A chromosomal-level reference genome of the widely utilized Coccidioides posadasii laboratory strain "Silveira".

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Heat Inactivation of Coccidioides posadasii and Coccidioides immitis for Use in Lower Biosafety Containment.

INTRODUCTION: The difficulty involved in obtaining sufficient intact genomic deoxyribonucleic acid (DNA) from Coccidioides spp for downstream applications using published protocols prompted the exploration of inactivating mycelia and arthroconidia using heat under biosafety level 3 containment. This was followed by optimizing DNA extraction from mycelia using various methods at lower containment. METHODS: Various exposure times and temperatures were examined to identify an effective heat inactivation procedure for arthroconidia and mycelia from both C immitis and C posadasii. Heat inactivation of mycelia was followed by DNA extraction using 2 commercially available kits, as well as a phenol:chloroform-based extraction procedure to determine DNA integrity and quantity among extraction methods using both live and heat-inactivated mycelia. RESULTS: Ten-minute and 30-minute exposure times at 80°C were sufficient to inactivate Coccidioides spp arthroconidia and mycelia, respectively. DNA yield between live versus heat-inactivated mycelia was similar for each extraction procedure. However, DNA obtained using phenol:chloroform was of higher quantity and integrity compared with DNA obtained using the commercially available kits, which was highly fragmented. CONCLUSION: The ability to heat-inactivate Coccidioides cultures for processing at a lower level of containment greatly increased the efficiency of DNA extractions. Therefore, this is an ideal method for obtaining Coccidioides spp DNA and inactivated arthroconidia.