Characterizing reduced coverage regions through comparison of exome and genome sequencing data across 10 centers.

PURPOSE: As massively parallel sequencing is increasingly being used for clinical decision making, it has become critical to understand parameters that affect sequencing quality and to establish methods for measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and describe a set of standards for variant calling in clinical sequencing applications. METHODS: To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCID) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in 10 projects at sequencing centers in the National Human Genome Research Institute/National Cancer Institute Clinical Sequencing Exploratory Research Consortium. RESULTS: This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers. CONCLUSION: This report provides a process road map for clinical sequencing centers looking to perform similar analyses on their data.

Comparative genome analysis and genome evolution of members of the magnaporthaceae family of fungi.

BACKGROUND: Magnaporthaceae, a family of ascomycetes, includes three fungi of great economic importance that cause disease in cereal and turf grasses: Magnaporthe oryzae (rice blast), Gaeumannomyces graminis var. tritici (take-all disease), and Magnaporthe poae (summer patch disease). Recently, the sequenced and assembled genomes for these three fungi were reported. Here, the genomes were compared for orthologous genes in order to identified genes that are unique to the Magnaporthaceae family of fungi. In addition, ortholog clustering was used to identify a core proteome for the Magnaporthaceae, which was examined for diversifying and purifying selection and evidence of two-speed genome evolution. RESULTS: A genome-scale comparative study was conducted across 74 fungal genomes to identify clusters of orthologous genes unique to the three Magnaporthaceae species as well as species specific genes. We found 1149 clusters that were unique to the Magnaporthaceae family of fungi with 295 of those containing genes from all three species. Gene clusters involved in metabolic and enzymatic activities were highly represented in the Magnaporthaceae specific clusters. Also highly represented in the Magnaporthaceae specific clusters as well as in the species specific genes were transcriptional regulators. In addition, we examined the relationship between gene evolution and distance to repetitive elements found in the genome. No correlations between diversifying or purifying selection and distance to repetitive elements or an increased rate of evolution in secreted and small secreted proteins were observed. CONCLUSIONS: Taken together, these data show that at the genome level, there is no evidence to suggest multi-speed genome evolution or that proximity to repetitive elements play a role in diversification of genes.

Phylogenetic analysis and classification of the fungal bHLH domain.

The basic Helix-Loop-Helix (bHLH) domain is an essential highly conserved DNA-binding domain found in many transcription factors in all eukaryotic organisms. The bHLH domain has been well studied in the Animal and Plant Kingdoms but has yet to be characterized within Fungi. Herein, we obtained and evaluated the phylogenetic relationship of 490 fungal-specific bHLH containing proteins from 55 whole genome projects composed of 49 Ascomycota and 6 Basidiomycota organisms. We identified 12 major groupings within Fungi (F1-F12); identifying conserved motifs and functions specific to each group. Several classification models were built to distinguish the 12 groups and elucidate the most discerning sites in the domain. Performance testing on these models, for correct group classification, resulted in a maximum sensitivity and specificity of 98.5% and 99.8%, respectively. We identified 12 highly discerning sites and incorporated those into a set of rules (simplified model) to classify sequences into the correct group. Conservation of amino acid sites and phylogenetic analyses established that like plant bHLH proteins, fungal bHLH-containing proteins are most closely related to animal Group B. The models used in these analyses were incorporated into a software package, the source code for which is available at www.fungalgenomics.ncsu.edu.

Accurate discrimination of bHLH domains in plants, animals, and fungi using biologically meaningful sites.

BACKGROUND: The highly conserved bHLH (basic Helix-Loop-Helix) domain, found in many transcription factors, has been well characterized separately in Plants, Animals, and Fungi. While conserved, even functionally constrained sites have varied since the Eukarya split. Our research identifies those slightly variable sites that were highly characteristic of Plants, Animals, or Fungi. RESULTS: Through discriminant analysis, we identified five highly discerning DNA-binding amino acid sites. Additionally, by incorporating Kingdom specific HMMs, we were able to construct a tool to quickly and accurately identify and classify bHLH sequences using these sites. CONCLUSIONS: We conclude that highly discerning sites identified through our analysis were likely under functional constraints specific to each Kingdom. We also demonstrated the utility of our tool by identifying and classifying previously unknown bHLH domains in both characterized genomes and from sequences in a large environmental sample.

