for colorectal-cancer screening. N Engl J Med. 2014;370(14):1287–1297. doi: 10.1056/NEJMoa1311194. - DOI - PubMed
    1. Bultman SJ. Emerging roles of the microbiome in cancer. Carcinogenesis. 2014;35(2):249–255. doi: 10.1093/carcin/bgt392. - DOI - PMC - PubMed
    1. Garrett WS. Cancer and the microbiota. Science (New York, NY) 2015;348(6230):80–86. doi: 10.1126/science.aaa4972. - DOI - PMC - PubMed

LinkOut - more resources