Microbial Ecology

, Volume 61, Issue 2, pp 353–362 | Cite as

The Effects from DNA Extraction Methods on the Evaluation of Microbial Diversity Associated with Human Colonic Tissue

  • Páraic Ó Cuív
  • Daniel Aguirre de Cárcer
  • Michelle Jones
  • Eline S. Klaassens
  • Daniel L. Worthley
  • Vicki L. J. Whitehall
  • Seungha Kang
  • Christopher S. McSweeney
  • Barbara A. Leggett
  • Mark Morrison
Environmental Microbiology

Abstract

Potentially valuable sources of DNA have been extracted from human colonic tissues and are retained in biobanks throughout the world, and might be re-examined to better understand host–microbe interactions in health and disease. However, the published protocols for DNA extraction typically used by gastroenterologists have not been systematically compared in terms of their recovery of the microbial fraction associated with colonic tissue. For this reason, we examined how three different tissue DNA extraction methods (the QIAGEN AllPrep DNA/RNA kit, salting out and high molecular weight (HMW) methods of DNA extraction) employed in past clinical trials, and the repeated bead beating and column (RBB+C) method might impact the recovery of microbial DNA from colonic tissue, using a custom designed phylogenetic microarray for gut bacteria and archaea. All four methods produced very similar profiles of the microbial diversity, but there were some differences in probe signal intensities, with the HMW method producing stronger probe intensities for a subset of the Firmicutes probes including Clostridium and Streptococcus spp. Real-time PCR analysis revealed that the HMW and RBB+C extracted DNA contained significantly more DNA of Firmicutes origin and that the different DNA extraction methods also gave variable results in terms of host DNA recovery. All of the methods tested recovered DNA from the archaeal community although there were some differences in probe signal intensity. Based on these findings, we conclude that while all four methods are efficacious at releasing microbial DNA from biopsy tissue samples, the HMW and RBB+C methods of DNA extraction may release more DNA from some of the Firmicutes bacteria associated with colonic tissue. Thus, DNA archived in biobanks could be suitable for retrospective profiling analyses, provided the caveats with respect to the DNA extraction method(s) used are taken into account.

Notes

Acknowledgements

This research was funded in part by CSIRO’s Preventative Health Flagship Research Program (Colorectal Cancer and Gut Health Theme), CSIRO's Transformational Biology Capability Platform, the OCE Science Leader award (to MM) and the OCE Postdoctoral Fellow program (awarded to EK). We thank Antonio Reverter and Maree O’Sullivan for providing suggestions with the statistical analyses used in this research and also thank Michael Conlon and Sean McWilliam for providing critical reading and suggestions to improve the manuscript. PÓC and DAC contributed equally to this work.

