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/据估计，全球每年新发隐球菌性脑膜炎（cryptococcal meningitis, CM）病例约100万例，其中绝大多数由新生隐球菌格鲁比变种（Cryptococcus neoformans var. grubii）感染引起，且患者多为艾滋病病毒（HIV）感染者。这100万例病例每年导致约62.5万例死亡。此外，新生隐球菌亦是医源性免疫抑制患者的重要致病菌，这类人群包括实体器官移植受者、骨髓移植受者，以及因慢性炎症性疾病接受生物制剂治疗的患者。既往认为，免疫功能正常宿主的隐球菌病多由格特隐球菌（C. gattii）感染所致——该相关菌种在热带、亚热带地区仅为罕见致病菌。但近年来，该菌种已被证实与加拿大西部及美国太平洋西北地区的大规模疾病暴发相关。在越南胡志明市（HCMC）的热带病医院与华侨医院（Cho Ray Hospital），每年可接诊150例CM病例。不同寻常的是，其中约30例患者为HIV阴性者，且大多数（85%）未发现明确的免疫抑制诱因。这些病例中，10%~20%由格特隐球菌感染引起；而令人意外的是，剩余病例的致病菌为新生隐球菌格鲁比变种。通过扩增片段长度多态性（Amplified Fragment Length Polymorphism, AFLP）与多位点序列分型（multi-locus sequence typing, MLST）技术，研究人员已证实，免疫功能正常患者的隐球菌病由特定进化枝（即单一MLST型别ST5）的新生隐球菌格鲁比变种感染所致。这与HIV感染者的情况形成鲜明对比：后者可被15种本地序列型别（autochthonous STs）中的任意一种菌株感染。伴有其他潜在免疫抑制基础疾病的HIV阴性患者，其感染菌株的序列型别多样性与HIV感染者相似。真菌的致病性需要一系列相互关联的特性支撑，目前已明确的隐球菌致病因子仅为致病所需基因中的一小部分。本研究团队（SH）已对288株越南分离菌株完成全基因组测序，这些菌株分别来自HIV感染者、HIV阴性者以及环境样本，旨在更深入地解析该菌株的群体结构与演化特征，并鉴定与致病相关的遗传标记。本次测序的覆盖度介于10~60倍之间。初步分析结果显示，ST5型菌株（可感染HIV感染者与免疫功能正常者）与亲缘关系最近的ST4、ST6型菌株（仅感染免疫抑制患者）之间存在显著差异，包括约100kbp的插入缺失序列（INDELS）、多个基因（包括假想基因与已知功能基因）的缺失，以及40000个单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）。不过，目前所有基因组均已比对至已发表的H99标准菌株（H99 type strain，源自一名美国淋巴瘤患者的分离株）的参考序列，而该菌株与本次研究的越南菌株存在显著的遗传差异。若通过PacBio测序技术获取越南四大主要进化枝的高质量参考基因组，将有助于获得更可靠的全基因组组装结果。本数据集属于预出版发布数据。如需了解威康桑格研究所（Wellcome Trust Sanger Institute）共享的预出版数据的规范使用方法，请访问http://www.sanger.ac.uk/datasharing/