Development and evaluation of a custom bait design based on 469 single-copy protein-coding genes for exon capture of isopods (Philosciidae: Haloniscus).

Danielle N Stringer,Steven J B Cooper,Simon M Tierney,Mohammad Javidkar,Christoph Mayer,Andrew D Austin,Karen Meusemann,Terry Bertozzi,Michelle T Guzik,Andreas Zwick,Steven Delean,Feng Zhang

doi:10.1371/journal.pone.0256861

Abstract

Transcriptome-based exon capture approaches, along with next-generation sequencing, are allowing for the rapid and cost-effective production of extensive and informative phylogenomic datasets from non-model organisms for phylogenetics and population genetics research. These approaches generally employ a reference genome to infer the intron-exon structure of targeted loci and preferentially select longer exons. However, in the absence of an existing and well-annotated genome, we applied this exon capture method directly, without initially identifying intron-exon boundaries for bait design, to a group of highly diverse Haloniscus (Philosciidae), paraplatyarthrid and armadillid isopods, and examined the performance of our methods and bait design for phylogenetic inference. Here, we identified an isopod-specific set of single-copy protein-coding loci, and a custom bait design to capture targeted regions from 469 genes, and analysed the resulting sequence data with a mapping approach and newly-created post-processing scripts. We effectively recovered a large and informative dataset comprising both short (<100 bp) and longer (>300 bp) exons, with high uniformity in sequencing depth. We were also able to successfully capture exon data from up to 16-year-old museum specimens along with more distantly related outgroup taxa, and efficiently pool multiple samples prior to capture. Our well-resolved phylogenies highlight the overall utility of this methodological approach and custom bait design, which offer enormous potential for application to future isopod, as well as broader crustacean, molecular studies.

Highlights

Phylogenetic and population genetic research on non-model organisms has largely relied on a limited selection of readily available genetic markers to address fundamental, and often difficult, evolutionary questions
Reduced representation approaches include RAD sequencing that targets unspecified loci associated with restriction enzyme sites [8], and those targeting highly specific loci with designed DNA or RNA baits, which are complementary to targeted DNA regions, including ultra-conserved element (UCE) sequencing [9], anchored hybrid enrichment (AHE) [10, 11], and transcriptome-based exon capture [12]
Transcriptome sequences were used without initially distinguishing intron-exon boundaries prior to bait design, which resulted in the recovery of numerous coding exons of various lengths, together with a considerable amount of non-coding flanking data

Summary

Introduction

Transcriptome-based exon capture, in particular, uses the transcript sequences assigned to clusters of orthologous groups (OGs) to infer custom baits, which target protein-coding exons across taxa, and is especially useful for generating sequence data from non-model organisms lacking reference genomes [7, 12,13,14,15,16,17,18] This method can further be employed to obtain genomic data from historical museum specimens, which may be critical for phylogenetics and taxonomic research, but typically contain degraded DNA, making it difficult to produce meaningful data using traditional Sanger sequencing techniques [19,20,21,22]. Becomes difficult when references are too divergent from the taxon of interest due to issues associated with aligning exons and since intron-exon structure may not be preserved in distantly related species [23]

Methods

Results

Conclusion