Assembly and Annotation of Pneumocystis jirovecii from the Human Lung Microbiome

Abstract

Pneumocystis jirovecii is a fungus that causes Pneumocystis pneumonia in immunosuppressed patients and has been closely associated with AIDS since the beginning of the AIDS epidemic. Because in vitro cultivation of P. jirovecii is not possible, progress has been hindered in our understanding of its life cycle, mode of transmission, metabolic function, and genome. Limited amounts of P. jirovecii can be obtained from infected patients, but the occurrence of bacteria, other fungi, and human cells in clinical samples presents new challenges for whole-genome sequencing and downstream bioinformatic analysis. In a recent article, Cissé et al. used cell immunoprecipitation enrichment together with whole-genome amplification to generate sufficient quantities of DNA for Roche 454 and Illumina sequencing [O. H. Cissé, M. Pagni, and P. M. Hauser, mBio 4(1):e00428-12, 2012, doi:10.1128/mBio.00428-12]. In addition, a bioinformatic pipeline was devised to sort and assemble lung microbiome reads, thereby generating an 8.1-Mb P. jirovecii genome comprised of 356 contigs with an N50 (median length of all contigs) of 41.6 kb. Knowledge of this genome will open new avenues of research, including the identification of nutritional requirements for in vitro cultivation as well as the identification of new and novel drug and vaccine targets.

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis

“…2b, Supplementary Table 9), the number of rDNA copies is approximately 10, and the RIR17 assembly covers almost all of the rDNA copies. This AMF rDNA copy number is the lowest among eukaryotes 48 other than pneumonia-causing Pneumocystis (one rDNA) 49 and malaria-causing Plasmodium (seven rDNAs) 50 .

Fig. 2Physical maps of rDNA structures and copy numbers in RIR17.

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Multilocus Microsatellite Genotyping Array for Investigation of Genetic Epidemiology of Pneumocystis jirovecii

“…In order to minimize bias in analyses of population structure, we tested for linkage between microsatellite loci (62). Because the Pneumocystis jirovecii draft genome is in 356 contigs (32,63) rather than in chromosomes, we could not rely on genetic maps and instead tested linkage disequilibrium between markers. Bonferroni-corrected perpopulation pairwise tests of linkage disequilibrium showed no significant linkage disequilibrium between any locus pairs in any of the three populations (results not shown).…”

Investigating Fungal Outbreaks in the 21st Century

“…The other significant limitation of current WGST is the difficulty in detecting and identifying fungal DNA directly in human clinical samples against the human DNA background. Proteomic and metagenomic methods have been used for culture-independent detection and typing of viral and bacterial pathogens [ 18 – 20 ], and these methods are being adapted for fungi [ 21 ]. Accumulation of fungal genomic data and the development of a WGST database for fungal pathogens will provide a necessary framework for developing metagenomic tools for detection and typing of fungi in clinical samples.…”