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J. Thompson, L. Marcelino, M. Polz (2002)
Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'.Nucleic acids research, 30 9
J. Huber, D. Welch, H. Morrison, Susan Huse, Phillip Neal, D. Butterfield, M. Sogin (2007)
Microbial Population Structures in the Deep Marine BiosphereScience, 318
M. Polz, C. Cavanaugh (1998)
Bias in Template-to-Product Ratios in Multitemplate PCRApplied and Environmental Microbiology, 64
Brent Ewing, Philip Green (1998)
Base-calling of automated sequencer traces using phred. II. Error probabilities.Genome research, 8 3
F. Rainey, N. Ward, L. Sly, E. Stackebrandt (1994)
Dependence on the taxon composition of clone libraries for PCR amplified, naturally occurring 16S rDNA, on the primer pair and the cloning system usedExperientia, 50
S. Acinas, R. Sarma-Rupavtarm, V. Klepac‐Ceraj, M. Polz (2005)
PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same SampleApplied and Environmental Microbiology, 71
B. Kleter, L. Doorn, Lianne Schrauwen, K. Krimpen, W. Quint (1998)
Technical Advance Novel Short-Fragment PCR Assay for Highly Sensitive Broad-Spectrum Detection of Anogenital Human Papillomaviruses
F. Wintzingerode, U. Göbel, E. Stackebrandt (1997)
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis.FEMS microbiology reviews, 21 3
S. Giovannoni, T. Britschgi, C. Moyer, K. Field (1990)
Genetic diversity in Sargasso Sea bacterioplanktonNature, 345
Y. Hongoh, Hiroe Yuzawa, M. Ohkuma, T. Kudo (2003)
Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment.FEMS microbiology letters, 221 2
T. Lueders, M. Friedrich (2003)
Evaluation of PCR Amplification Bias by Terminal Restriction Fragment Length Polymorphism Analysis of Small-Subunit rRNA and mcrA Genes by Using Defined Template Mixtures of Methanogenic Pure Cultures and Soil DNA ExtractsApplied and Environmental Microbiology, 69
K. Ishii, M. Fukui (2001)
Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCRApplied and Environmental Microbiology, 67
K. Ashelford, N. Chuzhanova, J. Fry, A. Jones, A. Weightman (2006)
New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain ChimerasApplied and Environmental Microbiology, 72
P. Schloss, J. Handelsman (2006)
Introducing SONS, a Tool for Operational Taxonomic Unit-Based Comparisons of Microbial Community Memberships and StructuresApplied and Environmental Microbiology, 72
A. Reysenbach, L. Giver, G. Wickham, N. Pace (1992)
Differential amplification of rRNA genes by polymerase chain reactionApplied and Environmental Microbiology, 58
X. Qiu, Liyou Wu, Heshu Huang, P. McDonel, A. Palumbo, J. Tiedje, Jizhong Zhou (2001)
Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based CloningApplied and Environmental Microbiology, 67
R. Sipos, Anna Székely, M. Palatinszky, Sára Révész, K. Márialigeti, M. Nikolausz (2007)
Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis.FEMS microbiology ecology, 60 2
K. Ashelford, N. Chuzhanova, J. Fry, A. Jones, A. Weightman (2005)
At Least 1 in 20 16S rRNA Sequence Records Currently Held in Public Repositories Is Estimated To Contain Substantial AnomaliesApplied and Environmental Microbiology, 71
W. Ludwig, O. Strunk, Ralf Westram, L. Richter, H. Meier, Yadhukumar, Arno Buchner, Tina Lai, Susanne Steppi, Gangolf Jobb, Wolfram Förster, Igor Brettske, Stefan Gerber, Anton Ginhart, Oliver Gross, Silke Grumann, Stefan Hermann, R. Jost, A. König, T. Liß, Ralph Lüssmann, M. May, Björn Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, Norbert Stuckmann, Alexander Vilbig, M. Lenke, T. Ludwig, A. Bode, K. Schleifer (2004)
ARB: a software environment for sequence data.Nucleic acids research, 32 4
B. Arezi, Weimei Xing, J. Sorge, H. Hogrefe (2003)
Amplification efficiency of thermostable DNA polymerases.Analytical biochemistry, 321 2
Q. Chou (1992)
Minimizing deletion mutagenesis artifact during Taq DNA polymerase PCR by E. coli SSB.Nucleic acids research, 20 16
Wen Liu, T. Marsh, Hans Cheng, L. Forney (1997)
Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNAApplied and Environmental Microbiology, 63
Marcelino Suzuki, S. Giovannoni (1996)
Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCRApplied and Environmental Microbiology, 62
M. Sogin, H. Morrison, J. Huber, D. Welch, Susan Huse, Phillip Neal, J. Arrieta, G. Herndl (2006)
Microbial diversity in the deep sea and the underexplored “rare biosphere”Proceedings of the National Academy of Sciences, 103
J. Cole, Benli Chai, Ryan Farris, Qiong Wang, A. Kulam-Syed-Mohideen, Donna McGarrell, A. Bandela, Erick Cardenas, G. Garrity, J. Tiedje (2006)
The ribosomal database project (RDP-II): introducing myRDP space and quality controlled public dataNucleic Acids Research, 35
Marcelino Suzuki, M. Rappé, S. Giovannoni (1998)
Kinetic Bias in Estimates of Coastal Picoplankton Community Structure Obtained by Measurements of Small-Subunit rRNA Gene PCR Amplicon Length HeterogeneityApplied and Environmental Microbiology, 64
Kleter (1998)
Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of Anogenital Human PapillomavirusesAm J Pathol, 153
P. Schloss, J. Handelsman (2005)
Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species RichnessApplied and Environmental Microbiology, 71
Bland Ewing, L. Hillier, M. Wendl, Philip Green (1998)
Base-calling of automated sequencer traces using phred. I. Accuracy assessment.Genome research, 8 3
C. Osborne, M. Galić, Parveen Sangwan, P. Janssen (2005)
PCR-generated artefact from 16S rRNA gene-specific primers.FEMS microbiology letters, 248 2
Robert Edgar (2004)
MUSCLE: a multiple sequence alignment method with reduced time and space complexityBMC Bioinformatics, 5
G. Muyzer, E. Waal, A. Uitterlinden (1993)
Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNAApplied and Environmental Microbiology, 59
Susan Huse, L. Dethlefsen, J. Huber, D. Welch, D. Relman, M. Sogin (2008)
Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag SequencingPLoS Genetics, 4
Sven Becker, P. Böger, Ralfh Oehlmann, A. Ernst (2000)
PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial CommunitiesApplied and Environmental Microbiology, 66
V. Farrelly, F. Rainey, E. Stackebrandt (1995)
Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial speciesApplied and Environmental Microbiology, 61
Summary PCR‐based surveys of microbial communities commonly use regions of the small‐subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)‐ and taxonomy‐based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.
Environmental Microbiology – Wiley
Published: May 1, 2009
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