Sequencing and Comparative Analysis of \u3ci\u3ede novo\u3c/i\u3e Genome Assemblies of \u3ci\u3eStreptomyces aureofaciens\u3c/i\u3e ATCC 10762

Streptomyces aureofaciens is a Gram-positive Actinomycete used for commercial antibiotic production. Although it has been the subject of many biochemical studies, no public genome resource was available prior to this project. To address this need, the genome of S. aureofaciens ATCC 10762 was sequenced using a combination of sequencing platforms (Illumina and 454-shotgun). Multiple de novo assembly methods (SGA, IDBA, Trinity, SOAPdenovo2, MIRA, Velvet and SPAdes) as well as combinations of these methods were assessed to determine which provided the most robust assembly. Combination strategies led to a consistent overestimation of the total genome size. Empirical data from targeted PCR of predicted gap regions provided a robust validation framework for our de novo assemblies. Overall, the best assembly was generated using SPAdes. The total assembly length was 9.47 Mb and the average G+C content was 71.15 %. We annotated this assembly using the NCBI Prokaryotic Genome Annotation Pipeline, revealing 8,073 total genes, including a total of 7,627 protein coding sequences. Additional functional analysis using the KEGG GENES database provided functional predictions for over 1,400 of these sequences whose functions were not initially inferred by NCBI. The information provided from multiple independent assemblies allowed us to close 200 scaffold gaps present in our first hybrid assembly. Comparative genomic and phylogenetic analyses suggested S. aureofaciens ATCC 10762 may be more closely related to the genus Kitasatospora than to neighboring Streptomyces species. Our results highlight the need for, and the value of, multiple assemblies when attempting to produce high quality prokaryotic genome sequences. Advisor: Etsuko Moriyam

Sequencing and Comparative Analysis of \u3ci\u3ede novo\u3c/i\u3e Genome Assemblies of \u3ci\u3eStreptomyces aureofaciens\u3c/i\u3e ATCC 10762

Streptomyces aureofaciens is a Gram-positive Actinomycete used for commercial antibiotic production. Although it has been the subject of many biochemical studies, no public genome resource was available prior to this project. To address this need, the genome of S. aureofaciens ATCC 10762 was sequenced using a combination of sequencing platforms (Illumina and 454-shotgun). Multiple de novo assembly methods (SGA, IDBA, Trinity, SOAPdenovo2, MIRA, Velvet and SPAdes) as well as combinations of these methods were assessed to determine which provided the most robust assembly. Combination strategies led to a consistent overestimation of the total genome size. Empirical data from targeted PCR of predicted gap regions provided a robust validation framework for our de novo assemblies. Overall, the best assembly was generated using SPAdes. The total assembly length was 9.47 Mb and the average G+C content was 71.15 %. We annotated this assembly using the NCBI Prokaryotic Genome Annotation Pipeline, revealing 8,073 total genes, including a total of 7,627 protein coding sequences. Additional functional analysis using the KEGG GENES database provided functional predictions for over 1,400 of these sequences whose functions were not initially inferred by NCBI. The information provided from multiple independent assemblies allowed us to close 200 scaffold gaps present in our first hybrid assembly. Comparative genomic and phylogenetic analyses suggested S. aureofaciens ATCC 10762 may be more closely related to the genus Kitasatospora than to neighboring Streptomyces species. Our results highlight the need for, and the value of, multiple assemblies when attempting to produce high quality prokaryotic genome sequences. Advisor: Etsuko Moriyam

Towards Defining Nutrient Conditions Encountered by the Rice Blast Fungus during Host Infection

Fungal diseases cause enormous crop losses, but defining the nutrient conditions encountered by the pathogen remains elusive. Here, we generated a mutant strain of the devastating rice pathogen Magnaporthe oryzae impaired for de novo methionine biosynthesis. The resulting methionine-requiring strain grew strongly on synthetic minimal media supplemented with methionine, aspartate or complex mixtures of partially digested proteins, but could not establish disease in rice leaves. Live-cell-imaging showed the mutant could produce normal appressoria and enter host cells but failed to develop, indicating the availability or accessibility of aspartate and methionine is limited in the plant. This is the first report to demonstrate the utility of combining biochemical genetics, plate growth tests and live-cell-imaging to indicate what nutrients might not be readily available to the fungal pathogen in rice host cells

Genome Sequence of \u3ci\u3eStreptomyces aureofaciens\u3c/i\u3e ATCC Strain 10762

Streptomyces aureofaciens is a Gram-positive actinomycete that produces the antibiotics tetracycline and chlortetracycline. Here, we report the assembly and initial annotation of the draft genome sequence of S. aureofaciens ATCC strain 10762

Complete Genome Sequence of \u3ci\u3eSulfolobus solfataricus\u3c/i\u3e Strain 98/2 and Evolved Derivatives

Sulfolobus solfataricus strain 98/2 is a thermoacidophilic chemoheterotrophic crenarcheote that grows optimally at 80°C and pH3.0 (1). The S. solfataricus 98/2 genome reported in 2009 (Gen- Bank accession no. CP001800.1, RefSeq NC_017274.1, GI: 261600703) is a deletion derivative of strain 98/2 called PBL2025 (2). It is often misconstrued as the wild-type strain 98/2, yet lacks a 50-kb region encoding numerous genes involved in sugar metabolism. A new closed and complete genome sequence for wildtype strain 98/2 referred to as SULA is presented here as GenBank accession no. CP011057 using locus tag SULA. This strain has been deposited at the Japan Collection of Microorganisms. Two additional closed and complete genomes derived from strain 98/2 are also presented and include SULB (GenBank accession no. CP011055) and SULC (GenBank accession no. CP011056). SULB and SULC resulted from extensive passage during selection for the biological trait of increased acid resistance (unpublished data)

