Angiosperms353 Hyb-seq of Leycesteria formosa
DNA was extracted using a modified CTAB protocol (Doyle and Doyle, 1987) mostly from silica dried leaf material. Sample concentration was assessed using a Qubit 2.0 (Life Technologies, Carlsbad, California, USA), and, when necessary, samples were re-concentrated to meet the minimum recommended target mass of 100 ng of genomic DNA in a 52.5 uL reaction volume (myBaits Hybridization Capture for Targeted NGS version 4.01, Daicel Arbor Biosciences, Ann Arbor, Michigan, USA). We sheared samples using a Covaris M220 Focused-ultrasonicator (Covaris, Woburn, Massachusetts, USA) following the manufacturers protocol for 550 bp insert size. Fragment lengths were assessed using a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, California, USA). We size selected fragments via a SPRI-bead cleanup with Ampure XP magnetic beads (Beckman Coulter Life Sciences, Indianapolis, Indiana, USA). Finally, end repair and A-tailing was completed using KAPA End Repair & A-Tailing Buffer and Enzyme (Roche, Basel, Switzerland) following the manufacturers instructions. Libraries were constructed using a KAPA Hyper Prep Kit with PCR Library Amplification/Illumina series KR0961 version 5.16 (Roche, Basel, Switzerland) following the manufacturers instructions, but with 1/3 reaction volumes at all steps. After adapter ligation, samples were amplified for 12 cycles with a target of 100 ng DNA for hybridization. After amplification, we combined samples into eight equimolar hybridization reaction pools. To minimize possible enrichment biases due to shared genomic variation (e.g., GC content, locus dropout), we pooled our taxa based on presumed phylogenetic relationships. We then proceeded with hybridization capture using the myBaits Target Capture Kit Angiosperm 353 version 1 (product #308108, Daicel Arbor Biosciences, Ann Arbor, Michigan, USA) following the manufacturers protocol (Hybridization Capture for Targeted NGS version 4.01), but with 1/4 baits volume (1.375 uL instead of 5.5 uL) during hybridization. Hybridization reactions were held at 65C for 17.5 h. The resulting enriched products were amplified using KAPA HiFi 2X HotStart ReadyMix for 12 cycles. Final PCR product quality was assessed using a 2100 Bioanalyzer Instrument and sequenced on an Illumina MiSeq with version 3 (600-cycle) chemistry (Illumina, San Diego, California, USA) in the DNA Discovery Center at the Field Museum of Natural History (Chicago, Illinois, USA).