The Quantification of Representative Sequences pipeline for amplicon sequencing: case study on within‐population ITS1 sequence variation in a microparasite infecting Daphnia

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 http://code.google.com/p/quantification-representative-sequences.

A from‐benchtop‐to‐desktop workflow for validating HTS data and for taxonomic identification in diet metabarcoding studies

“…Due to PCR biases, the number of reads is a very poor estimator of abundance in metabarcoding studies (Elbrecht & Leese, ), and only presence/absence of MOTUs can be obtained with clustering‐based approaches, since different alleles coming from the same taxon are confounded in the same MOTU. Alternatively, several studies highlighted the pertinence of determining the number of distinct sequences belonging to the same taxon when assessing genetic diversity (González‐Tortuero et al., ; Shokralla et al., ) or prey biomass (Jo et al., ). In this study, thanks to rigorous filtering procedures we produced a robust data set of variants and contigs that is reliable and relatively free from false positives and artefacts.…”

Comparison of DNA metabarcoding and morphological identification for stream macroinvertebrate biodiversity assessment and monitoring

“…Whereas many previous amplicon-based HTS studies have focused on microbial or planktonic taxa (e.g., Kermarrec et al, 2013, González-Tortuero et al, 2015, Hajibabaei et al, 2012, Tang et al, 2012, Zimmermann et al, 2015), we assessed environmental samples of diverse size with representatives having relatively large body size (>0.6 mm) which allowed us to directly compare the HTS data from conventional morphological identification. The positive correlation between the abundance of morphological taxa and sequence read abundance of the metabarcoding taxa among the merged taxonomic groups suggests that metabarcoding could be used to quantify relative species abundance based on depth information.…”

Genetic diversity of two Daphnia-infecting microsporidian parasites, based on sequence variation in the internal transcribed spacer region

“…The bioinformatic analyses (and subsequent statistical tests) were run separately for each of the two parasite species. The raw datasets were analysed using the Quantification of Representative Sequences (QRS) pipeline [ 42 ]. The pipeline was run with default parameters, unless indicated otherwise.…”