Genome Sequence of \u3ci\u3eStreptomyces aureofaciens\u3c/i\u3e ATCC Strain 10762

Streptomyces aureofaciens is a Gram-positive actinomycete that produces the antibiotics tetracycline and chlortetracycline. Here, we report the assembly and initial annotation of the draft genome sequence of S. aureofaciens ATCC strain 10762

Advantages of an Improved Rhesus Macaque Genome for Evolutionary Analyses

The rhesus macaque (Macaca mulatta) is widely used in molecular evolutionary analyses, particularly to identify genes under adaptive or unique evolution in the human lineage. For such studies, it is necessary to align nucleotide sequences of homologous protein-coding genes among multiple species. The validity of these analyses is dependent on high quality genomic data. However, for most mammalian species (other than humans and mice), only draft genomes are available. There has been concern that some results obtained from evolutionary analyses using draft genomes may not be correct. The rhesus macaque provides a unique opportunity to determine whether an improved genome (MacaM) yields better results than a draft genome (rheMac2) for evolutionary studies. We compared protein-coding genes annotated in the rheMac2 and MacaM genomes with their human orthologs. We found many genes annotated in rheMac2 had apparently spurious sequences not present in genes derived from MacaM. The rheMac2 annotations also appeared to inflate a frequently used evolutionary index, ω (the ratio of nonsynonymous to synonymous substitution rates). Genes with these spurious sequences must be filtered out from evolutionary analyses to obtain correct results. With the MacaM genome, improved sequence information means many more genes can be examined for indications of selection. These results indicate how upgrading genomes from draft status to a higher level of quality can improve interpretation of evolutionary patterns

Advantages of an Improved Rhesus Macaque Genome for Evolutionary Analyses.

The rhesus macaque (Macaca mulatta) is widely used in molecular evolutionary analyses, particularly to identify genes under adaptive or unique evolution in the human lineage. For such studies, it is necessary to align nucleotide sequences of homologous protein-coding genes among multiple species. The validity of these analyses is dependent on high quality genomic data. However, for most mammalian species (other than humans and mice), only draft genomes are available. There has been concern that some results obtained from evolutionary analyses using draft genomes may not be correct. The rhesus macaque provides a unique opportunity to determine whether an improved genome (MacaM) yields better results than a draft genome (rheMac2) for evolutionary studies. We compared protein-coding genes annotated in the rheMac2 and MacaM genomes with their human orthologs. We found many genes annotated in rheMac2 had apparently spurious sequences not present in genes derived from MacaM. The rheMac2 annotations also appeared to inflate a frequently used evolutionary index, ω (the ratio of nonsynonymous to synonymous substitution rates). Genes with these spurious sequences must be filtered out from evolutionary analyses to obtain correct results. With the MacaM genome, improved sequence information means many more genes can be examined for indications of selection. These results indicate how upgrading genomes from draft status to a higher level of quality can improve interpretation of evolutionary patterns

Complete Genome Sequence of an Evolved \u3ci\u3eThermotoga maritima\u3c/i\u3e Isolate

Thermotoga maritima MSB8 genomovar DSM3109 is a hyperthermophilic anaerobic bacterium (1) that grows at 80°C, producing a maximum of 4 mol of H2 per mol of glucose (2). There are a variety of duplicated genes and direct repeats in its genome, suggesting the potential for genome instability. Genome resequencing of T. maritima MSB8 genomovar DSM3109 in 2011 and 2013 (3, 4) indicated that the earlier sequenced T. maritima MSB8 reported by Nelson et al. (NC_000853.1) (5) was an evolved laboratory variant with an approximately 8-kb deletion located between TM1847 and TM1848 (annotation according to reference 5). The 8-kb deletion may have resulted from genome instability. To assess the potential for additional instability, a cell line harboring a chromosomally integrated kanamycin-resistant suicide plasmid was allowed to segregate without drug addition but with selection for maltose catabolism as part of ongoing studies involving experimental microbial evolution (unpublished data). The initially sequenced genome of T. maritima by Nelson et al. (5) (NC_000853.1) was used to describe the genome changes in the resulting strains. Of 50 clonal isolates screened, 10 underwent deletion formation, including a 10-kb loss between TM1322 and TM1332. One of these 10-kb deletion isolates was named Tma200. The deleted region in Tma200 encoded five hypothetical proteins, two AstB/ChuR-related proteins, one LacI family transcriptional regulator, and three ABC transporter ATP-binding proteins. In addition, two distinct repeat sequences of 921 bp and 313 bp, respectively, were identified in TM1322 (coordinates 1340942 to 1341862 and 1342246 to 1342558) and TM1332 (1350970 to 1351890 and 1352274 to 1352586). Crossover between the 921-bp direct repeats deleted the intervening region (1341863 to 1351890). Genome resequencing of this T. maritima derivative expands the understanding of factors underlying genome instability in this lineage

Towards Defining Nutrient Conditions Encountered by the Rice Blast Fungus during Host Infection

Fungal diseases cause enormous crop losses, but defining the nutrient conditions encountered by the pathogen remains elusive. Here, we generated a mutant strain of the devastating rice pathogen Magnaporthe oryzae impaired for de novo methionine biosynthesis. The resulting methionine-requiring strain grew strongly on synthetic minimal media supplemented with methionine, aspartate or complex mixtures of partially digested proteins, but could not establish disease in rice leaves. Live-cell-imaging showed the mutant could produce normal appressoria and enter host cells but failed to develop, indicating the availability or accessibility of aspartate and methionine is limited in the plant. This is the first report to demonstrate the utility of combining biochemical genetics, plate growth tests and live-cell-imaging to indicate what nutrients might not be readily available to the fungal pathogen in rice host cells