Cryptococcus_neoformans_reference_PacBio_sequencing. Cryptococcus_neoformans_reference_PacBio_sequencing

There are an estimated 1 million cases of cryptococcal meningitis (CM) each year, the majority due to infection with Cryptococcus neoformans var. grubii and occurring in HIV infected patients. These 1 million cases result in around 625,000 deaths. Further, the C. neoformans is an important pathogen of patients with iatrogenic immunosuppression, including solid organ transplant recipients, marrow transplant recipients, and those in receipt of biological agents for chronic inflammatory conditions. Disease in immunocompetent patients was previously thought to be driven by infection with C. gattii, a related species which is a rare cause of disease in the tropics and subtropics. More recently, this species has been associated with a significant outbreak of disease in Western Canada and the Pacific Northwest of the USA. In HCMC, Vietnam, 150 cases of CM are seen each year at the Hospital for Tropical Disease and Cho Ray Hospital. Unusually, around 30 of these cases are in HIV-uninfected patients, the majority of whom (85%) do not have any recognized cause of immunosuppression. 10-20% of these are due to infection with C. gattii; unexpectedly the rest are due to infection with C. neoforman s var. grubii. Using AFLP and multi-locus sequence typing it has been demonstrated that disease in immunocompetent patients is due to infection with a particular clade (and single MLST – ST5) of C. neoformans var. grubii. This contrasts with HIV infected patients who can be infected by any one of 15 autochthonous STs. HIV uninfected patients who have other potentially immunosuppressive underlying disease have similar diversity of infecting sequence types as HIV infected patients. Fungi require a minimum set of interacting properties for pathogenicity and it is clear that for Cryptococcus the pathogenicity factors identified to date represent only a fraction of the genes vital to cause disease. We (SH) have sequenced 288 Vietnamese strains from HIV infected patients, HIV uninfected patients and the environment, in order to better understand the population structure and evolution of the strains, and to identify genetic markers associated with pathogenesis. We have achieved sequencing coverage rates between 10 and 60 fold. Our initial analysis suggests that there are significant difference between the ST5 strains (that can infect both HIV infected and uninfected patients), and the most closely related ST4 and ST6 strains that infect only immunosuppressed patients. These include approx. 100kbp of INDELS), multiple gene (hypothetical and of known function) deletions and 40,000 SNPs. However, the genomes have been mapped to the published sequence of the H99 type strain (a strain derived from an American patient diagnosed with lymphoma) which is significantly genetically divergent. Higher quality reference genomes for the 4 main Vietnamese clades, delivered by PACBIO sequencing, will enable more robust genome assemblies This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute please see http://www.sanger.ac.uk/datasharing/