Next generation sequencing (NGS) platforms are replacing traditional molecular biology protocols like cloning and Sanger sequencing. However, accuracy of NGS platforms has rarely been measured when quantifying relative frequencies of genotypes or taxa within populations. Here we developed a new bioinformatic pipeline (QRS) that pools similar sequence variants and estimates their frequencies in NGS data sets from populations or communities. We tested whether the estimated frequency of representative sequences, generated by 454 amplicon sequencing, differs significantly from that obtained by Sanger sequencing of cloned PCR products. This was performed by analysing sequence variation of the highly variable first internal transcribed spacer (ITS1) of the ichthyosporean Caullerya mesnili, a microparasite of cladocerans of the genus Daphnia. This analysis also serves as a case example of the usage of this pipeline to study within-population variation. Additionally, a public Illumina data set was used to validate the pipeline on community-level data. Overall, there was a good correspondence in absolute frequencies of C. mesnili ITS1 sequences obtained from Sanger and 454 platforms. Furthermore, analyses of molecular variance (amova) revealed that population structure of C. mesnili differs across lakes and years independently of the sequencing platform. Our results support not only the usefulness of amplicon sequencing data for studies of within-population structure but also the successful application of the QRS pipeline on Illumina-generated data. The QRS pipeline is freely available together with its documentation under GNU Public Licence version 3 at http://code.google.com/p/quantification-representative-sequences.

Department of Biology 2 Ludwig Maximilians University Großhaderner Straße 2 82512 Planegg Martinsried Germany

Department of Ecology Faculty of Science Charles University Prague Viničná 7 128 44 Prague Czech Republic

Department of Ecosystem Research Leibniz Institute of Freshwater Ecology and Inland Fisheries Königin Luise Straße 6 8 14195 Berlin Germany Department of Biology 2 Ludwig Maximilians University Großhaderner Straße 2 82512 Planegg Martinsried Germany

Department of Ecosystem Research Leibniz Institute of Freshwater Ecology and Inland Fisheries Müggelseedamm 301 12587 Berlin Germany

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