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Journal of Clinical Microbiology, November 2009, p. 3562-3568, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00973-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Direct 16S rRNA Gene Sequencing from Clinical Specimens, with Special Focus on Polybacterial Samples and Interpretation of Mixed DNA Chromatograms {triangledown}

Øyvind Kommedal,1,2* Kristine Kvello,2 Rune Skjåstad,1 Nina Langeland,3,4 and Harald G. Wiker1,2

Department of Microbiology and Immunology, Haukeland University Hospital, Bergen, Norway,1 Section for Microbiology and Immunology, the Gade Institute, University of Bergen, Bergen, Norway,2 Institute of Medicine, University of Bergen, Bergen, Norway,3 Department of Medicine, Haukeland University Hospital, Bergen, Norway4

Received 15 May 2009/ Returned for modification 23 July 2009/ Accepted 1 September 2009

RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Haukeland University Hospital, Paradisleitet 12, 5231 Paradis, Bergen, Norway. Phone: 47 48 19 26 19. Fax: 47 94 76 59 36. E-mail: oyvind.kommedal{at}isentio.com

{triangledown} Published ahead of print on 9 September 2009.

Journal of Clinical Microbiology, November 2009, p. 3562-3568, Vol. 47, No. 11
0095-1137/09/$08.00+0     doi:10.1128/JCM.00973-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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