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

Abstract

Despite the increasing use of high-throughput sequencing in phylogenetics, many phylogenetic relationships remain difficult to resolve because of conflict between gene trees and species trees. Selection of different types of markers (i.e. protein-coding exons, non-coding introns, ultraconserved elements) is becoming important to alleviate these phylogenomic challenges. For evolutionary studies in frogs, we introduce the new publicly available FrogCap suite of genomic resources, which is a large and flexible collection of probes corresponding to ~15,000 markers that unifies previous frog sequencing work. FrogCap is designed to be modular, such that subsets of markers can be selected based on the phylogenetic scale of the intended study. FrogCap uses a variety of molecular marker types that include newly obtained exons and introns, previously sequenced UCEs, and Sanger-sequencing markers, which span a range of alignment lengths (100-12,000 base pairs). We tested three probe sets from FrogCap using 105 samples across five phylogenetic scales, comparing probes designed using a consensus-or genome-based approach. We also tested the effects of using different bait kit sizes on depth of coverage and missing data. We found that larger bait kits did not result in lowered depth of coverage or increased missing data. We also found that sensitivity, specificity, and missing data are not related to genetic distance in the consensus-based probe design, suggesting that this approach has greater success and overcomes a major hurdle in probe design. We observed sequence capture success (in terms of missing data, quantity of sequence data, recovered marker length, and number of informative sites) and compared them at all phylogenetic scales. The incorporation of different molecular marker types allowed recovery of the variation required for resolving difficult phylogenetic relationships and for performing population genetic studies. Altogether, FrogCap is a valuable and adaptable resource for performing high-throughput sequencing projects across variable timescales.

Gene flow creates a mirage of cryptic species in a Southeast Asian spotted stream frog complex

“…Accordingly, we undertook the present study, using a newly developed target‐capture protocol specifically designed for anurans (FrogCap; Hutter et al., 2019) and obtained more than 12,000 informative loci consisting of exons, introns, and ultraconserved elements (UCEs) from representative populations across the distributional range of P. picturata to determine whether deep divergences among clades and observed geographically structured genetic variation correspond with statistically defensible cryptic species boundaries. Specifically, we test for gene flow among genetically structured populations and assess its effects on inferences of phylogenetic and species boundaries to determine whether species delimitation based on phylogenetic arrangement and genetic divergence can accurately estimate cryptic species diversity.…”

Improving sustainable use of genetic resources in biodiversity archives

“…We first conducted a preliminary experiment to determine if coarse visual assessment of tissue mass (i.e., the "eyeball" approach to tissue quantification used by most biodiversity collections staff) is capable of sampling tissues that result in consistent DNA yield which are sufficient for modern downstream DNA sequencing applications. The concentration and amount of DNA required for sequencing depends on the sequencing method used, ranging from less than 10 ng of DNA for Sanger sequencing a single DNA fragment to 500 ng for Illumina Truseq-style library preparation (Hutter et al, 2019) to over 1,000 ng for high coverage sequencing of an entire vertebrate genome via the Illumina platform (Arbor Biosciences, 2019). Because 1,000 ng is at the high end of the amount used for standard sequencing methods applied to typical vertebrate genomes (including whole genome sequencing and popular methods such as RADseq and probe capture), we used this amount as our threshold for establishing extraction success.…”

New targets acquired: improving locus recovery from the Angiosperms353 probe set

“…Such kits typically require a larger number of baits to encompass the sequence diversity potentially found between samples at each locus. Larger kits are more costly (Hutter et al, 2019;Couvreur et al, 2019), and therefore to keep costs manageable universal bait kits balance the number of baits synthesised, and hence bait sequence diversity for each locus, against the total number of RNA baits strictly required to fully capture diversity at each locus. Incomplete representation of sample sequence diversity in the synthesised baits is in part compensated for by the high affinity of the biochemical interaction in the hybridisation reaction binding the RNA-bait to the DNA-target.…”