Molecular Analysis of the Microbiome in Colorectal Cancer

  • Fiona Clegg
  • Susan H. Berry
  • Richard Hansen
  • Georgina L. Hold
Part of the Methods in Molecular Biology book series (MIMB, volume 1765)


The human gut microbiota plays a major role in the development of colorectal cancer (CRC). Many studies have attempted to define links between microbiota residents, their function and disease development. We now have incredible molecular tools to allow us to study the gut microbiome however in order to make best use of these sophisticated approaches we need to ensure that samples are collected and processed using standardized and reproducible protocols. Here we provide an overview of molecular analysis methods and describe protocols for collecting and processing clinical samples for subsequent microbiome analysis.

Key words

Colorectal cancer Microbiome Microbial diversity Clinical samples Stool Biopsy DNA RNA Bacteria Fungi Molecular microbiology 



This work was supported by grants from The Chief Scientist Office, Friends of Anchor, NHS Grampian Endowments, NHS Grampian Gastroenterology Endowment funds as well as donations from CRC patients.


  1. 1.
    Weisburger JH, Reddy BS, Narisawa T et al (1975) Germ-free status and colon tumor induction by N-methyl-N′-nitro-N-nitrosoguanidine. Proc Soc Exp Biol Med 148(4):1119–1121CrossRefPubMedGoogle Scholar
  2. 2.
    Louis P, Hold GL, Flint HJ (2014) The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev Microbiol 12(10):661–672CrossRefPubMedGoogle Scholar
  3. 3.
    Hold GL, Garrett WS (2015) Gut microbiota: microbiota organisation a key to understanding CRC development. Nat Rev Gastroenterol Hepatol 12(3):128–129CrossRefPubMedGoogle Scholar
  4. 4.
    Wang T, Cai G, Qiu Y et al (2012) Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers. ISME J 6(2):320–329CrossRefPubMedGoogle Scholar
  5. 5.
    Boleij A, van Gelder MM, Swinkels DW et al (2011) Clinical Importance of Streptococcus gallolyticus infection among colorectal cancer patients: systematic review and meta-analysis. Clin Infect Dis 53(9):870–878CrossRefPubMedGoogle Scholar
  6. 6.
    Kostic AD, Chun E, Robertson L et al (2013) Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 14(2):207–215CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Sears CL (2009) Enterotoxigenic Bacteroides fragilis: a rogue among symbiotes. Clin Microbiol Rev 22(2):349–369CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Martin HM, Campbell BJ, Hart CA et al (2004) Enhanced Escherichia coli adherence and invasion in Crohn’s disease and colon cancer. Gastroenterology 127(1):80–93CrossRefPubMedGoogle Scholar
  9. 9.
    Swidsinski A, Khilkin M, Kerjaschki D et al (1998) Association between intraepithelial Escherichia coli and colorectal cancer. Gastroenterology 115(2):281–286CrossRefPubMedGoogle Scholar
  10. 10.
    Arthur JC, Perez-Chanona E, Mühlbauer M et al (2012) Intestinal inflammation targets cancer-inducing activity of the microbiota. Science 338(6103):120–123CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Olsen GJ, Lane DJ, Giovannoni SJ et al (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol 40(1):337–365CrossRefPubMedGoogle Scholar
  12. 12.
    Thomson J, Hansen R, Berry S et al (2011) Enterohepatic helicobacter in ulcerative colitis: potential pathogenic entities? PLoS One 6(2):e17184CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59(3):695–700PubMedPubMedCentralGoogle Scholar
  14. 14.
    Zoetendal EG, Akkermans AD, De Vos WM (1998) Temperature gradient gel electrophoresis analysis of 16S rRNA from human fecal samples reveals stable and host-specific communities of active bacteria. Appl Environ Microbiol 64(10):3854–3859PubMedPubMedCentralGoogle Scholar
  15. 15.
    Weisburg WG, Barns SM, Pelletier DA et al (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Giovannoni SJ, Britschgi TB, Moyer CL et al (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345(6270):60CrossRefPubMedGoogle Scholar
  17. 17.
    Hold GL, Pryde SE, Russell VJ et al (2002) Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. FEMS Microbiol Ecol 39(1):33–39CrossRefPubMedGoogle Scholar
  18. 18.
    Margulies M, Egholm M, Altman WE et al (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437(7057):376–380CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Rajilić-Stojanović M, Heilig HG, Molenaar D et al (2009) Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults. Environ Microbiol 11(7):1736–1751CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Claesson MJ, O’Sullivan O, Wang Q et al (2009) Comparative analysis of pyrosequencing and a phylogenetic microarray for exploring microbial community structures in the human distal intestine. PLoS One 4(8):e6669CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Hansen R, Russell RK, Reiff C et al (2012) Microbiota of de-novo pediatric IBD: increased Faecalibacterium Prausnitzii and reduced bacterial diversity in Crohn's but not in ulcerative colitis. Am J Gastroenterol 107(12):1913–1922CrossRefPubMedGoogle Scholar
  22. 22.
    Watt E, Gemmell MR, Berry S et al (2016) Extending colonic mucosal microbiome analysis—assessment of colonic lavage as a proxy for endoscopic colonic biopsies. Microbiome 4(1):61CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Wheeler DA, Srinivasan M, Egholm M et al (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452(7189):872–876CrossRefPubMedGoogle Scholar
  24. 24.
    Qin J, Li R, Raes J et al (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464(7285):59–65CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Frank JA, Reich CI, Sharma S et al (2008) Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl Environ Microbiol 74(8):2461–2470CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mukhopadhya I, Hansen R, Meharg C et al (2015) The fungal microbiota of de-novo paediatric inflammatory bowel disease. Microbes Infect 17(4):304–310CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Ghannoum MA, Jurevic RJ, Mukherjee PK et al (2010) Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog 6(1):e1000713CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Fiona Clegg
    • 1
  • Susan H. Berry
    • 1
  • Richard Hansen
    • 2
  • Georgina L. Hold
    • 1
  1. 1.Gastrointestinal Research GroupSchool of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of AberdeenAberdeenUK
  2. 2.Department of Paediatric GastroenterologyRoyal Hospital for ChildrenGlasgowUK

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