Skip to main content
SpringerLink
Log in

Next-Generation Sequencing in the Analysis of Human Microbiota

Essential Considerations for Clinical Application

  • Review Article
  • Published:
Molecular Diagnosis & Therapy Aims and scope Submit manuscript

Abstract

The development of next-generation sequencing (NGS) presents an unprecedented opportunity to investigate the complex microbial communities that are associated with the human body. It offers for the first time a basis for detailed temporal and spatial analysis, with the potential to revolutionize our understanding of many clinically important systems. However, while advances continue to be made in areas such as PCR amplification for NGS, sequencing protocols, and data analysis, in many cases the quality of the data generated is undermined by a failure to address fundamental aspects of experimental design. While little is added in terms of time or cost by the analysis of repeat samples, the exclusion of DNA from dead bacterial cells and the extracellular matrix, the use of efficient nucleic acid extraction methodologies, and the implementation of safeguards to minimize the introduction of contaminating nucleic acids, such considerations are essential in achieving an accurate representation of the system being studied. In this review, the chronic bacterial infections that characterize lower respiratory tract infections in cystic fibrosis patients are used as an example system to examine the implications of a failure to address these issues when designing NGS-based analysis of human-associated microbiota. Further, ways in which the impact of these factors can be minimized are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Coakley RD, Boucher RC. Pathophysiology: epithelial cell biology and ion channel function in the lung, sweat gland and pancreas. In: Hodson M, Geddes D, Bush A, editors. Cystic fibrosis. 3rd ed. London: Hodder Arnold, 2007: 59–68

    Google Scholar 

  2. Rogers GB, Carroll MP, Serisier DJ, et al. Characterization of bacterial community diversity in cystic fibrosis lung infections by use of 16S ribosomal DNA terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2004 Nov; 42(11): 5176–83

    Article  PubMed  CAS  Google Scholar 

  3. Rogers GB, Daniels TT, Tuck A, et al. Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches. BMC Pulm Med 2009 Apr 15; 9(1): 14

    Article  PubMed  Google Scholar 

  4. Gest H. Scotoma in contemporary microbiology [comment]. Microbiology Today 2008 Nov; 35(4): 220 [online]. Available from URL: http://www.sgm.ac.uk/pubs/micro_today/pdf/110813.pdf [Accessed 2010 Dec 15]

    Google Scholar 

  5. Suau A, Bonnet R, Sutren M, et al. Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 1999 Nov; 65(11): 4799–807

    PubMed  CAS  Google Scholar 

  6. Eckburg PB, Bik EM, Bernstein CN, et al. Diversity of the human intestinal microbial flora. Science 2005 Jun 10; 308(5728): 1635–8

    Article  PubMed  Google Scholar 

  7. Flint HJ, Duncan SH, Scott KP, et al. Interactions and competition within the microbial community of the human colon: links between diet and health. Environ Microbiol 2007 May; 9(5): 1101–11

    Article  PubMed  CAS  Google Scholar 

  8. Socransky SS, Gibbons RJ, Dale AC, et al. The microbiota of the gingival crevice area of man: I. Total microscopic and viable counts and counts of specific organisms. Arch Oral Biol 1963 May–Jun; 8: 275–8

    Article  PubMed  CAS  Google Scholar 

  9. Woese CR. Bacterial evolution. Microbiol Rev 1987 Jun; 51(2): 221–71

    PubMed  CAS  Google Scholar 

  10. Kolbert CP, Persing DH. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr Opin Microbiol 1999 Jun; 2(3): 299–305

    Article  PubMed  CAS  Google Scholar 

  11. Balfour-Lynn IA, Elborn JS. Respiratory disease: infection. In: Hodson M, Geddes D, Bush A, editors. Cystic fibrosis. 3rd ed. London: Hodder Arnold, 2007: 137–58

    Google Scholar 

  12. Rogers GB, Hart CA, Mason JR, et al. Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling. J Clin Microbiol 2003 Aug; 41(8): 3548–58

    Article  PubMed  CAS  Google Scholar 

  13. Petrosino JF, Highlander S, Luna RA, et al. Metagenomic pyrosequencing and microbial identification. Clin Chem 2009 May; 55(5): 856–66

