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Applied and Environmental Microbiology, August 2009, p. 5227-5236, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00592-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Comparison of Species Richness Estimates Obtained Using Nearly Complete Fragments and Simulated Pyrosequencing-Generated Fragments in 16S rRNA Gene-Based Environmental Surveys{triangledown} ,{dagger}

Noha Youssef,1 Cody S. Sheik,2 Lee R. Krumholz,2 Fares Z. Najar,3 Bruce A. Roe,3 and Mostafa S. Elshahed1*

Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74074,1 Department of Botany and Microbiology and Institute for Energy and the Environment, University of Oklahoma, Norman, Oklahoma 73019,2 Department of Chemistry and Biochemistry and the Advanced Center for Genome Technology, University of Oklahoma, Norman, Oklahoma 730193

Received 11 March 2009/ Accepted 19 June 2009

Pyrosequencing-based 16S rRNA gene surveys are increasingly utilized to study highly diverse bacterial communities, with special emphasis on utilizing the large number of sequences obtained (tens to hundreds of thousands) for species richness estimation. However, it is not yet clear how the number of operational taxonomic units (OTUs) and, hence, species richness estimates determined using shorter fragments at different taxonomic cutoffs correlates with the number of OTUs assigned using longer, nearly complete 16S rRNA gene fragments. We constructed a 16S rRNA clone library from an undisturbed tallgrass prairie soil (1,132 clones) and used it to compare species richness estimates obtained using eight pyrosequencing candidate fragments (99 to 361 bp in length) and the nearly full-length fragment. Fragments encompassing the V1 and V2 (V1+V2) region and the V6 region (generated using primer pairs 8F-338R and 967F-1046R) overestimated species richness; fragments encompassing the V3, V7, and V7+V8 hypervariable regions (generated using primer pairs 338F-530R, 1046F-1220R, and 1046F-1392R) underestimated species richness; and fragments encompassing the V4, V5+V6, and V6+V7 regions (generated using primer pairs 530F-805R, 805F-1046R, and 967F-1220R) provided estimates comparable to those obtained with the nearly full-length fragment. These patterns were observed regardless of the alignment method utilized or the parameter used to gauge comparative levels of species richness (number of OTUs observed, slope of scatter plots of pairwise distance values for short and nearly complete fragments, and nonparametric and parametric species richness estimates). Similar results were obtained when analyzing three other datasets derived from soil, adult Zebrafish gut, and basaltic formations in the East Pacific Rise. Regression analysis indicated that these observed discrepancies in species richness estimates within various regions could readily be explained by the proportions of hypervariable, variable, and conserved base pairs within an examined fragment.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Oklahoma State University, 1110 S. Innovation Way, Stillwater, OK 74074. Phone: (405) 744-3005. Fax: (405) 744-1112. E-mail: Mostafa{at}okstate.edu

{triangledown} Published ahead of print on 26 June 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2009, p. 5227-5236, Vol. 75, No. 16
0099-2240/09/$08.00+0     doi:10.1128/AEM.00592-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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