The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia.
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 Ecosystem Research, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301, 12587, Berlin, Germany.|Berlin Centre for Genomics in Biodiversity Research (BeGenDiv), Königin-Luise-Straße 6-8, 14195, Berlin, Germany.|Department of Biology II, Ludwig Maximilians University, Großhaderner Straße 2, 82512, Planegg-Martinsried, Germany.
E
E
González-Tortuero
Department of Biology II, Ludwig Maximilians University, Großhaderner Straße 2, 82512, Planegg-Martinsried, Germany.
J
J
Rusek
Department of Ecology, Faculty of Science, Charles University in Prague, Viničná 7, 128 44, Prague, Czech Republic.
A
A
Petrusek
Department of Biology II, Ludwig Maximilians University, Großhaderner Straße 2, 82512, Planegg-Martinsried, Germany.
S
S
Gießler
Department of Biology II, Ludwig Maximilians University, Großhaderner Straße 2, 82512, Planegg-Martinsried, Germany.
D
D
Lyras
Department of Biology II, Ludwig Maximilians University, Großhaderner Straße 2, 82512, Planegg-Martinsried, Germany.
S
S
Grath
Department of Ecosystem Research, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301, 12587, Berlin, Germany.
F
F
Castro-Monzón
Department of Ecosystem Research, Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Müggelseedamm 301, 12587, Berlin, Germany.
J
J
Wolinska
D000818:Animals; D019295:Computational Biology / Q000379:methods*; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D003621:Daphnia / Q000469:parasitology*; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D050298:Mesomycetozoea / Q000145:classification* / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012984:Software
D021903:DNA, Ribosomal Spacer
Caullerya mesnili; ITS1 region; Ichthyosporea; Next Generation Sequencing; amplicon sequencing; pipeline