References

  1. 1.
    Anchordoquy TJ, Molina MC (2007) Preservation of DNA. Cell Preserve Tech 5:180–188CrossRefGoogle Scholar
  2. 2.
    Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI (2005) Host-bacterial mutualism in the human intestine. Science 307:1915–1920CrossRefPubMedGoogle Scholar
  3. 3.
    Bas A, Forsberg G, Hammarstrom S, Hammarstrom ML (2004) Utility of the housekeeping genes 18S rRNA, b-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scand J Immunol 59:566–573CrossRefPubMedGoogle Scholar
  4. 4.
    Baty F, Facompre M, Wiegand J, Schwager J, Brutsche MH (2006) Analysis with respect to instrumental variables for the exploration of microarray data structures. BMC Bioinformatics 7:422CrossRefPubMedGoogle Scholar
  5. 5.
    Benson AK, Kelly SA, Legge R, Ma F, Low SJ, Kim J, Zhang M, Oh PL, Nehrenberg D, Hua K, Kachman SD, Moriyama EN, Walter J, Peterson DA, Pomp D (2010) Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.1007028107 Google Scholar
  6. 6.
    Bibiloni R, Mangold M, Madsen KL, Fedorak RN, Tannock GW (2006) The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn’s disease and ulcerative colitis patients. J Med Microbiol 55:1141–1149CrossRefPubMedGoogle Scholar
  7. 7.
    Campieri M, Gionchetti P (2001) Bacteria as the cause of ulcerative colitis. Gut 48:132–135CrossRefPubMedGoogle Scholar
  8. 8.
    Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Meth 7:335–336CrossRefGoogle Scholar
  9. 9.
    Chalmers NI, Palmer RJ Jr, Cisar JO, Kolenbrander PE (2008) Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque. J Bacteriol 190:8145–8154CrossRefPubMedGoogle Scholar
  10. 10.
    Culhane AC, Perriere G, Considine EC, Cotter TG, Higgins DG (2002) Between-group analysis of microarray data. Bioinformatics 18:1600–1608CrossRefPubMedGoogle Scholar
  11. 11.
    Denman SE, McSweeney CS (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol 58:572–582CrossRefPubMedGoogle Scholar
  12. 12.
    Dray S, Dufour A-B (2007) The ade4 package: implementing the duality diagram for ecologists. J Stat Soft 22:1–20Google Scholar
  13. 13.
    Dridi B, Henry M, El Khéchine A, Raoult D, Drancourt M (2009) High prevalence of Methanobrevibacter smithii and Methanosphaera stadtmanae detected in the human gut using an improved DNA detection protocol. PLoS ONE 4:e7063CrossRefPubMedGoogle Scholar
  14. 14.
    Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA (2005) Diversity of the human intestinal microbial flora. Science 308:1635–1638CrossRefPubMedGoogle Scholar
  15. 15.
    Fellenberg K, Hauser NC, Brors B, Neutzner A, Hoheisel JD, Vingron M (2001) Correspondence analysis applied to microarray data. Proc Natl Acad Sci U S A 98:10781–10786CrossRefPubMedGoogle Scholar
  16. 16.
    Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Liggett CM, Nelson KE (2006) Metagenomic analysis of the human distal gut microbiome. Science 312:1355–1359CrossRefPubMedGoogle Scholar
  17. 17.
    Griffin ME, McMahon KD, Mackie RI, Raskin L (1998) Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids. Biotechnol Bioeng 57:342–355CrossRefPubMedGoogle Scholar
  18. 18.
    Guarner F, Malagelada JR (2003) Gut flora in health and disease. Lancet 361:512–519CrossRefPubMedGoogle Scholar
  19. 19.
    Hershberger KL, Barns SM, Reysenbach A-L, Dawson SC, Pace NR (1996) Wide diversity of Crenarchaeota. Nature 384:420CrossRefPubMedGoogle Scholar
  20. 20.
    Janssen PH, Kirs M (2008) Structure of the archaeal community of the rumen. Appl Environ Microbiol 74:3619–3625CrossRefPubMedGoogle Scholar
  21. 21.
    Kang S, Denman SE, Morrison M, Yu Z, Dore J, Leclerc M, McSweeney CS (2010) Dysbiosis of fecal microbiota in Crohn’s disease patients as revealed by a custom phylogenetic microarray. Inflamm Bowel Dis. doi: 10.1002/ibd.21319 Google Scholar
  22. 22.
    Khachatryan ZA, Ktsoyan ZA, Manukyan GP, Kelly D, Ghazaryan KA, Aminov RI (2008) Predominant role of host genetics in controlling the composition of gut microbiota. PLoS ONE 3:e3064CrossRefPubMedGoogle Scholar
  23. 23.
    Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol 12:118–123CrossRefPubMedGoogle Scholar
  24. 24.
