Targeted Exon Sequencing (HybSeq) in Non-Model Organisms: Best Practices for Probe Design and Data Analysis with HybPiper

Targeted sequencing of exon regions and flanking introns (also known as HybSeq) has emerged as a powerful method for phylogenetics in non-model organisms, but designing probes and extracting sequence information without a reliable reference genome is challenging. Using examples from plants, we discuss strategies for designing probe sequences that can be used on a variety of phylogenetic scales-- from within a genus to an entire phylum. After sequencing, the construction of datasets suitable for phylogenetic analysis requires re-assembly of sequencing reads into contiguous sequences and extraction of exons. We describe HybPiper, a modular user-friendly Python pipeline for efficient generation of phylogenetic datasets from high-throughput sequencing reads. HybPiper can also assemble flanking intron regions, detect putative paralgous sequences, and includes data visualization scripts to quickly infer the success of a targeted sequencing project.