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Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?

View ORCID ProfileTristan Cumer, Charles Pouchon, Frédéric Boyer, Glenn Yannic, Delphine Rioux, Aurélie Bonin, Thibaut Capblancq
doi: https://doi.org/10.1101/445122
Tristan Cumer
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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  • ORCID record for Tristan Cumer
Charles Pouchon
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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Frédéric Boyer
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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Glenn Yannic
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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Delphine Rioux
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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Aurélie Bonin
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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Thibaut Capblancq
1Univ. Grenoble Alpes, Univ. Savoie Mont-Blanc, CNRS, LECA, Grenoble, France
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  • For correspondence: thibaut.capblancq@univ-grenoble-alpes.fr
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ABSTRACT

  1. Next-generation sequencing technologies have opened a new era of research in genomics. Among these, restriction enzyme-based techniques such as restriction-site associated DNA sequencing (RADseq) or double-digest RAD-sequencing (ddRADseq) are now widely used in many population genomics fields. From DNA sampling to SNP calling, both wet and dry protocols have been discussed in the literature to identify key parameters for an optimal loci reconstruction.

  2. The impact of these parameters on downstream analyses and biological results drawn from RADseq or ddRADseq data has however not been fully explored yet. In this study, we tackled this issue by investigating the effects of ddRADseq laboratory (i.e. wet protocol) and bioinformatics (i.e. dry protocol) settings on loci reconstruction and inferred biological signal at two evolutionary scale using two systems: a complex of butterfly species (Coenonympha sp.) and populations of Common beech (Fagus sylvatica).

  3. Results suggest an impact of wet protocol parameters (DNA quantity, number of PCR cycles during library preparation) on the number of recovered reads and SNPs, the number of unique alleles and individual heterozygosity. We also found that bioinformatic settings (i.e. clustering and minimum coverage thresholds) impact loci reconstruction (e.g. number of loci, mean coverage) and SNP calling (e.g. number of SNPs, heterozygosity). We however do not detect an impact of parameter settings on three types of analysis performed with ddRADseq data: measure of genetic differentiation, estimation of individual admixture, and demographic inferences. In addition, our work demonstrates the high reproducibility and low rate of genotyping inconsistencies of the ddRADseq protocol.

  4. Thus, our study highlights the impact of wet parameters on ddRADseq protocol with strong consequences on experimental success and biological conclusions. Dry parameters affects loci reconstruction and descriptive statistics but not biological conclusion for the two studied systems. Overall, this study illustrates, with others, the relevance of ddRADseq for population and evolutionary genomics at the inter- or intraspecific scales.

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  • ↵* co-first authors

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted October 16, 2018.
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Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?
Tristan Cumer, Charles Pouchon, Frédéric Boyer, Glenn Yannic, Delphine Rioux, Aurélie Bonin, Thibaut Capblancq
bioRxiv 445122; doi: https://doi.org/10.1101/445122
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Double-digest RAD-sequencing: do wet and dry protocol parameters impact biological results?
Tristan Cumer, Charles Pouchon, Frédéric Boyer, Glenn Yannic, Delphine Rioux, Aurélie Bonin, Thibaut Capblancq
bioRxiv 445122; doi: https://doi.org/10.1101/445122

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