Genome reconstruction and comparative genomics of pneumocystis jirovecii

Pneumocystis jirovecii is a fungus belonging to a basal lineage of the Ascomycotina, the Taphrinomycotina subphylum. It is a parasite specific to humans that dwells primarily in the lung and can cause severe pneumonia in individuals with debilitated immune system. Despite its clinical importance, many aspects of its biology remain poorly understood, at least in part because of the lack of a continuous in vitro cultivation system. The present thesis consists in the genome reconstruction and comparative genomics of P. jirovecii. It is made of three parts: (i) the de novo sequencing of P. jirovecii genome starting from a single broncho- alveolar lavage fluid of a single patient (ii) the de novo sequencing of the genome of the plant pathogen Taphrina deformans, a fungus closely related to P. jirovecii, and (iii) the genome scale comparison of P. jirovecii to other Taphrinomycotina members. Enrichment in P. jirovecii cells by immuno-precipitation, whole DNA random amplification, two complementary high throughput DNA sequencing methods, and in silico sorting and assembly of sequences were used for the de novo reconstruction of P. jirovecii genome from the microbiota of a single clinical specimen. An iterative ad hoc pipeline as well as numerical simulations was used to recover P. jirovecii sequences while purging out contaminants and assembly or amplification chimeras. This strategy produced a 8.1 Mb assembly, which encodes 3,898 genes. Homology searches, mapping on biochemical pathways atlases, and manual validations revealed that this genome lacks (i) most of the enzymes dedicated to the amino acids biosyntheses, and (ii) most virulence factors observed in other fungi, e.g. the glyoxylate shunt pathway and specific peptidases involved in the degradation of the host cell membrane. The same analyses applied to the available genomic sequences from Pneumocystis carinii the species infecting rats and Pneumocystis murina the species infecting mice revealed the same deficiencies. The genome sequencing of T. deformans yielded a 13 Mb assembly, which encodes 5,735 genes. T. deformans possesses enzymes involved plant cell wall degradation, secondary metabolism, the glyoxylate cycle, detoxification, sterol biosynthesis, as well as the biosyntheses of plant hormones such as abscisic acid or indole-3-acetic acid. T. deformans also harbors gene subsets that have counterparts in plant saprophytes or pathogens, which is consistent with its alternate saprophytic and pathogenic lifestyles. Mating genes were also identified. The homothallism of this fungus suggests a mating-type switching mechanism. Comparative analyses indicated that 81% of P. jirovecii genes are shared with eight other Taphrinomycotina members, including T. deformans, P. carinii and P. murina. These genes are mostly involved in housekeeping activities. The genes specific to the Pneumocystis genus represent 8%, and are involved in RNA metabolism and signaling. The signaling is known to be crucial for interaction of Pneumocystis spp with their environment. Eleven percent are unique to P. jirovecii and encode mostly proteins of unknown function. These genes in conjunction with other ones (e.g. the major surface glycoproteins) might govern the interaction of P. jirovecii with its human host cells, and potentially be responsible of the host specificity. P. jirovecii exhibits a reduced genome in size with a low GC content, and most probably scavenges vital compounds such as amino acids and cholesterol from human lungs. Consistently, its genome encodes a large set of transporters (ca. 22% of its genes), which may play a pivotal role in the acquisition of these compounds. All these features are generally observed in obligate parasite of various kingdoms (bacteria, protozoa, fungi). Moreover, epidemiological studies failed to evidence a free-living form of the fungus and Pneumocystis spp were shown to co-evolved with their hosts. Given also the lack of virulence factors, our observations strongly suggest that P. jirovecii is an obligate parasite specialized in the colonization of human lungs, and which causes disease only in individuals with compromised immune system. The same conclusion is most likely true for all other Pneumocystis spp in their respective mammalian host. - Pneumocystis jirovecii est un champignon appartenant à ine branche basale des Ascomycotina, le sous-embranchement des Taphrinomycotina. C'est un parasite spécifique aux humains qui réside principalement dans les poumons, et qui peut causer des pneumonies sévères chez des individus ayant un