    Lane DS (1990) 16S and 23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–148Google Scholar
  25. 25.
    Lauber CL, Zhou N, Gordon JI, Knight R, Fierer N (2010) Effect of storage conditions on the assessment of bacterial community structure in soil and human-associated samples. FEMS Microbiol Lett 307:80–86CrossRefPubMedGoogle Scholar
  26. 26.
    Layton A, McKay L, Williams D, Garrett V, Gentry R, Sayler G (2006) Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water. Appl Environ Microbiol 72:4214–4224CrossRefPubMedGoogle Scholar
  27. 27.
    Lepage P, Seksik P, Sutren M, de la Cochetiere MF, Jian R, Marteau P, Dore J (2005) Biodiversity of the mucosa-associated microbiota is stable along the distal digestive tract in healthy individuals and patients with IBD. Inflamm Bowel Dis 11:473–480CrossRefPubMedGoogle Scholar
  28. 28.
    Li F, Hullar MA, Lampe JW (2007) Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota. J Microbiol Methods 68:303–311CrossRefPubMedGoogle Scholar
  29. 29.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  30. 30.
    Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218CrossRefGoogle Scholar
  31. 31.
    Matsuki T, Watanabe K, Fujimoto J, Miyamoto Y, Takada T, Matsumoto K, Oyaizu H, Tanaka R (2002) Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces. Appl Environ Microbiol 68:5445–5451CrossRefPubMedGoogle Scholar
  32. 32.
    McMahon KD, Zheng D, Stams AJM, Mackie RI, Raskin L (2004) Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge. Biotechnol Bioeng 87:823–834CrossRefPubMedGoogle Scholar
  33. 33.
    McOrist AL, Jackson M, Bird AR (2002) A comparison of five methods for extraction of bacterial DNA from human faecal samples. J Microbiol Methods 50:131–139CrossRefPubMedGoogle Scholar
  34. 34.
    Mihajlovski A, Alric M, Brugère J-F (2008) A putative new order of methanogenic Archaea inhabiting the human gut, as revealed by molecular analyses of the mcrA gene. Res Microbiol 159:516–521CrossRefPubMedGoogle Scholar
  35. 35.
    Miller SA, Dykes DD, Polesky HF (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215CrossRefPubMedGoogle Scholar
  36. 36.
    Mondot S, Kang S, Furet J, Aguirre de Carcer D, McSweeney C, Morrison M, Marteau P, Doré J, Leclerc M (2010) Highlighting new phylogenetic specificities of Crohn’s disease microbiota. Inflamm Bowel Dis. doi: 10.1002/ibd.21436 PubMedGoogle Scholar
  37. 37.
    Nagashima K, Hisada T, Sato M, Mochizuki J (2003) Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. Appl Environ Microbiol 69:1251–1262CrossRefPubMedGoogle Scholar
  38. 38.
    Nam YD, Chang HW, Kim KH, Roh SW, Kim MS, Jung MJ, Lee SW, Kim JY, Yoon JH, Bae JW (2008) Bacterial, archaeal, and eukaryal diversity in the intestines of Korean people. J Microbiol 46:491–501CrossRefPubMedGoogle Scholar
  39. 39.
    Nechvatal JM, Ram JL, Basson MD, Namprachan P, Niec SR, Badsha KZ, Matherly LH, Majumdar AP, Kato I (2008) Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces. J Microbiol Methods 72:124–132CrossRefPubMedGoogle Scholar
  40. 40.
    Osborne CA, Galic M, Sangwan P, Janssen PH (2005) PCR-generated artefact from 16S rRNA gene-specific primers. FEMS Microbiol Lett 248:183–187CrossRefPubMedGoogle Scholar
  41. 41.
    Ott SJ, Musfeldt M, Timmis KN, Hampe J, Wenderoth DF, Schreiber S (2004) In vitro alterations of intestinal bacterial microbiota in fecal samples during storage. Diagn Microbiol Infect Dis 50:237–245CrossRefPubMedGoogle Scholar
  42. 42.
    Oxley APA, Lanfranconi MP, Würdemann D, Ott S, Schreiber S, McGenity TJ, Timmis KN, Nogales B (2010) Halophilic archaea in the human intestinal mucosa. Environ Microbiol 12:2398–2410CrossRefPubMedGoogle Scholar
  43. 43.
    Palmer C, Bik EM, Eisen MB, Eckburg PB, Sana TR, Wolber PK, Relman DA, Brown PO (2006) Rapid quantitative profiling of complex microbial populations. Nucleic Acids Res 34:e5CrossRefPubMedGoogle Scholar
  44. 44.
    Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, Sicheritz-Ponten T, Turner K, Zhu H, Yu C, Li S, Jian M, Zhou Y, Li Y, Zhang X, Li S, Qin N, Yang H, Wang J, Brunak S, Dore J, Guarner F, Kristiansen K, Pedersen O, Parkhill J, Weissenbach J, Bork P, Ehrlich SD, Wang J (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464:59–65CrossRefPubMedGoogle Scholar
