1887

Journal of Medical Microbiology logo

Volume 63, Issue 3

Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples Free

Abstract

Although 16S rRNA gene (rDNA) sequencing is the gold standard for categorizing bacteria or characterizing microbial communities its clinical utility is limited by bias in metagenomic studies, in either the experiments or the data analyses. To evaluate the efficiency of current metagenomic methods, we sequenced seven simulated samples of ten bacterial species mixed at different concentrations. The V3 region of 16S rDNA was targeted and used to determine the distribution of bacterial species. The number of target sequences in individual simulated samples was in the range 1–1000 to provide a better reflection of natural microbial communities. However, for a given bacterial species present in the same proportion but at different concentrations, the observed percentage of 16S rDNAs was similar, except at very low concentrations that cannot be detected by real-time PCR. These results confirmed that the comparative microbiome in a sample characterized by 16S rDNA sequencing is sufficient to detect not only potential infectious pathogens, but also the relative proportion of 16S rDNA in the sample.

  • Received:
  • Accepted:
  • Published Online:
© 2014 SGM
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2014-03-01
2024-04-25
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References

  1. Barriuso J., Valverde J. R., Mellado R. P. 2011; Estimation of bacterial diversity using next generation sequencing of 16S rDNA: a comparison of different workflows. BMC Bioinformatics 12:473 [View Article][PubMed]
     [Google Scholar]
  2. Charlson E. S., Chen J., Custers-Allen R., Bittinger K., Li H., Sinha R., Hwang J., Bushman F. D., Collman R. G. 2010; Disordered microbial communities in the upper respiratory tract of cigarette smokers. PLoS ONE 5:e15216 [View Article][PubMed]
     [Google Scholar]
  3. Claesson M. J., Wang Q., O’Sullivan O., Greene-Diniz R., Cole J. R., Ross R. P., O’Toole P. W. 2010; Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38:e200 [View Article][PubMed]
     [Google Scholar]
  4. Cole J. R., Wang Q., Cardenas E., Fish J., Chai B., Farris R. J., Kulam-Syed-Mohideen A. S., McGarrell D. M., Marsh T.& other authors ( 2009; The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:Database issueD141–D145 [View Article][PubMed]
     [Google Scholar]
  5. Eckburg P. B., Bik E. M., Bernstein C. N., Purdom E., Dethlefsen L., Sargent M., Gill S. R., Nelson K. E., Relman D. A. 2005; Diversity of the human intestinal microbial flora. Science 308:1635–1638 [View Article][PubMed]
     [Google Scholar]
  6. Fox G. E., Magrum L. J., Balch W. E., Wolfe R. S., Woese C. R. 1977; Classification of methanogenic bacteria by 16S ribosomal RNA characterization. Proc Natl Acad Sci U S A 74:4537–4541 [View Article][PubMed]
     [Google Scholar]
  7. Gilbert J. A., Steele J. A., Caporaso J. G., Steinbrück L., Reeder J., Temperton B., Huse S., McHardy A. C., Knight R.& other authors ( 2012; Defining seasonal marine microbial community dynamics. ISME J 6:298–308 [View Article][PubMed]
     [Google Scholar]
  8. Giongo A., Crabb D. B., Davis-Richardson A. G., Chauliac D., Mobberley J. M., Gano K. A., Mukherjee N., Casella G., Roesch L. F.& other authors ( 2010; PANGEA: pipeline for analysis of next generation amplicons. ISME J 4:852–861 [View Article][PubMed]
     [Google Scholar]
  9. Griffen A. L., Beall C. J., Firestone N. D., Gross E. L., Difranco J. M., Hardman J. H., Vriesendorp B., Faust R. A., Janies D. A., Leys E. J. 2011; CORE: a phylogenetically-curated 16S rDNA database of the core oral microbiome. PLoS ONE 6:e19051 [View Article][PubMed]
     [Google Scholar]
  10. Hamady M., Knight R. 2009; Microbial community profiling for human microbiome projects: tools, techniques, and challenges. Genome Res 19:1141–1152 [View Article][PubMed]
     [Google Scholar]
  11. Hong S., Bunge J., Leslin C., Jeon S., Epstein S. S. 2009; Polymerase chain reaction primers miss half of rRNA microbial diversity. ISME J 3:1365–1373 [View Article][PubMed]
     [Google Scholar]
  12. Huttenhower C., Gevers D., Knight R., Abubucker S., Badger J. H., Chinwalla A. T., Creasy H. H., Earl A. M., FitzGerald M. G.& other authors ( 2012; Structure, function and diversity of the healthy human microbiome. Nature 486:207–214 [View Article][PubMed]
     [Google Scholar]