    Article  PubMed  CAS  Google Scholar 

  14. Metzker ML. Emerging technologies in DNA sequencing. Genome Res 2005 Dec; 15(12): 1767–76

    Article  PubMed  CAS  Google Scholar 

  15. Tringe SG, Hugenholtz P. A renaissance for the pioneering 16S rRNA gene. Curr Opin Microbiol 2008 Oct; 11(5): 442–6

    Article  PubMed  CAS  Google Scholar 

  16. Guss AM, Roeselers G, Newton IL, et al. Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis. ISME J 2011 Jan; 5(1): 20–9

    Article  PubMed  Google Scholar 

  17. Rogers GB, Stressmann FA, Koller G, et al. Assessing the diagnostic importance of nonviable bacterial cells in respiratory infections. Diagn Microbiol Infect Dis 2008 Oct; 62(2): 133–41

    Article  PubMed  CAS  Google Scholar 

  18. Sibley CD, Parkins MD, Rabin HR, et al. A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients. Proc Natl Acad Sci U S A 2008 Sep 30; 105(39): 15070–5

    Article  PubMed  CAS  Google Scholar 

  19. Engelbrektson A, Kunin V, Wrighton KC, et al. Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J 2010 May; 4(5): 642–7

    Article  PubMed  CAS  Google Scholar 

  20. Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 2005 Dec; 71(12): 8228–35

    Article  PubMed  CAS  Google Scholar 

  21. Huse SM, Dethlefsen L, Huber JA, et al. Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing. PLoS Genet 2008 Nov; 4(11): e1000255

    Article  PubMed  Google Scholar 

  22. Kunin V, Engelbrektson A, Ochman H, et al. Wrinkles in the rare biosphere: pyrosequencing errors lead to artificial inflation of diversity estimates. Environ Microbiol 2009 Aug 27; 12: 118–23

    Article  PubMed  Google Scholar 

  23. Hamady M, Lozupone C, Knight R. Fast UniFrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data. ISME J 2009 Aug 27; 4: 17–27

    Article  PubMed  Google Scholar 

  24. Kuczynski J, Liu Z, Lozupone C, et al. Microbial community resemblance methods differ in their ability to detect biologically relevant patterns. Nat Methods 2010 Oct; 7(10): 813–9

    Article  PubMed  CAS  Google Scholar 

  25. vonWintzingerode F, Gobel UB, Stackebrandt E. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev 1997 Nov; 21(3): 213–29

    Article  Google Scholar 

  26. Rogers GB, Skelton S, Serisier DJ, et al. Determining CF lung microbiology: a comparison of spontaneous and serially induced sputum samples using T-RFLP profiling. J Clin Microbiol 2009 Nov 11; 48: 78–86

    Article  PubMed  Google Scholar 

  27. Prosser JI. Replicate or lie. Environ Microbiol 2010 Jul; 12(7): 1806–1

    Article  PubMed  CAS  Google Scholar 

  28. DePaoli P. Bio-banking in microbiology: from sample collection to epidemiology, diagnosis and research. FEMS Microbiol Rev 2005 Nov; 29(5): 897–91

    Article  PubMed  Google Scholar 

  29. Ranasinha C, Assoufi B, Shak S, et al. Efficacy and safety of short-term administration of aerosolised recombinant human DNase I in adults with stable stage cystic fibrosis. Lancet 1993 Jul 24; 342(8865): 199–202

    Article  PubMed  CAS  Google Scholar 

  30. Brandt T, Breitenstein S, von der Hardt H, et al. DNA concentration and length in sputum of patients with cystic fibrosis during inhalation with recombinant human DNase. Thorax 1995 Aug; 50(8): 880–2

    Article  PubMed  CAS  Google Scholar 

  31. Ulmer JS, Herzka A, Toy KJ, et al. Engineering actin-resistant human DNase I for treatment of cystic fibrosis. Proc Natl Acad Sci U S A 1996 Aug 6; 93(16): 8225–9

    Article  PubMed  CAS  Google Scholar 

  32. Rogers GB, Carroll MP, Serisier DJ, et al. Bacterial activity in cystic fibrosis lung infections. Respir Res 2005 Jun 1; 6: 49

    Article  PubMed  Google Scholar 

  33. Nocker A, Cheung CY, Camper AK. Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 2006 Nov; 67(2): 310–2

    Article  PubMed  CAS  Google Scholar 

  34. Nocker A, Sossa-Fernandez P, Burr MD, et al. Use of propidium monoazide for live/dead distinction in microbial ecology. Appl Environ Microbiol 2007 Aug; 73(16): 5111–7

