Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics

Abstract

Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. •

Methods and results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. •

Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Keywords: Hyb-Seq; genome skimming; nuclear loci; phylogenomics; species tree; target enrichment.

Fig. 1.

Histogram of exon sequence divergence between the species used for probe design, Asclepias syriaca, and four other species: the most divergent species of Asclepias, A. flava; another member of Asclepiadinae (Asclepiadeae: Asclepiadoideae), Calotropis procera; a member of Gonolobinae (Asclepiadeae: Asclepiadoideae), Matelea cynanchoides; and a member of a different subfamily, Catharanthus roseus (Rauvolfioideae). Note that a maximum sequence divergence of 75% was allowed for BLAT and that exons with >10% divergence were less likely to be observed in Calotropis and Matelea because they were less likely to be enriched by the probes, while the Catharanthus data were from transcriptome sequences of multiple tissues and not subject to target enrichment bias.

Fig. 2.

Comparison of the species tree of Asclepias based on 761 putatively single-copy loci and the whole plastome phylogeny. The MP-EST tree is shown at left, and the difference between this topology and that recovered through an analysis of the concatenated nuclear gene data set is indicated by the red arrow. Solid lines connect each species to its placement in the plastome phylogeny (right). Values near the branches are bootstrap support values (* = 100%). Colors reflect the plastid clades of Fishbein et al. (2011): temperate North America (green), unplaced (orange), highland Mexico (purple), series Incarnatae sensu Fishbein (pink), Sonoran Desert (blue), and outgroup (black).