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Accurate determination of microbial diversity from 454 pyrosequencing data


We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.

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Figure 1: OTU number as a function of percentage sequence difference for 90 pyrosequenced 16S rRNA gene clones of known sequence.
Figure 2: Proportion of sequences assigned to the correct OTU as a function of percentage sequence difference for pyrosequenced 16S rRNA gene clones of known sequence.

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We thank K. Ashelford (University of Liverpool) for the preliminary bioinformatics analysis of the sequencing data and for making the Mallard code publicly available, S. Huse (Marine Biological Laboratory, Woods Hole) for providing the quickdist algorithm and R. Knight for commenting on the manuscript. C.Q. is supported by a Lord Kelvin Glasgow University fellowship, W.T.S. by an Engineering and Physical Sciences Research Council Advanced Fellowship, R.J.D. by a Royal Society fellowship. The Natural Environment Research Council funded sequencing.

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Authors and Affiliations



T.P.C., R.J.D., I.M.H., C.Q. and W.T.S. designed the study. C.Q. devised algorithms and wrote software. A.L. and C.Q. performed analysis. R.J.D. and L.F.R. performed experiments. N.H. oversaw sequencing. T.P.C., R.J.D., N.H., I.M.H., A.L., C.Q., L.F.R. and W.T.S. wrote the paper.

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Correspondence to Christopher Quince.

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Quince, C., Lanzén, A., Curtis, T. et al. Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods 6, 639–641 (2009).

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