    Article  PubMed  CAS  Google Scholar 

  35. Kobayashi H, Oethinger M, Tuohy MJ, et al. Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis. J Orthop Res 2009 Sep; 27(9): 1243–7

    Article  PubMed  CAS  Google Scholar 

  36. Nocker A, Richter-Heitmann T, Montijn R, et al. Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing. Int Microbiol 2010 Jun; 13(2): 59–65

    PubMed  CAS  Google Scholar 

  37. Rawsthorne H, Dock CN, Jaykus LA. PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores. Appl Environ Microbiol 2009 May; 75(9): 2936–9

    Article  PubMed  CAS  Google Scholar 

  38. Kralik P, Nocker A, Pavlik I. Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int J Food Microbiol 2010 Jul 31; 141Suppl. 1: S80–6

    Article  PubMed  CAS  Google Scholar 

  39. Rogers GB, Marsh P, Stressmann AF, et al. The exclusion of dead bacterial cells is essential for accurate molecular analysis of clinical samples. Clin Microbiol Infect 2010 Nov; 16(11): 1656–8

    Article  PubMed  CAS  Google Scholar 

  40. Wu GD, Lewis JD, Hoffmann C, et al. Sampling and pyrosequencing methods for characterizing bacterial communities in the human gut using 16S sequence tags. BMC Microbiol 2010 Jul 30; 1: 206

    Article  Google Scholar 

  41. Feinstein LM, Sul WJ, Blackwood CB. Assessment of bias associated with incomplete extraction of microbial DNA from soil. Appl Environ Microbiol 2009 Aug; 75(16): 5428–33

    Article  PubMed  CAS  Google Scholar 

  42. Carrigg C, Rice O, Kavanagh S, et al. DNA extraction method affects microbial community profiles from soils and sediment. Appl Microbiol Biotechnol 2007 Dec; 77(4): 955–64

    Article  PubMed  CAS  Google Scholar 

  43. Webster G, Newberry CJ, Fry JC, et al. Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale. J Microbiol Methods 2003 Oct; 55(1): 155–64

    Article  PubMed  CAS  Google Scholar 

  44. Morgan JL, Darling AE, Eisen JA. Metagenomic sequencing of an in vitro-simulated microbial community. PLoS One 2010 Apr 16; 5(4): e10209

    Article  PubMed  Google Scholar 

  45. Rantakokko-Jalava K, Nikkari S, Jalava J, et al. Direct amplification of rRNA genes in diagnosis of bacterial infections. J Clin Microbiol 2000 Jan; 38(1): 32–9

    PubMed  CAS  Google Scholar 

  46. Kitchin PA, Szotyori Z, Fromholc C, et al. Avoidance of PCR false positives [published erratum appears in Nature 1990 Mar 29; 344 (6265): 388]. Nature 1990 Mar 15; 344(6263): 201

    Article  PubMed  CAS  Google Scholar 

  47. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989 May 18; 339(6221): 237–8

    Article  PubMed  CAS  Google Scholar 

  48. Bottger EC. Frequent contamination of Taq polymerase with DNA. Clin Chem 1990 Jun; 36(6): 1258–9

    PubMed  CAS  Google Scholar 

  49. Corless CE, Guiver M, Borrow R, et al. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol 2000 May; 38(5): 1747–52

    PubMed  CAS  Google Scholar 

  50. Hughes MS, Beck LA, Skuce RA. Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 1994 Aug; 32(8): 2007–8

    PubMed  CAS  Google Scholar 

  51. Newsome T, Li BJ, Zou N, et al. Presence of bacterial phage-like DNA sequences in commercial Taq DNA polymerase reagents. J Clin Microbiol 2004 May; 42(5): 2264–7

    Article  PubMed  CAS  Google Scholar 

  52. Rand KH, Houck H. Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes 1990 Dec; 4(6): 445–5

    Article  PubMed  CAS  Google Scholar 

  53. Schmidt TM, Pace B, Pace NR. Detection of DNA contamination in Taq polymerase. BioTechniques 1991 Aug; 11(2): 176–7

    PubMed  CAS  Google Scholar 

  54. Maiwald M, Ditton HJ, Sonntag HG, et al. Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. Mol Cell Probes 1994 Feb; 8(1): 11–4