  45. 45.
    Quince C, Lanzen A, Curtis TP, Davenport RJ, Hall N, Head IM, Read LF, Sloan WT (2009) Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods 6:639–641CrossRefPubMedGoogle Scholar
  46. 46.
    Reeder J, Knight R (2010) Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat Methods 7:668–669CrossRefPubMedGoogle Scholar
  47. 47.
    Rieu-Lesme F, Delbes C, Sollelis L (2005) Recovery of partial 16S rDNA sequences suggests the presence of Crenarchaeota in the human digestive ecosystem. Curr Microbiol 51:317–321CrossRefPubMedGoogle Scholar
  48. 48.
    Salonen A, Nikkilä J, Jalanka-Tuovinen J, Immonen O, Rajilic-Stojanovic M, Kekkonen RA, Palva A, de Vos WM (2010) Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. J Microbiol Methods 81:127–134CrossRefPubMedGoogle Scholar
  49. 49.
    Scanlan PD, Shanahan F, Clune Y, Collins JK, O’Sullivan GC, O'Riordan M, Holmes E, Wang Y, Marchesi JR (2008) Culture-independent analysis of the gut microbiota in colorectal cancer and polyposis. Environ Microbiol 10:789–798CrossRefPubMedGoogle Scholar
  50. 50.
    Scanlan PD, Shanahan F, Marchesi JR (2008) Human methanogen diversity and incidence in healthy and diseased colonic groups using mcrA gene analysis. BMC Microbiol 8:79CrossRefPubMedGoogle Scholar
  51. 51.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefPubMedGoogle Scholar
  52. 52.
    Stevenson DM, Weimer PJ (2007) Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl Microbiol Biotechnol 75:165–174CrossRefPubMedGoogle Scholar
  53. 53.
    Tap J, Mondot S, Levenez F, Pelletier E, Caron C, Furet J-P, Ugarte E, Muñoz-Tamayo R, Paslier DLE, Nalin R, Dore J, Leclerc M (2009) Towards the human intestinal microbiota phylogenetic core. Environ Microbiol 11:2574–2584CrossRefPubMedGoogle Scholar
  54. 54.
    Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M, Henrissat B, Heath AC, Knight R, Gordon JI (2009) A core gut microbiome in obese and lean twins. Nature 457:480–484CrossRefPubMedGoogle Scholar
  55. 55.
    Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1031CrossRefPubMedGoogle Scholar
  56. 56.
    Wang RF, Beggs ML, Erickson BD, Cerniglia CE (2004) DNA microarray analysis of predominant human intestinal bacteria in fecal samples. Mol Cell Probes 18:223–234CrossRefPubMedGoogle Scholar
  57. 57.
    Willing B, Halfvarson J, Dicksved J, Rosenquist M, Jarnerot G, Engstrand L, Tysk C, Jansson JK (2009) Twin studies reveal specific imbalances in the mucosa-associated microbiota of patients with ileal Crohn’s disease. Inflamm Bowel Dis 15:653–660CrossRefPubMedGoogle Scholar
  58. 58.
    Yu Z, Morrison M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. Biotechniques 36:808–812PubMedGoogle Scholar
  59. 59.
    Zoetendal EG, Ben-Amor K, Akkermans AD, Abee T, de Vos WM (2001) DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract. Syst Appl Microbiol 24:405–410CrossRefPubMedGoogle Scholar
  60. 60.
    Zoetendal EG, von Wright A, Vilpponen-Salmela T, Ben-Amor K, Akkermans AD, de Vos WM (2002) Mucosa-associated bacteria in the human gastrointestinal tract are uniformly distributed along the colon and differ from the community recovered from feces. Appl Environ Microbiol 68:3401–3407CrossRefPubMedGoogle Scholar

Copyright information

© Scientific & Industrial Research Organisation 2010

Authors and Affiliations

  • Páraic Ó Cuív
    • 1
  • Daniel Aguirre de Cárcer
    • 1
  • Michelle Jones
    • 1
  • Eline S. Klaassens
    • 1
  • Daniel L. Worthley
    • 2
  • Vicki L. J. Whitehall
    • 2
  • Seungha Kang
    • 1
  • Christopher S. McSweeney
    • 1
  • Barbara A. Leggett
    • 2
  • Mark Morrison
    • 1
    • 3
  1. 1.CSIRO Preventative Health Flagship Research Program and Division of Livestock IndustriesQueensland Biosciences PrecinctQueenslandAustralia
  2. 2.The Conjoint Gastroenterology LaboratoryRoyal Brisbane and Women’s Hospital Foundation Clinical Research Centre and the Queensland Institute of Medical ResearchQueenslandAustralia
  3. 3.The Ohio State UniversityColumbusUSA

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