système immunitaire déficient. En dépit de son importance clinique, de nombreux aspects de sa biologie demeurent,largement méconnus, au moins en partie à cause de l'absence d'un système de culture in vitro continu. Cette thèse traite de la reconstruction du génome et de la génomique comparative de P. jirovecii. Elle comporte trois parties: (i) le séquençage de novo du génome de P. jirovecii à partir d'un lavage broncho-alvéolaire provenant d'un seul patient, (ii) le séquençage de novo du génome d'un champignon pathogène de plante Taphrina deformans qui est phylogénétiquement proche de P. jirovecii, et (iii) la comparaison du génome de P. jirovecii à celui d'autres membres du sous-embranchement des Taphrinomycotina. Un enrichissement en cellules de P. jirovecii par immuno-précipitation, une amplification aléatoire des molécules d'ADN, deux méthodes complémentaires de séquençage à haut débit, un tri in silico et un assemblage des séquences ont été utilisés pour reconstruire de novo le génome de P. jirovecii à partir du microbiote d'un seul échantillon clinique. Un pipeline spécifique ainsi que des simulations numériques ont été utilisés pour récupérer les séquences de P. jirovecii tout en éliminant les séquences contaminants et les chimères d'amplification ou d'assemblage. Cette stratégie a produit un assemblage de 8.1 Mb, qui contient 3898 gènes. Les recherches d'homologies, de cartographie des voies métaboliques et des validations manuelles ont révélé que ce génome est dépourvu (i) de la plupart des enzymes dédiées à la biosynthèse des acides aminés, et (ii) de la plupart des facteurs de virulence observés chez d'autres champignons, par exemple, le cycle du glyoxylate ainsi que des peptidases spécifiques impliquées dans la dégradation de la membrane de la cellule hôte. Les analyses appliquées aux données génomiques disponibles de Pneumocystis carinii, l'espèce infectant les rats, et de Pneumocystis murina, l'espèce infectant les souris, ont révélé les mêmes déficiences. Le séquençage du génome de T. deformans a généré un assemblage de 13.3 Mb qui contient 5735 gènes. T. deformans possède les gènes codant pour les enzymes impliquées dans la dégradation des parois cellulaires des plantes, le métabolisme secondaire, le cycle du glyoxylate, la détoxification, la biosynthèse des stérols ainsi que la biosynthèse d'hormones de plantes telles que l'acide abscissique ou l'acide indole 3-acétique. T. deformans possède également des sous-ensembles de gènes présents exclusivement chez des saprophytes ou des pathogènes de plantes, ce qui est consistent avec son mode de vie alternatif saprophyte et pathogène. Des gènes impliqués dans la conjugaison ont été identifiés. L'homothallisme de ce champignon suggère mécanisme de permutation du type conjuguant. Les analyses comparatives ont démontré que 81% des gènes de P. jirovecii sont présent chez les autres membres du sous-embranchement des Taphrinomycotina. Ces gènes sont essentiellement impliqués dans le métabolisme basai. Les gènes spécifiques au genre Pneumocystis représentent 8%, et sont impliqués dans le métabolisme de l'ARN et la signalisation. La signalisation est connue pour être cruciale pour l'interaction des espèces de Pneumocystis avec leur environnement. Les gènes propres à P. jirovecii représentent 11% et codent en majorité pour des protéines dont la fonction est inconnue. Ces gènes en conjonction avec d'autres (par exemple, les glycoprotéines de surface), pourraient être déterminants dans l'interaction de P. jirovecii avec les cellules de l'hôte humain, et être potentiellement responsable de la spécificité d'hôte. P. jirovecii possède un génome de taille réduite à faible pourcentage en GC et récupère très probablement des composés vitaux comme les acides aminés et le cholestérol à partir des poumons humains. De manière consistante, son génome code pour de nombreux transporteurs (22% de ses gènes), qui pourraient jouer un rôle essentiel dans l'acquisition de ces composés. Ces caractéristiques sont généralement observées chez les parasites obligatoires de plusieurs règnes (bactéries, protozoaires, champignons). De plus, les études épidémiologiques n'ont pas réussi à prouver l'existence d'ime forme vivant librement du champignon. Etant donné également l'absence de facteurs de virulence, nos observations suggèrent que P. jirovecii est un parasite obligatoire spécialisé dans la colonisation des poumons humains, ne causant une maladie que chez des individus ayant un système immunitaire compromis. La même conclusion est très probablement applicable à toutes les autres espèces de Pneumocystis dans leur hôte mammifère respectif.