    Article  PubMed  CAS  Google Scholar 

  55. Ehricht R, Hotzel H, Sachse K, et al. Residual DNA in thermostable DNA polymerases: a cause of irritation in diagnostic PCR and microarray assays. Biologicals 2007 Apr; 35(2): 145–7

    Article  PubMed  CAS  Google Scholar 

  56. van der Zee A, Peeters M, de Jong C, et al. Qiagen DNA extraction kits for sample preparation for Legionella PCR are not suitable for diagnostic purposes. J Clin Microbiol 2002 Mar; 40(3): 1126

    Article  PubMed  Google Scholar 

  57. Evans GE, Murdoch DR, Anderson TP, et al. Contamination of Qiagen DNA extraction kits with Legionella DNA. J Clin Microbiol 2003 Jul; 41(7): 3452–3

    Article  PubMed  CAS  Google Scholar 

  58. Mohammadi T, Reesink HW, Vandenbroucke-Grauls CM, et al. Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. J Microbiol Methods 2005 May; 61(2): 285–8

    Article  PubMed  CAS  Google Scholar 

  59. Sarkar G, Sommer S. More light on PCR contamination. Nature 1990 Sep 27; 347(6291): 340–1

    Article  PubMed  CAS  Google Scholar 

  60. Sarkar G, Sommer SS. Shedding light on PCR contamination. Nature 1990 Jan 4; 343(6253): 27

    Article  PubMed  CAS  Google Scholar 

  61. Furrer B, Candrian U, Wieland P, et al. Improving PCR efficiency. Nature 1990 Jul 26; 346(6282): 324

    Article  PubMed  CAS  Google Scholar 

  62. Jinno Y, Yoshiura K, Niikawa N. Use of psoralen as extinguisher of contaminated DNA in PCR. Nucleic Acids Res 1990 Nov 25; 18(22): 6739

    Google Scholar 

  63. Meier A, Persing DH, Finken M, et al. Elimination of contaminating DNA within polymerase chain reaction reagents: implications for a general approach to detection of uncultured pathogens. J Clin Microbiol 1993 Mar; 31(3): 646–52

    PubMed  CAS  Google Scholar 

  64. Tseng CP, Cheng JC, Tseng CC, et al. Broad-range ribosomal RNA real-time PCR after removal of DNA from reagents: melting profiles for clinically important bacteria. Clin Chem 2003 Feb; 49(2): 306–9

    Article  PubMed  CAS  Google Scholar 

  65. Cherkaoui A, Emonet S, Ceroni D, et al. Development and validation of a modified broad-range 16S rDNA PCR for diagnostic purposes in clinical microbiology. J Microbiol Methods 2009 Nov; 79(2): 227–31

    Article  PubMed  CAS  Google Scholar 

  66. Glushkov SA, Bragin AG, Dymshits GM. Decontamination of polymerase chain reaction reagents using DEAE-cellulose. Anal Biochem 2009 Oct 1; 393(1): 135–7

    Article  PubMed  CAS  Google Scholar 

  67. Philipp S, Huemer HP, Irschick EU, et al. Obstacles of multiplex real-time PCR for bacterial 16S rDNA: primer specifity and DNA decontamination of Taq polymerase. Transfus Med Hemother 2010 Feb; 37(1): 21–8

    Article  PubMed  Google Scholar 

  68. Klaschik S, Lehmann LE, Raadts A, et al. Comparison of different decontamination methods for reagents to detect low concentrations of bacterial 16S DNA by real-time-PCR. Mol Biotechnol 2002 Nov; 22(3): 231–42

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors declare no conflicts of interest, and no funding support was used in the writing of this article.

Author information

Authors and Affiliations

  1. Molecular Microbiology Research Laboratory, Pharmaceutical Science Division, King’s College London, London, UK

    Geraint B. Rogers & Kenneth D. Bruce

Authors
  1. Geraint B. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenneth D. Bruce
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Geraint B. Rogers.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rogers, G.B., Bruce, K.D. Next-Generation Sequencing in the Analysis of Human Microbiota. Mol Diag Ther 14, 343–350 (2010). https://doi.org/10.1007/BF03256391

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF03256391

Keywords

Search

Navigation