FrogCap: A modular sequence capture probe‐set for phylogenomics and population genetics for all frogs, assessed across multiple phylogenetic scales

Hutter, Carl R.; Cobb, Kerry A.; Portik, Daniel M.; Travers, Scott L.; Wood, Perry L.; Brown, Rafe M.

doi:10.1111/1755-0998.13517

Cited by 24 publications

(19 citation statements)

References 114 publications

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“…These contrasting results demonstrate the additional challenges of comparing GBS and similar datasets collected from different studies. Targeted sequence capture approaches may be preferable to increase comparability across studies, though they remain more expensive and require more initial effort to design probe kits ( Harvey et al 2016 ; Singhal et al 2017a ; Hutter et al 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Predictors of genomic diversity within North American squamates

Larkin

Myers

Carstens

et al. 2023

Journal of Heredity

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Comparisons of intraspecific genetic diversity across species can reveal the roles of geography, ecology, and life history in shaping biodiversity. The wide availability of mitochondrial DNA (mtDNA) sequences in open-access databases makes this marker practical for conducting analyses across several species in a common framework, but patterns may not be representative of overall species diversity. Here, we gather new and existing mtDNA sequences and genome-wide nuclear data (genotyping-by-sequencing; GBS) for 30 North American squamate species sampled in the Southeastern and Southwestern United States. We estimated mtDNA nucleotide diversity for two mtDNA genes, COI (22 species alignments; average 16 sequences) and cytb (22 species; average 58 sequences), as well as nuclear heterozygosity and nucleotide diversity from GBS data for 118 individuals (30 species; four individuals and 6,820–44,309 loci per species). We showed that nuclear genomic diversity estimates were highly consistent across individuals for some species, while other species showed large differences depending on the locality sampled. Range size was positively correlated with both cytb diversity (Phylogenetically Independent Contrasts: R 2 = 0.31, p = 0.007) and GBS diversity (R 2 = 0.21; p = 0.006), while other predictors differed across the top models for each dataset. Mitochondrial and nuclear diversity estimates were not correlated within species, although sampling differences in the data available made these datasets difficult to compare. Further study of mtDNA and nuclear diversity sampled across species’ ranges is needed to evaluate the roles of geography and life history in structuring diversity across a variety of taxonomic groups.

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Section: Discussionmentioning

confidence: 99%

Predictors of genomic diversity within North American squamates

Larkin

Myers

Carstens

et al. 2023

Journal of Heredity

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“…As a comparative basis for evaluating the quality of phylogenetic analyses performed with RADseq data, nuclear loci were also sampled through a Hybrid-Enrichment protocol, using a modified version of the FrogCap sequence capture probe set (Hutter et al 2022). In brief, our goal was to create a set of universal amphibian markers that could be targeted using a single set of probes.…”

Section: Hybrid-enrichment Analysesmentioning

confidence: 99%

“…In brief, our goal was to create a set of universal amphibian markers that could be targeted using a single set of probes. We started by using the FrogCap markers (Hutter et al 2022; https://github.com/chutter/FrogCap-Sequence-Capture) that were successfully captured broadly across Anura in the data from Hutter et al (2022). Next, these markers were matched against the Salamander Ambystoma mexicanum genome (Keinath et al 2018) and retained if they matched with ≥65% similarity.…”

Section: Hybrid-enrichment Analysesmentioning

confidence: 99%

“…To further investigate these issues, we generated phylogenomic data sets of four European amphibian genera (Discoglossus, Lissotriton, Rana, Triturus), using double-digest RADseq (ddRADseq; Peterson et al 2012) as well as Hybrid-Enrichment Capture (HE, based on the FrogCap probe set; Hutter et al 2022). As the latter consists of genome-verified single-copy markers exempt of the biases of de novo clustering, we use it as a reference to evaluate the accuracy of inferences based on RADseq de novo assemblies produced using various CT values.…”

Section: Introductionmentioning

confidence: 99%

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Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians

Rancilhac

Sylvestre

Hutter

et al. 2023

Preprint

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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.

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“…The first advantage of the FLc-Capture method is that it saves both cost and time compared to commercially synthesized probe sets. Previous transcriptome-based capture studies normally used transcriptome data to design capture probes and ordered those probe sets from commercial companies (e.g., Bi et al, 2012;Bragg et al, 2016;Portik et al, 2016;Quek et al, 2020;Peakall et al, 2021;Hutter et al, 2022). The whole process to synthesize a custom probe kit typically takes several weeks and costs $2,400-$5,000, depending on the supplier (Peñalba et al, 2014).…”

Section: The Merits Of Full-length Cdna-capturementioning

confidence: 99%

Simultaneously collecting coding and non-coding phylogenomic data using homemade full-length cDNA probes, tested by resolving the high-level relationships of Colubridae

Liang²,

Zhang³

2022

Front. Ecol. Evol.

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Resolving intractable phylogenetic relationships often requires simultaneously analyzing a large number of coding and non-coding orthologous loci. To gather both coding and non-coding data, traditional sequence capture methods require custom-designed commercial probes. Here, we present a cost-effective sequence capture method based on homemade probes, to capture thousands of coding and non-coding orthologous loci simultaneously, suitable for all organisms. This approach, called “FLc-Capture,” synthesizes biotinylated full-length cDNAs from mRNA as capture probes, eliminates the need for costly commercial probe design and synthesis. To demonstrate the utility of FLc-Capture, we prepared full-length cDNA probes from mRNA extracted from a common colubrid snake. We performed capture experiments with these homemade cDNA probes and successfully obtained thousands of coding and non-coding genomic loci from 24 Colubridae species and 12 distantly related snake species of other families. The average capture specificity of FLc-Capture across all tested snake species is 35%, similar to the previously published EecSeq method. We constructed two phylogenomic data sets, one including 1,075 coding loci (∼817,000 bp) and the other including 1,948 non-coding loci (∼1,114,000 bp), to study the phylogeny of Colubridae. Both data sets yielded highly similar and well-resolved trees, with 85% of nodes having >95% bootstrap support. Our experimental tests show that FLc-Capture is a flexible, fast, and cost-effective sequence capture approach for simultaneously gathering coding and non-coding phylogenomic data sets to study intractable phylogenetic questions. We hope that this method will serve as a new data collection tool for evolutionary biologists working in the era of phylogenomics.

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FrogCap: A modular sequence capture probe‐set for phylogenomics and population genetics for all frogs, assessed across multiple phylogenetic scales

Cited by 24 publications

References 114 publications

Predictors of genomic diversity within North American squamates

Predictors of genomic diversity within North American squamates

Exploring the impact of read clustering thresholds on RADseq-based systematics: an empirical example from European amphibians

Simultaneously collecting coding and non-coding phylogenomic data using homemade full-length cDNA probes, tested by resolving the high-level relationships of Colubridae

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