Genome-wide characterization of methylguanosine-capped and polyadenylated small RNAs in the rice blast fungus Magnaporthe oryzae.

Small RNAs are well described in higher eukaryotes such as mammals and plants; however, knowledge in simple eukaryotes such as filamentous fungi is limited. In this study, we discovered and characterized methylguanosine-capped and polyadenylated small RNAs (CPA-sRNAs) by using differential RNA selection, full-length cDNA cloning and 454 transcriptome sequencing of the rice blast fungus Magnaporthe oryzae. This fungus causes blast, a devastating disease on rice, the principle food staple for over half the world's population. CPA-sRNAs mapped primarily to the transcription initiation and termination sites of protein-coding genes and were positively correlated with gene expression, particularly for highly expressed genes including those encoding ribosomal proteins. Numerous CPA-sRNAs also mapped to rRNAs, tRNAs, snRNAs, transposable elements and intergenic regions. Many other 454 sequence reads could not be mapped to the genome; however, inspection revealed evidence for non-template additions and chimeric sequences. CPA-sRNAs were independently confirmed using a high affinity variant of eIF-4E to capture 5'-methylguanosine-capped RNA followed by 3'-RACE sequencing. These results expand the repertoire of small RNAs in filamentous fungi.

Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae.

BACKGROUND: Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited. RESULTS: Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae. Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively. CONCLUSIONS: Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.

Genome Sequences of Three Phytopathogenic Species of the Magnaporthaceae Family of Fungi.

Magnaporthaceae is a family of ascomycetes that includes three fungi of great economic importance: Magnaporthe oryzae, Gaeumannomyces graminis var. tritici, and Magnaporthe poae. These three fungi cause widespread disease and loss in cereal and grass crops, including rice blast disease (M. oryzae), take-all disease in wheat and other grasses (G. graminis), and summer patch disease in turf grasses (M. poae). Here, we present the finished genome sequence for M. oryzae and draft sequences for M. poae and G. graminis var. tritici. We used multiple technologies to sequence and annotate the genomes of M. oryzae, M. poae, and G. graminis var. tritici. The M. oryzae genome is now finished to seven chromosomes whereas M. poae and G. graminis var. tritici are sequenced to 40.0× and 25.0× coverage respectively. Gene models were developed by the use of multiple computational techniques and further supported by RNAseq data. In addition, we performed preliminary analysis of genome architecture and repetitive element DNA.

Genetic codes as evolutionary filters: subtle differences in the structure of genetic codes result in significant differences in patterns of nucleotide substitution.

The codon-degeneracy model (CDM) predicts that patterns of nucleotide substitution in protein-coding genes are largely determined by the relative frequencies of four-fold (4f), two-fold, and non-degenerate sites, the attributes of which are determined by the structure of the governing genetic code. The CDM thus further predicts that genetic codes with alternative structures will "filter" molecular evolution differentially. A method, therefore, is presented by which the CDM may be applied to the unique structure of any genetic code. The mathematical relationship between the proportion of transitions at 4f degenerate nucleotide sites and the transition-to-transversion ratio is described. Predictions for five individual genetic codes, relative to the relationship between code structure and expected patterns of nucleotide substitution, are clearly defined. To test this "filter" hypothesis of genetic codes, simulated DNA sequence data sets were generated with a variety of input parameter values to estimate the relationship between patterns of nucleotide substitution and best-fit estimates of transition bias at 4f degenerate sites for both the universal genetic code and the vertebrate mitochondrial genetic code. These analyses confirm the prediction of the CDM that, all else being equal, even small differences in the structure of alternative genetic codes may result in significant shifts in the overall pattern of nucleotide substitution.