Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales

Abstract

Background: To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species.

Results: We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified.

Conclusions: Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life.

Figure 1

An overall work flow of this study. The Tamias phylogenetic tree is modified from [13] by replacing the outgroup species with T.striatus. The Tamias species that were not under investigation in the present study are not shown.

Figure 2

Specificity of exon capture in the four chipmunk species. Specificity denotes the percentage of cleaned reads aligned within the targets over the total reads aligned to the consensus assemblies. The number of individual libraries barcoded and pooled on the same array varies for different species, while the overall specificity among the 31 specimens is similar. Each column represents an individual library.

Figure 3

Sequence coverage of target exons enriched in the four chipmunk species. The columns show the distribution of average base coverage per exon. Coverage is shown on the X-axis, binned percentage of exons at each coverage on the Y-axis (left). The green line and right Y-axis show the cumulative coverage as a percent of total exons.

Figure 4

Coverage-exon distance distributions. Exons that ranged between 201–600 bp were used for generating the plot. Each target exon was split into 20-bp bins depicted by the red bar (X-axis). The average base coverage within each exon bin is shown on the Y-axis.

Figure 5

Capture efficiency vs. sequence divergence. The captured reads from all species libraries (Tamias alpinus, T. amoenus, T. ruficaudus, and T. striatus) derived from T. alpinus exon and Ictidomys tridecemlineatus genomic interval targets were combined to generate the plot. Outliers are not shown in the plot. Capture efficiency is represented by normalized base coverage. Sequence divergence between the targets and the corresponding in-target assemblies (X-axis) were placed in 1% bins.