GENCODE: the reference human genome annotation for The ENCODE Project.

The GENCODE Consortium aims to identify all gene features in the human genome using a combination of computational analysis, manual annotation, and experimental validation. Since the first public release of this annotation data set, few new protein-coding loci have been added, yet the number of alternative splicing transcripts annotated has steadily increased. The GENCODE 7 release contains 20,687 protein-coding and 9640 long noncoding RNA loci and has 33,977 coding transcripts not represented in UCSC genes and RefSeq. It also has the most comprehensive annotation of long noncoding RNA (lncRNA) loci publicly available with the predominant transcript form consisting of two exons. We have examined the completeness of the transcript annotation and found that 35% of transcriptional start sites are supported by CAGE clusters and 62% of protein-coding genes have annotated polyA sites. Over one-third of GENCODE protein-coding genes are supported by peptide hits derived from mass spectrometry spectra submitted to Peptide Atlas. New models derived from the Illumina Body Map 2.0 RNA-seq data identify 3689 new loci not currently in GENCODE, of which 3127 consist of two exon models indicating that they are possibly unannotated long noncoding loci. GENCODE 7 is publicly available from gencodegenes.org and via the Ensembl and UCSC Genome Browsers.

Draft genome of the red harvester ant Pogonomyrmex barbatus.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus. The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans (Apis mellifera and Nasonia vitripennis) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Genome organization and gene expression shape the transposable element distribution in the Drosophila melanogaster euchromatin.

The distribution of transposable elements (TEs) in a genome reflects a balance between insertion rate and selection against new insertions. Understanding the distribution of TEs therefore provides insights into the forces shaping the organization of genomes. Past research has shown that TEs tend to accumulate in genomic regions with low gene density and low recombination rate. However, little is known about the factors modulating insertion rates across the genome and their evolutionary significance. One candidate factor is gene expression, which has been suggested to increase local insertion rate by rendering DNA more accessible. We test this hypothesis by comparing the TE density around germline- and soma-expressed genes in the euchromatin of Drosophila melanogaster. Because only insertions that occur in the germline are transmitted to the next generation, we predicted a higher density of TEs around germline-expressed genes than soma-expressed genes. We show that the rate of TE insertions is greater near germline- than soma-expressed genes. However, this effect is partly offset by stronger selection for genome compactness (against excess noncoding DNA) on germline-expressed genes. We also demonstrate that the local genome organization in clusters of coexpressed genes plays a fundamental role in the genomic distribution of TEs. Our analysis shows that-in addition to recombination rate-the distribution of TEs is shaped by the interaction of gene expression and genome organization. The important role of selection for compactness sheds a new light on the role of TEs in genome evolution. Instead of making genomes grow passively, TEs are controlled by the forces shaping genome compactness, most likely linked to the efficiency of gene expression or its complexity and possibly their interaction with mechanisms of TE silencing.

Genometrics as an essential tool for the assembly of whole genome sequences: the example of the chromosome of Bifidobacterium longum NCC2705.

