Exploring Angiosperms353: developing and applying a universal toolkit for flowering plant phylogenomics

Special Issue Introduction. Target enrichment represents a useful, cost-effective method for researchers working on the phylogenomics of non-model organisms (e.g., Cronn et al., 2012; Hale et al., 2020). The ability to sequence a customizable predefined genomic subset for several dozens or even hundreds of taxa allows in-depth analyses and the testing of phylogenetic hypotheses in ways that were not previously possible (reviewed in McKain et al., 2018). The most popular methods for targeted sequencing of genomic loci in phylogenomics include (long-)amplicon sequencing (Rothfels et al., 2017) and hybridization capture (Mandel et al., 2014; Weitemier et al., 2014). Targeted amplicon sequencing is based on single-fragment PCR amplification or by using multiplexing methods such as a microfluidic PCR-based amplification of multiple pre-selected genomic regions (e.g., Zhang and Ozdemir, 2009; Ho et al., 2014), which can then be pooled and sequenced. Massively parallel amplicon sequencing was first used in medical diagnostics (Turner et al., 2009) and was later applied to metazoan phylogenetics (Bybee et al., 2011; O’Neill et al., 2013). Microfluidic PCR and long-amplicon sequencing were subsequently applied in plant systematics (Uribe-Convers et al., 2014, 2016; Gostel et al., 2015). Amplicon-based methods can be time consuming as they require careful optimization and validation of primers. These methods are also susceptible to many of the common problems in PCR (such as nonspecific products, inability to amplify large loci in their entirety, or simply no products). Recently, amplicon approaches have been largely supplanted by hybridization-based targeted enrichment, which allows for relatively rapid probe design with reference to a few related transcriptomes or genomes, and allows simultaneous and efficient recovery of many hundreds of genes.