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Applied and Environmental Microbiology, December 2005, p. 8966-8969, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8966-8969.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample{dagger}

Silvia G. Acinas, Ramahi Sarma-Rupavtarm, Vanja Klepac-Ceraj, and Martin F. Polz*

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 31 March 2005/ Accepted 26 August 2005

The contribution of PCR artifacts to 16S rRNA gene sequence diversity from a complex bacterioplankton sample was estimated. Taq DNA polymerase errors were found to be the dominant sequence artifact but could be constrained by clustering the sequences into 99% sequence similarity groups. Other artifacts (chimeras and heteroduplex molecules) were significantly reduced by employing modified amplification protocols. Surprisingly, no skew in sequence types was detected in the two libraries constructed from PCR products amplified for different numbers of cycles. Recommendations for modification of amplification protocols and for reporting diversity estimates at 99% sequence similarity as a standard are given.

* Corresponding author. Mailing address: Massachusetts Institute of Technology, 48-421, 77 Massachusetts Avenue, Cambridge, MA 02139. Phone: (617) 253-7128. Fax: (617) 258-8850. E-mail: mpolz{at}mit.edu.

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

Applied and Environmental Microbiology, December 2005, p. 8966-8969, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8966-8969.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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