Pyrosequencing of 16S rRNA genes allows for in-depth characterization of complex microbial communities. Although it is known that primer selection can influence the profile of a community generated by sequencing, the extent and severity of this bias on deep-sequencing methodologies is not well elucidated. We tested the hypothesis that the hypervariable region targeted for sequencing and primer degeneracy play important roles in influencing the composition of 16S pyrotag communities. Subgingival plaque from deep sites of current smokers with chronic periodontitis was analyzed using Sanger sequencing and pyrosequencing using 4 primer pairs. Greater numbers of species were detected by pyrosequencing than by Sanger sequencing. Rare taxa constituted nearly 6% of each pyrotag community and less than 1% of the Sanger sequencing community. However, the different target regions selected for pyrosequencing did not demonstrate a significant difference in the number of rare and abundant taxa detected. The genera Prevotella, Fusobacterium, Streptococcus, Granulicatella, Bacteroides, Porphyromonas and Treponema were abundant when the V1–V3 region was targeted, while Streptococcus, Treponema, Prevotella, Eubacterium, Porphyromonas, Campylobacer and Enterococcus predominated in the community generated by V4–V6 primers, and the most numerous genera in the V7–V9 community were Veillonella, Streptococcus, Eubacterium, Enterococcus, Treponema, Catonella and Selenomonas. Targeting the V4–V6 region failed to detect the genus Fusobacterium, while the taxa Selenomonas, TM7 and Mycoplasma were not detected by the V7–V9 primer pairs. The communities generated by degenerate and non-degenerate primers did not demonstrate significant differences. Averaging the community fingerprints generated by V1–V3 and V7–V9 primers providesd results similar to Sanger sequencing, while allowing a significantly greater depth of coverage than is possible with Sanger sequencing. It is therefore important to use primers targeted to these two regions of the 16S rRNA gene in all deep-sequencing efforts to obtain representational characterization of complex microbial communities.
References
[1]
Diaz PI, Chalmers NI, Rickard AH, Kong C, Milburn CL, et al. (2006) Molecular characterization of subject-specific oral microflora during initial colonization of enamel. Appl Environ Microbiol 72: 2837–2848.
[2]
de Lillo A, Ashley FP, Palmer RM, Munson MA, Kyriacou L, et al. (2006) Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets. Oral Microbiol Immunol 21: 61–68.
Gomes SC, Piccinin FB, Oppermann RV, Susin C, Nonnenmacher CI, et al. (2006) Periodontal status in smokers and never-smokers: clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens. J Periodontol 77: 1483–1490.
[5]
Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43: 5721–5732.
[6]
Hutter G, Schlagenhauf U, Valenza G, Horn M, Burgemeister S, et al. (2003) Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens. Microbiology 149: 67–75.
[7]
Kroes I, Lepp PW, Relman DA (1999) Bacterial diversity within the human subgingival crevice. Proc Natl Acad Sci U S A 96: 14547–14552.
[8]
Kumar PS, Griffen AL, Moeschberger ML, Leys EJ (2005) Identification of candidate periodontal pathogens and beneficial species by quantitative 16S clonal analysis. J Clin Microbiol 43: 3944–3955.
[9]
Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, et al. (2001) Bacterial diversity in human subgingival plaque. J Bacteriol 183: 3770–3783.
[10]
Kunin V, Engelbrektson A, Ochman H, Hugenholtz P. Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol 12: 118–123.
[11]
Zaura E, Keijser BJ, Huse SM, Crielaard W (2009) Defining the healthy “core microbiome” of oral microbial communities. BMC Microbiol 9: 259.
[12]
Keijser BJ, Zaura E, Huse SM, van der Vossen JM, Schuren FH, et al. (2008) Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res 87: 1016–1020.
[13]
Li L, Hsiao WW, Nandakumar R, Barbuto SM, Mongodin EF, et al. Analyzing Endodontic Infections by Deep Coverage Pyrosequencing. J Dent Res.
[14]
Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, et al. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A 107: 11971–11975.
[15]
Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55: 541–555.
[16]
Wang Y, Qian PY (2009) Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS One 4: e7401.
[17]
Chakravorty S, Helb D, Burday M, Connell N, Alland D (2007) A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J Microbiol Methods 69: 330–339.
[18]
Nossa CW, Oberdorf WE, Yang L, Aas JA, Paster BJ, et al. rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. World J Gastroenterol 16: 4135–4144.
[19]
Youssef N, Sheik CS, Krumholz LR, Najar FZ, Roe BA, et al. (2009) Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys. Appl Environ Microbiol 75: 5227–5236.
[20]
Claesson MJ, Wang Q, O'Sullivan O, Greene-Diniz R, Cole JR, et al. 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.
[21]
Riggio MP, Lennon A, Rolph HJ, Hodge PJ, Donaldson A, et al. (2008) Molecular identification of bacteria on the tongue dorsum of subjects with and without halitosis. Oral Dis 14: 251–258.
[22]
Dowd SE, Sun Y, Wolcott RD, Domingo A, Carroll JA (2008) Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: bacterial diversity in the ileum of newly weaned Salmonella-infected pigs. Foodborne Pathog Dis 5: 459–472.
[23]
Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ.Removing noise from pyrosequenced amplicons. BMC Bioinformatics 12: 38.
[24]
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, et al. (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72: 5069–5072.
[25]
Price MN, Dehal PS, Arkin AP (2009) FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix. Molecular Biology and Evolution 26: 1641–1650. %R 1610.1093/molbev/msp1077.
[26]
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, et al. QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7: 335–336.
[27]
Shchipkova AY, Nagaraja HN, Kumar PS (2010) Subgingival Microbial Profiles of Smokers with Periodontitis. J Dent Res.
[28]
Osborne JW (2002) Notes on the use of data transformations. Practical Assessment, Research and Evaluation.
[29]
Clarridge JE 3rd (2004) Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clin Microbiol Rev 17: 840–862. table of contents.
Shannon CE (1997) The mathematical theory of communication. 1963. MD Comput 14: 306–317.
[32]
Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM (2007) Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol 8: R143.
[33]
Kumar PS, Leys EJ, Bryk JM, Martinez FJ, Moeschberger ML, et al. (2006) Changes in periodontal health status are associated with bacterial community shifts as assessed by quantitative 16S cloning and sequencing. J Clin Microbiol 44: 3665–3673.
[34]
Huber JA, Morrison HG, Huse SM, Neal PR, Sogin ML, et al. (2009) Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure. Environ Microbiol 11: 1292–1302.
[35]
Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64: 3724–3730.
[36]
Kleter B, van Doorn LJ, ter Schegget J, Schrauwen L, van Krimpen K, et al. (1998) Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses. Am J Pathol 153: 1731–1739.
[37]
Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62: 625–630.
[38]
Anton AI, Martinez-Murcia AJ, Rodriguez-Valera F, Dalet F (2001) Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains. Clin Microbiol Infect 7: 345–351.
[39]
Martinez-Murcia AJ, Anton AI, Rodriguez-Valera F (1999) Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli. Int J Syst Bacteriol 49 Pt 2: 601–610.
[40]
Suzuki M, Rappe MS, Giovannoni SJ (1998) Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity. Appl Environ Microbiol 64: 4522–4529.
