G2545a hybridization oven

We sheared gDNA to 200–300 bp fragments using a Qsonica 800R sonicator (Millard & Muriel Jacobs Genetics and Genomics Lab, CalTech; Qsonica, LLC., Newton, CT, USA). We chose this relatively shorter fragment size to maximize the number of on-target reads (Hodges et al., 2007 (link)). We prepared Illumina libraries following the protocol of Sass et al. (2016) (link) at the Evolutionary Genetics Laboratory at UC, Berkeley. We quantified libraries using a Qubit Fluorometer and pooled them at equimolar ratios and checked fragment sizes with an Agilent Bioanalyzer (Agilent Technologies, USA). We carried out TSC of plastid genomes following Hodges et al. (2009) (link). Briefly, we hybridized libraries at 65˚C for 65 h in an Agilent G2545A Hybridization Oven, eluted hybridized DNA, enriched the pool via PCR amplification (Phusion® High-Fidelity DNA Polymerase; ThermoFisher Scientific, Waltham, Massachusetts, USA), and then sequenced in a lane of 100 bp paired-end reads on an Illumina HiSeq2000 at the QB3 Vincent J Coates Genomic Sequencing Facility at UC, Berkeley (http://qb3.berkeley.edu/gsl). We cleaned the captured read data as above, with the only difference being that we skipped the removal step of low complexity reads, as plastid genomes often contain low-complexity, AT-rich intergenic regions.