PacBio sequence reads of the Malassezia pachydermatis 44378 genome
M. pachydermatis strain 20-44378 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).