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Double-digest RAD-sequencing: do pre- and post-sequencing protocol parameters impact biological results?

Tristan Cumer1,2, Charles Pouchon1, Frédéric Boyer1

  • 1Laboratoire D'Ecologie Alpine (LECA), Univ. Grenoble Alpes, Univ. Savoie Mont Blanc, CNRS, 38000, Grenoble, France.

Molecular Genetics and Genomics : MGG
|January 20, 2021
PubMed
Summary

Pre-sequencing choices significantly affect double-digest RAD-sequencing (ddRAD-seq) data quality and SNP recovery. Post-sequencing parameters influence loci and SNP calling but minimally impact population genetics analyses, confirming ddRAD-seq robustness.

Keywords:
BioinformaticsLaboratory protocolMolecular biologyNext-generation sequencing

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Area of Science:

  • Population genetics
  • Genomics
  • Molecular biology

Background:

  • Next-generation sequencing advanced population genetics research.
  • Restriction enzyme-based genotyping, like double-digest RAD-sequencing (ddRAD-seq), is increasingly used.
  • The impact of ddRAD-seq parameters on downstream analyses is not well-documented.

Purpose of the Study:

  • To investigate the effects of pre- and post-sequencing parameters on ddRAD-seq results.
  • To assess the sensitivity and robustness of ddRAD-seq for population genetics studies.
  • To evaluate parameter impacts on two distinct biological systems: butterflies and beech populations.

Main Methods:

  • Utilized double-digest RAD-sequencing (ddRAD-seq) on butterfly (Coenonympha sp.) and common beech (Fagus sylvatica) populations.
  • Varied pre-sequencing (DNA quantity, PCR cycles) and post-sequencing (clustering, coverage thresholds) parameters.
  • Performed replication analyses to confirm reproducibility.

Main Results:

  • Pre-sequencing parameters significantly influenced read counts, SNP numbers, unique alleles, and heterozygosity.
  • Post-sequencing parameters affected loci reconstruction and SNP calling but had minor effects on genetic differentiation, admixture, and demographic inferences.
  • Replication analyses validated the reproducibility of the ddRAD-seq procedure.

Conclusions:

  • ddRAD-seq data are sensitive to pre-sequencing protocols, affecting initial data output.
  • Post-sequencing parameters have a limited impact on higher-level population genetic inferences.
  • ddRAD-seq is a robust method for population genetics research despite parameter sensitivities.