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

The nuclear genome is a largely untapped resource for plant systematics and evolution studies that can readily be accessed through target enrichment and high throughput sequencing (Hyb-Seq). Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were utilized to design target capture probes for 3385 exons from 768 genes (ca. 1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of ten Asclepias species and two related genera resulted in at least partial assembly of 92.6% of exons and 99.7% of genes on average and a total average assembly length over 2 Mbp. Whole plastomes and nrDNA cistrons were assembled from off-target reads. Phylogenomic analyses demonstrated signal conflict among genomes. This protocol results in targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of the high copy fraction of genome, to efficiently produce genome-scale data sets for phylogenomics in plants.