Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Restriction site-Associated DNA sequencing (RADseq) has great potential for genome-wide systematics studies of non-model organisms. However, accurately assembling RADseq reads into orthologous loci remains a major challenge in the absence of a reference genome. Traditional assembly pipelines cluster putative orthologous sequences based on a user-defined clustering threshold. Because improper clustering of orthologs is expected to affect results in downstream analyses, it is crucial to design pipelines for empirically optimizing the clustering threshold. While this issue has been largely discussed from a population genomics perspective, it remains understudied in the context of phylogenomics and coalescent species delimitation. To address this issue, we generated RADseq assemblies of representatives of the amphibian genera Discoglossus, Rana, Lissotriton and Triturus using a wide range of clustering thresholds. Particularly, we studied the effects of the intra-sample Clustering Threshold (iCT) and between-sample Clustering Threshold (bCT) separately, as both are expected to differ in multi-species data sets. The obtained assemblies were used for downstream inference of concatenation-based phylogenies, and multi-species coalescent species trees and species delimitation. The results were evaluated in the light of a reference genome-wide phylogeny calculated from newly generated Hybrid-Enrichment markers, as well as extensive background knowledge on the species’ systematics. Overall, our analyses show that the inferred topologies and their resolution are resilient to changes of the iCT and bCT, regardless of the analytical method employed. Except for some extreme clustering thresholds, all assemblies yielded identical, well-supported inter-species relationships that were mostly congruent with those inferred from the reference Hybrid-Enrichment data set. Similarly, coalescent species delimitation was consistent among similarity threshold values. However, we identified a strong effect of the bCT on the branch lengths of concatenation and species trees, with higher bCTs yielding trees with shorter branches, which might be a pitfall for downstream inferences of evolutionary rates. Our results suggest that the choice of assembly parameters for RADseq data in the context of shallow phylogenomics might be less challenging than previously thought. Finally, we propose a pipeline for empirical optimization of the iCT and bCT, implemented in optiRADCT, a series of scripts readily usable for future RADseq studies.
Authors 17
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Loïs Rancilhac corresponding
Uppsala University · Technische Universität Braunschweig
Affiliation as printed
Animal Ecology, Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden
Zoological Institute, Technische Universität Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany
Zoological Institute, Technische Universität Braunschweig, Mendelssohnstr. 4, 3806 Braunschweig, Germany
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Affiliation as printed
Département de biologie, Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, 1030 Avenue de la Médecine, G1V 0A6 Quebec, Canada
Département de biologie, Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, 100 Avenue de la Médecine, G1V 0A6 Quebec, Canada
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Affiliation as printed
Museum of Natural Sciences and Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA
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Affiliation as printed
Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands
Naturalis Biodiversity Center, P.O. Box 917, 2300 RA Leiden, The Netherlands
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Affiliation as printed
Institute of Environmental Sciences, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland
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Centre National de la Recherche Scientifique · École Pratique des Hautes Études · Centre d'Écologie Fonctionnelle et Évolutive · Institut de Recherche pour le Développement
Affiliation as printed
CEFE, CNRS, Univ Montpellier, EPHE, IRD, Montpellier, France
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Affiliation as printed
AHPAM (Association Herpétologique de Provence Alpes Méditerranée), Maison des associations, 384 route de Caderousse, 84100 Orange, France
AHPAM (Association Herpétologique de Provence Alpes Méditerranée), Maison des associations, 34 route de Caderousse, 4100 Orange, France
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Affiliation as printed
Alcedo Faune et Flore, 85, impasse Baslaval, 07110 Sanilhac, France
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Affiliation as printed
Grupo de Investigación en Bioloxía Evolutiva (GIBE), Departamento de Bioloxía, Facultade de Ciencias, Universidade da Coruña,15071 A Coruña, Spain
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Affiliation as printed
Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, ul. Gronostajowa 9, 30-387 Kraków, Poland
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Affiliation as printed
7, impasse de la Collinette, 13124 Peypin, France
7, impasse de la Collinette, 134 Peypin, France
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Affiliation as printed
Statipop-Scientific consulting, 34190 Cazilhac, France
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Affiliation as printed
GELIFES-Groningen Institute for Evolutionary Life Sciences, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
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Affiliation as printed
School of Biodiversity, One Health & Veterinary Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
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Affiliation as printed
School of Biodiversity, One Health & Veterinary Medicine, College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
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Affiliation as printed
LASER, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
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Technische Universität Braunschweig
Affiliation as printed
Zoological Institute, Technische Universität Braunschweig, Mendelssohnstr. 4, 38106 Braunschweig, Germany
Zoological Institute, Technische Universität Braunschweig, Mendelssohnstr. 4, 3806 Braunschweig, Germany
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