The quantification of representative sequences pipeline for amplicon sequencing : case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia

Gonzalez Tortuero, E; Rusek, J; Petrusek, A; Gießler, S; Lyras, D; Grath, S; Castro‐Monzón, F; Wolinska, J

Abstract

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 <a href="http://code.google.com/p/quantification-representative-sequences"> http://code.google.com/p/quantification-representative-sequences</a>

Citation

Gonzalez Tortuero, E., Rusek, J., Petrusek, A., Gießler, S., Lyras, D., Grath, S., …Wolinska, J. (2015). The quantification of representative sequences pipeline for amplicon sequencing : case study on within-population ITS1 sequence variation in a microparasite infecting Daphnia. Molecular Ecology Resources, 15(6), 1385-1395. https://doi.org/10.1111/1755-0998.12396

• Journal Article Type: Article
• Acceptance Date: Feb 19, 2015
• Online Publication Date: Feb 25, 2015
• Publication Date: Nov 1, 2015
• Deposit Date: Feb 22, 2021
• Journal: Molecular Ecology Resources
• Print ISSN: 1755-098X
• Electronic ISSN: 1755-0998
• Publisher: Wiley
• Volume: 15
• Issue: 6
• Pages: 1385-1395
• DOI: https://doi.org/10.1111/1755-0998.12396
• Publisher URL: https://doi.org/10.1111/1755-0998.12396
• Related Public URLs: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1755-0998
• Additional Information: Funders : German Research Foundation (DFG‐SPP 1399 and DFG‐SNF);European Science Foundation;Czech Science Foundation;German Science Foundation (DFG); Grant Number: WO 1587/2‐2, WO 1587/3‐1; Grant Number: EEF/10/E022; Grant Number: ME 3134/4‐1