Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing

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.

[1]  S. Giovannoni,et al.  Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR , 1996, Applied and environmental microbiology.

[2]  C E Shannon,et al.  The mathematical theory of communication. 1963. , 1997, M.D. computing : computers in medical practice.

[3]  W. Quint,et al.  Technical Advance Novel Short-Fragment PCR Assay for Highly Sensitive Broad-Spectrum Detection of Anogenital Human Papillomaviruses , 1998 .

[4]  Martin F. Polz,et al.  Bias in Template-to-Product Ratios in Multitemplate PCR , 1998, Applied and Environmental Microbiology.

[5]  S. Giovannoni,et al.  Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length Heterogeneity , 1998, Applied and Environmental Microbiology.

[6]  F. Rodríguez-Valera,et al.  Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli. , 1999, International journal of systematic bacteriology.

[7]  D. Relman,et al.  Bacterial diversity within the human subgingival crevice. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[8]  W. König,et al.  Rapid Ribosequencing – an Effective Diagnostic Tool for Detecting Microbial Infection , 2001, Infection.

[9]  F. Rodríguez-Valera,et al.  Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains. , 2001, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[10]  F. Dewhirst,et al.  Bacterial Diversity in Human Subgingival Plaque , 2001, Journal of bacteriology.

[11]  J. Osborne.  Notes on the use of data transformations. , 2002 .

[12]  D. Cowan,et al.  Review and re-analysis of domain-specific 16S primers. , 2003, Journal of microbiological methods.

[13]  M. Horn,et al.  Molecular analysis of bacteria in periodontitis: evaluation of clone libraries, novel phylotypes and putative pathogens. , 2003, Microbiology.

[14]  J. Clarridge,et al.  Impact of 16 S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases , 2004 .

[15]  J. Clarridge,et al.  Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases , 2004, Clinical Microbiology Reviews.

[16]  J. A. Aas,et al.  Defining the Normal Bacterial Flora of the Oral Cavity , 2005, Journal of Clinical Microbiology.

[17]  M. Moeschberger,et al.  Identification of Candidate Periodontal Pathogens and Beneficial Species by Quantitative 16S Clonal Analysis , 2005, Journal of Clinical Microbiology.

[18]  R. Oppermann,et al.  Periodontal status in smokers and never-smokers: clinical findings and real-time polymerase chain reaction quantification of putative periodontal pathogens. , 2006, Journal of periodontology.

[19]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[20]  Sang Joon Kim,et al.  A Mathematical Theory of Communication , 2006 .

[21]  W. Wade,et al.  Novel subgingival bacterial phylotypes detected using multiple universal polymerase chain reaction primer sets. , 2006, Oral microbiology and immunology.

[22]  R. Palmer,et al.  Molecular Characterization of Subject-Specific Oral Microflora during Initial Colonization of Enamel , 2006, Applied and Environmental Microbiology.

[23]  M. Moeschberger,et al.  Changes in Periodontal Health Status Are Associated with Bacterial Community Shifts as Assessed by Quantitative 16S Cloning and Sequencing , 2006, Journal of Clinical Microbiology.

[24]  Susan M. Huse,et al.  Accuracy and quality of massively parallel DNA pyrosequencing , 2007, Genome Biology.

[25]  D. Alland,et al.  A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. , 2007, Journal of microbiological methods.

[26]  Susan M. Huse,et al.  Pyrosequencing analysis of the Oral Microflora of healthy adults , 2008, Journal of dental research.

[27]  A. Lennon,et al.  Molecular identification of bacteria on the tongue dorsum of subjects with and without halitosis. , 2008, Oral diseases.

[28]  S. Dowd,et al.  Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) for microbiome studies: bacterial diversity in the ileum of newly weaned Salmonella-infected pigs. , 2008, Foodborne pathogens and disease.

[29]  Susan M. Huse,et al.  Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure. , 2009, Environmental microbiology.

[30]  Adam P. Arkin,et al.  FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix , 2009, Molecular biology and evolution.

[31]  P. Qian,et al.  Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies , 2009, PloS one.

[32]  Susan M. Huse,et al.  Defining the healthy "core microbiome" of oral microbial communities , 2009, BMC Microbiology.

[33]  B. Roe,et al.  Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys , 2009, Applied and Environmental Microbiology.

[34]  Purnima S. Kumar,et al.  Response of Subgingival Bacteria to Smoking Cessation , 2010, Journal of Clinical Microbiology.

[35]  Russell J. Davenport,et al.  Removing Noise From Pyrosequenced Amplicons , 2011, BMC Bioinformatics.

[36]  R. Knight,et al.  Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns , 2010, Proceedings of the National Academy of Sciences.

[37]  William A. Walters,et al.  QIIME allows analysis of high-throughput community sequencing data , 2010, Nature Methods.

[38]  Marcus J. Claesson,et al.  Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions , 2010, Nucleic acids research.

[39]  H. Nagaraja,et al.  Subgingival Microbial Profiles of Smokers with Periodontitis , 2010, Journal of dental research.

[40]  E. Mongodin,et al.  Analyzing Endodontic Infections by Deep Coverage Pyrosequencing , 2010, Journal of dental research.

[41]  V. Kunin,et al.  Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. , 2009, Environmental microbiology.

[42]  J. A. Aas,et al.  Design of 16S rRNA gene primers for 454 pyrosequencing of the human foregut microbiome. , 2010, World journal of gastroenterology.