Comprehensive Cryptococcus neoformans Isolation and Sequencing

C. neoformans was isolated from HIV-infected individuals on location by plating CSF onto Sabourand Dextrose (SD) agar (Oxoid, Fisher Scientific), and growing at 30° for 48 hr. A representative sample of the C. neoformans population was taken by selecting a broad “sweep” of all colonies on the SD agar plate, which was stored in cryopreservative medium (80% SD broth, 20% glycerol) at −80° until further testing. This approach ensures that all genetic diversity is maintained through the process, and single colony picking only occurs at the final stage of liquid culture and DNA extraction.
Frozen stocks were plated onto SD agar and cultured for 72 hr. A single colony was inoculated into 6 ml Yeast Peptone Digest broth (Oxoid) supplemented with 0.5 M NaCl and cultured at 37° with agitation (165 rpm) for 40 hr, followed by genomic DNA extraction using the Masterpure Yeast DNA purification kit (Epicentre) modified by addition of two cycles of rapid bead beating (45 sec at 4.5 m/sec) using a FastPrep 24 homogenizer (MP Bio). Genomic DNA libraries were prepared using the TruSeq DNA v2 or TruSeq Nano DNA kit (Illumina), and WGS was performed on an Illumina HiSequation 2500 at the Medical Research Council Clinical Genomics Centre (Imperial College London) as previously described (Rhodes et al. 2014 (link)).