DOI: 10.5061/dryad.n2z34tn1z
title: Supplementary materials for: Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians

description: 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.
description: # Data from: Exploring the impact of read clustering thresholds on
 RADseq-based systematics: an empirical example from European amphibians.
 --- This repository contains genetic sequences obtained from
 Hybrid-Enrichment and RAD sequencing protocols of the amphibian genera
 *Discoglossus*, *Lissotriton*, *Rana* and *Triturus*, as well as
 phylogenetic trees inferred from the RADseq data. This data was generated
 for the manuscript "Exploring the impact of read clustering
 thresholds on RADseq-based systematics: an empirical example from European
 amphibians.", in which we tested the influence of the clustering
 threshold used to assemble RADseq data on downstream phylogenetic
 inferences. Details on the data generation and analyses can be found in
 the manuscript and related supplementary materials. ## Description of the
 data and file structure The repository includes the supplementary
 materials of the manuscript as well as two directories containing data:
 ***Hybrid-Enrichment*** - this directory contains single alignments for
 each of the Hybrid-Enrichment markers in fasta format. The data is
 provided separately for each of the four genera studied (*Discoglossus*,
 *Lissotriton*, *Rana*, *Triturus*). ***RADseq*** - this directory contains
 RADseq data assemblies and summary statistics, as well as downstream
 phylogenetic trees. The data is organized as follow: * *Assemblies* -
 contains complete loci sequences and SNP matrices, as well as spreadsheets
 with assembly metrics. Divided into "iCT" (assemblies produced
 with 23 different intra-sample Clustering Threshold [iCT] and a fixed
 between-samples Clustering Threshold [bCT]) and "bCT"
 (assemblies produced with a fixed iCT and 23 different bCTs). Both iCT and
 bCT are further divided in four sub-directories corresponding to the four
 genera: Discoglossus, Lissotriton, Rana, Triturus). * *Trees* -
 Phylogenetic trees inferred from the aforementionned assemblies. Divided
 into "iCT" (RAxML concatenation trees inferred from the
 assemblies with different iCTs) and "bCT" (RAxML concatenation
 trees and Tetrad species trees inferred from the assemblies with different
 bCTs). ## Code/Software Scripts used to generate this data are available
 at
 [https://github.com/rancilhac/optiRADCT](https://github.com/rancilhac/optiRADCT)