PacBio sequence reads of the Malassezia pachydermatis ATCC 14522 (CBS 1879) genome
M. pachydermatis strain ATCC 14522 (CBS 1879) was grown to saturation at 35C in liquid modified Dixon medium (ATCC Medium 2693). Cells were collected by centrifugation, washed in sterile water and resuspended in 3 ml 10 mM Tris-HCl pH 8.0 containing 100 mM NaCl, 10 mM MgCl2, and 2 mM 2-mercaptoethanol. An equal volume of phenol/chloroform (1:1) and a 0.5 ml equivalent of sterile glass beads (425-600 microns) were added to the tube. The tube was vortexed at top speed for 2 min, then centrifuged at 3000 x g to separate the phases. The aqueous phase was collected and DNA precipitated with 2 volumes of isopropanol. DNA was resuspended in 10 mM Tris-EDTA (pH 8.0), then treated with RNase A (Gold Biotechnology) at 37C for 1 h. Proteinase K (Gold Biotechnology) was added and incubated at 50C for 16 h. The preparation was extracted with phenol/chloroform and DNA precipitated with 2 volumes of isopropanol. Genomic DNA was sheared to an average size of 13 kb using a Megaruptor 3 (Diagenode). Sheared DNA was run on a Blue Pippin system (Sage Science) to select 7-50 kb fragments using a 0.75% agarose cassette and DNA marker S1. Sheared fragments were converted to a library using the SMRTbell prep kit 3.0 (Pacific Biosciences). The library was sequenced on a single-molecule real-time (SMRT) cell on a PacBio Revio system using the circular consensus sequencing (CCS) mode and a 30-h movie time. CCS analysis used SMRT Link v13.1 (ccs --min-passes 3 --min-rq 0.99).