BACKGROUND: Analysis of the first reported complete genome sequence of Bifidobacterium longum NCC2705, an actinobacterium colonizing the gastrointestinal tract, uncovered its proteomic relatedness to Streptomyces coelicolor and Mycobacterium tuberculosis. However, a rapid scrutiny by genometric methods revealed a genome organization totally different from all so far sequenced high-GC Gram-positive chromosomes. RESULTS: Generally, the cumulative GC- and ORF orientation skew curves of prokaryotic genomes consist of two linear segments of opposite slope: the minimum and the maximum of the curves correspond to the origin and the terminus of chromosome replication, respectively. However, analyses of the B. longum NCC2705 chromosome yielded six, instead of two, linear segments, while its dnaA locus, usually associated with the origin of replication, was not located at the minimum of the curves. Furthermore, the coorientation of gene transcription with replication was very low. Comparison with closely related actinobacteria strongly suggested that the chromosome of B. longum was misassembled, and the identification of two pairs of relatively long homologous DNA sequences offers the possibility for an alternative genome assembly proposed here below. By genometric criteria, this configuration displays all of the characters common to bacteria, in particular to related high-GC Gram-positives. In addition, it is compatible with the partially sequenced genome of DJO10A B. longum strain. Recently, a corrected sequence of B. longum NCC2705, with a configuration similar to the one proposed here below, has been deposited in GenBank, confirming our predictions. CONCLUSION: Genometric analyses, in conjunction with standard bioinformatic tools and knowledge of bacterial chromosome architecture, represent fast and straightforward methods for the evaluation of chromosome assembly.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene.

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ∼446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.

The European dimension for the mouse genome mutagenesis program.

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Clinical, polysomnographic and genome-wide association analyses of narcolepsy with cataplexy: a European Narcolepsy Network study.

The aim of this study was to describe the clinical and PSG characteristics of narcolepsy with cataplexy and their genetic predisposition by using the retrospective patient database of the European Narcolepsy Network (EU-NN). We have analysed retrospective data of 1099 patients with narcolepsy diagnosed according to International Classification of Sleep Disorders-2. Demographic and clinical characteristics, polysomnography and multiple sleep latency test data, hypocretin-1 levels, and genome-wide genotypes were available. We found a significantly lower age at sleepiness onset (men versus women: 23.74 ± 12.43 versus 21.49 ± 11.83, P = 0.003) and longer diagnostic delay in women (men versus women: 13.82 ± 13.79 versus 15.62 ± 14.94, P = 0.044). The mean diagnostic delay was 14.63 ± 14.31 years, and longer delay was associated with higher body mass index. The best predictors of short diagnostic delay were young age at diagnosis, cataplexy as the first symptom and higher frequency of cataplexy attacks. The mean multiple sleep latency negatively correlated with Epworth Sleepiness Scale (ESS) and with the number of sleep-onset rapid eye movement periods (SOREMPs), but none of the polysomnographic variables was associated with subjective or objective measures of sleepiness. Variant rs2859998 in UBXN2B gene showed a strong association (P = 1.28E-07) with the age at onset of excessive daytime sleepiness, and rs12425451 near the transcription factor TEAD4 (P = 1.97E-07) with the age at onset of cataplexy. Altogether, our results indicate that the diagnostic delay remains extremely long, age and gender substantially affect symptoms, and that a genetic predisposition affects the age at onset of symptoms.

Mutation identification by direct comparison of whole-genome sequencing data from mutant and wild-type individuals using k-mers.

Genes underlying mutant phenotypes can be isolated by combining marker discovery, genetic mapping and resequencing, but a more straightforward strategy for mapping mutations would be the direct comparison of mutant and wild-type genomes. Applying such an approach, however, is hampered by the need for reference sequences and by mutational loads that confound the unambiguous identification of causal mutations. Here we introduce NIKS (needle in the k-stack), a reference-free algorithm based on comparing k-mers in whole-genome sequencing data for precise discovery of homozygous mutations. We applied NIKS to eight mutants induced in nonreference rice cultivars and to two mutants of the nonmodel species Arabis alpina. In both species, comparing pooled F2 individuals selected for mutant phenotypes revealed small sets of mutations including the causal changes. Moreover, comparing M3 seedlings of two allelic mutants unambiguously identified the causal gene. Thus, for any species amenable to mutagenesis, NIKS enables forward genetics without requiring segregating populations, genetic maps and reference sequences.

A genome-wide approach accounting for body mass index identifies genetic variants influencing fasting glycemic traits and insulin resistance.

