Genome assembly of the bearded iris, Iris Pallida
For genomic DNA extraction, four 50 ml Falcon centrifuge tubes were each filled with 10 ml of extraction buffer (2% CTAB, 1.4M NaCl, 20 mM EDTA, 100mM Tris-HCl pH 8.0 and 0.2% beta-mercaptoethanol. The tubes were placed in a polystyrene floatation device and warmed to 60C. A large pestle and mortar were cooled overnight in a -20C freezer. The mortar was filled with liquid nitrogen to a depth of 3cm. Sterile sand was added to a depth of 0.5 cm. Five young iris leaves, approximately 20 cm in length, were cut from the plant and immediately cut into 2-cm lengths and submerged in liquid nitrogen. They were then ground into a fine powder, with additional liquid nitrogen carefully added as needed. Approximately 25% of the iris/sand powder was added to each warm extraction buffer tube and mixed by inversion 3-4 times. The mixture was incubated at 60C for 30 minutes and mixed by inversion every 5-10 minutes. Then,10 ml of 25:24:1 phenol:chloroform:isoamyl alcohol was added and gently mixed by continuous inversion for 1 minute. The sample was centrifuged at 6000 g for 10 minutes to separate the phases. The upper phase was carefully removed and transferred to a 50 ml phase-lock gel tube (Eppendorf), and 10 ml of 24:1 chloroform:isoamyl alcohol was added and gently mixed by inversion for 1 minute. The sample was centrifuged for 6000 g for 10 minutes, after which the aqueous phase above the phase-lock wax was removed and transferred to a fresh tube. An equal volume of isopropanol was added to the tube, and the sample was mixed by inversion until a gelatinous mixture of nucleic acids formed. This nucleic acid mixture was removed with a glass rod and washed three times with ice-cold 70% ethanol. The nucleic acid was allowed to air dry and then dissolved overnight in TE buffer. The sample was assigned the Novartis tracking ID AS_SAM_17_03QT. Library preparation used continuous long read methods for genomic DNA sequencing for the Sequel 1 instrument, as per manufacturers instructions (PacBio).