Supplementary materials for: Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians
Data files
Apr 26, 2023 version files 875.31 MB
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Hybrid-enrichment.zip
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RADseq.zip
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README.md
Jun 24, 2024 version files 875.31 MB
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Hybrid-enrichment.zip
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RADseq.zip
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README.md
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.
README: 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