Recent genome-wide association studies have described many loci implicated in type 2 diabetes (T2D) pathophysiology and β-cell dysfunction but have contributed little to the understanding of the genetic basis of insulin resistance. We hypothesized that genes implicated in insulin resistance pathways might be uncovered by accounting for differences in body mass index (BMI) and potential interactions between BMI and genetic variants. We applied a joint meta-analysis approach to test associations with fasting insulin and glucose on a genome-wide scale. We present six previously unknown loci associated with fasting insulin at P < 5 × 10(-8) in combined discovery and follow-up analyses of 52 studies comprising up to 96,496 non-diabetic individuals. Risk variants were associated with higher triglyceride and lower high-density lipoprotein (HDL) cholesterol levels, suggesting a role for these loci in insulin resistance pathways. The discovery of these loci will aid further characterization of the role of insulin resistance in T2D pathophysiology.

Genome-wide association analyses identify 18 new loci associated with serum urate concentrations.

Elevated serum urate concentrations can cause gout, a prevalent and painful inflammatory arthritis. By combining data from >140,000 individuals of European ancestry within the Global Urate Genetics Consortium (GUGC), we identified and replicated 28 genome-wide significant loci in association with serum urate concentrations (18 new regions in or near TRIM46, INHBB, SFMBT1, TMEM171, VEGFA, BAZ1B, PRKAG2, STC1, HNF4G, A1CF, ATXN2, UBE2Q2, IGF1R, NFAT5, MAF, HLF, ACVR1B-ACVRL1 and B3GNT4). Associations for many of the loci were of similar magnitude in individuals of non-European ancestry. We further characterized these loci for associations with gout, transcript expression and the fractional excretion of urate. Network analyses implicate the inhibins-activins signaling pathways and glucose metabolism in systemic urate control. New candidate genes for serum urate concentration highlight the importance of metabolic control of urate production and excretion, which may have implications for the treatment and prevention of gout.

Genome-wide association study of major recurrent depression in the U.K. population.

OBJECTIVE: Studies of major depression in twins and families have shown moderate to high heritability, but extensive molecular studies have failed to identify susceptibility genes convincingly. To detect genetic variants contributing to major depression, the authors performed a genome-wide association study using 1,636 cases of depression ascertained in the U.K. and 1,594 comparison subjects screened negative for psychiatric disorders. METHOD: Cases were collected from 1) a case-control study of recurrent depression (the Depression Case Control [DeCC] study; N=1346), 2) an affected sibling pair linkage study of recurrent depression (probands from the Depression Network [DeNT] study; N=332), and 3) a pharmacogenetic study (the Genome-Based Therapeutic Drugs for Depression [GENDEP] study; N=88). Depression cases and comparison subjects were genotyped at Centre National de Génotypage on the Illumina Human610-Quad BeadChip. After applying stringent quality control criteria for missing genotypes, departure from Hardy-Weinberg equilibrium, and low minor allele frequency, the authors tested for association to depression using logistic regression, correcting for population ancestry. RESULTS: Single nucleotide polymorphisms (SNPs) in BICC1 achieved suggestive evidence for association, which strengthened after imputation of ungenotyped markers, and in analysis of female depression cases. A meta-analysis of U.K. data with previously published results from studies in Munich and Lausanne showed some evidence for association near neuroligin 1 (NLGN1) on chromosome 3, but did not support findings at BICC1. CONCLUSIONS: This study identifies several signals for association worthy of further investigation but, as in previous genome-wide studies, suggests that individual gene contributions to depression are likely to have only minor effects, and very large pooled analyses will be required to identify them.

Visualization and quality assessment of de novo genome assemblies.

Recent technological progress has greatly facilitated de novo genome sequencing. However, de novo assemblies consist in many pieces of contiguous sequence (contigs) arranged in thousands of scaffolds instead of small numbers of chromosomes. Confirming and improving the quality of such assemblies is critical for subsequent analysis. We present a method to evaluate genome scaffolding by aligning independently obtained transcriptome sequences to the genome and visually summarizing the alignments using the Cytoscape software. Applying this method to the genome of the red fire ant Solenopsis invicta allowed us to identify inconsistencies in 7%, confirm contig order in 20% and extend 16% of scaffolds.Scripts that generate tables for visualization in Cytoscape from FASTA sequence and scaffolding information files are publicly available at https://github.com/ksanao/TGNet.