Onagraceae backbone target capture data for Oenothera_cavernae_RAL13.44
Genomic libraries with an insert size of 550 bp were prepared using the TruSeq Nano HT DNA Library Preparation Kit (Illumina San Diego, CA, USA) following manufacturers instructions, except that all reagent volumes (except PCR reagents) were cut in half beginning with the second addition of AMPure (SPRI) beads (Beckman Coulter, Beverly, MA). Successful library preparation was confirmed with the Qubit 2.0 fluorometer (Invitrogen Carlsbad, CA, USA) using the dsDNA HS Assay Kit, as well as BioAnalyzer 2100 traces (Agilent Technologies, Santa Clara, CA, USA) on a subset of samples. Target enrichment with liquid hybridization was performed using a MYbaits custom target enrichment kit (Mycroarray, Ann Arbor, MI, USA) designed for use in Oenothera [22, 23]. Libraries were multiplexed into pools containing 618 samples, roughly organized by taxonomic affiliation (e.g., Oenothera samples were hybridized together), with 100 ng of total starting library per sample in each pool. In the few cases where less than 100 ng was present, we used the total amount available (lowest successfully attempted ~25 ng). In all cases, we did not exceed 1.2 g of total DNA per pool as recommended by the manufacturer. Hybridization was performed at 65 C for ~18 hours and enriched library pools were amplified with 1418 PCR cycles as needed. No correlation was observed between PCR cycle number and ultimate target recovery by sample, suggesting that 18 cycles (or possibly more) results in little target loss through library bias under our multiplexing and sequencing parameters. In many cases, using higher PCR-cycle numbers was crucial for gaining sufficient product concentration for sequencing, especially for samples from older collections and herbarium vouchers. Each resulting PCR-amplified pool was then cleaned with a QiaQuick PCR Purification Kit (Qiagen, Hilden, Germany). Excess adapter, as revealed through the BioAnalyzer, was removed pre-sequencing with a 0.7 to 1 volume ratio of Ampure beads to product. Sequencing of enriched pools of libraries containing 6080 individual samples was carried out on the Illumina MiSeq System (600 cycle, v3 chemistry) with a final loading concentration of 16.5 pM (estimated from Qubit and BioAnalyzer output) and a 1% molar ratio of PhiX Control (Illumina). Individual sequencing runs resulted in approximately 28 million read-pairs passing Illumina quality filtering with an average of approximately 11.5% of reads assigned to each individual.