  13. Kaeberlein T., Lewis K., Epstein S. S. 2002; Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:1127–1129 [View Article][PubMed]
     [Google Scholar]
  14. Lozupone C., Hamady M., Knight R. 2006; UniFrac–an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7:371 [View Article][PubMed]
     [Google Scholar]
  15. Lozupone C., Lladser M. E., Knights D., Stombaugh J., Knight R. 2011; UniFrac: an effective distance metric for microbial community comparison. ISME J 5:169–172 [View Article][PubMed]
     [Google Scholar]
  16. Luna G. M., Dell’Anno A., Danovaro R. 2006; DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sediments. Environ Microbiol 8:308–320 [View Article][PubMed]
     [Google Scholar]
  17. Mardis E. R. 2008; Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402 [View Article][PubMed]
     [Google Scholar]
  18. Nelson K. E., Weinstock G. M., Highlander S. K., Worley K. C., Creasy H. H., Wortman J. R., Rusch D. B., Mitreva M., Sodergren E.& other authors ( 2010; A catalog of reference genomes from the human microbiome. Science 328:994–999 [View Article][PubMed]
     [Google Scholar]
  19. Peterson D. A., Frank D. N., Pace N. R., Gordon J. I. 2008; Metagenomic approaches for defining the pathogenesis of inflammatory bowel diseases. Cell Host Microbe 3:417–427 [View Article][PubMed]
     [Google Scholar]
  20. Roesch L. F., Fulthorpe R. R., Riva A., Casella G., Hadwin A. K., Kent A. D., Daroub S. H., Camargo F. A., Farmerie W. G., Triplett E. W. 2007; Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J 1:283–290[PubMed]
     [Google Scholar]
  21. Salonen A., Nikkilä J., Jalanka-Tuovinen J., Immonen O., Rajilić-Stojanović M., Kekkonen R. A., Palva A., de Vos W. M. 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 81:127–134 [View Article][PubMed]
     [Google Scholar]
  22. Schloss P. D., Handelsman J. 2006; Introducing SONS, a tool for operational taxonomic unit-based comparisons of microbial community memberships and structures. Appl Environ Microbiol 72:6773–6779 [View Article][PubMed]
     [Google Scholar]
  23. Schloss P. D., Gevers D., Westcott S. L. 2011; Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS ONE 6:e27310 [View Article][PubMed]
     [Google Scholar]
  24. Sun Y., Cai Y., Liu L., Yu F., Farrell M. L., McKendree W., Farmerie W. 2009; ESPRIT: estimating species richness using large collections of 16S rRNA pyrosequences. Nucleic Acids Res 37:e76 [View Article][PubMed]
     [Google Scholar]
  25. Terrat S., Christen R., Dequiedt S., Lelièvre M., Nowak V., Regnier T., Bachar D., Plassart P., Wincker P.& other authors ( 2012; Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure. Microb Biotechnol 5:135–141 [View Article][PubMed]
     [Google Scholar]
  26. von Bubnoff A. 2008; Next-generation sequencing: the race is on. Cell 132:721–723 [View Article][PubMed]
     [Google Scholar]
  27. Wang Y., Qian P. Y. 2009; Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS ONE 4:e7401 [View Article][PubMed]
     [Google Scholar]
  28. Ward D. M., Weller R., Bateson M. M. 1990; 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345:63–65 [View Article][PubMed]
     [Google Scholar]
  29. Woo P. C. Y., Lau S. K. P., Teng J. L. L., Tse H., Yuen K. Y. 2008; Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 14:908–934 [View Article][PubMed]
     [Google Scholar]
  30. Wooley J. C., Godzik A., Friedberg I. 2010; A primer on metagenomics. PLOS Comput Biol 6:e1000667 [View Article][PubMed]
     [Google Scholar]
  31. Yang S., Ramachandran P., Rothman R., Hsieh Y. H., Hardick A., Won H., Kecojevic A., Jackman J., Gaydos C. 2009; Rapid identification of biothreat and other clinically relevant bacterial species by use of universal PCR coupled with high-resolution melting analysis. J Clin Microbiol 47:2252–2255 [View Article][PubMed]
     [Google Scholar]
  32. Zhao J., Schloss P. D., Kalikin L. M., Carmody L. A., Foster B. K., Petrosino J. F., Cavalcoli J. D., VanDevanter D. R., Murray S.& other authors ( 2012; Decade-long bacterial community dynamics in cystic fibrosis airways. Proc Natl Acad Sci U S A 109:5809–5814 [View Article][PubMed]
     [Google Scholar]
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Comparative analysis of microbiome between accurately identified 16S rDNA and quantified bacteria in simulated samples
J. Med. Microbiol. 63, 433 (2014); https://doi.org/10.1099/jmm.0.060616-0
/content/journal/jmm/10.1099/jmm.0.060616-0
/content/journal/jmm/10.1099/jmm.0.060616-0
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