Brian Desany

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. NOTE: In the version of this article initially published, it should have been acknowledged that Jan F. Simons, in addition to Roman K. Thomas and Elizabeth Nickerson, contributed equally to this work. The error has been corrected in the HTML and PDF versions of the article.

Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium

Nitrogen (N2)-fixing marine cyanobacteria are an important source of fixed inorganic nitrogen that supports oceanic primary productivity and carbon dioxide removal from the atmosphere. A globally distributed, periodically abundant N2-fixing marine cyanobacterium, UCYN-A, was recently found to lack the oxygen-producing photosystem II complex of the photosynthetic apparatus, indicating a novel metabolism, but remains uncultivated. Here we show, from metabolic reconstructions inferred from the assembly of the complete UCYN-A genome using massively parallel pyrosequencing of paired-end reads, that UCYN-A has a photofermentative metabolism and is dependent on other organisms for essential compounds. We found that UCYN-A lacks a number of major metabolic pathways including the tricarboxylic acid cycle, but retains sufficient electron transport capacity to generate energy and reducing power from light. Unexpectedly, UCYN-A has a reduced genome (1.44 megabases) that is structurally similar to many chloroplasts and some bacteria, in that it contains inverted repeats of ribosomal RNA operons. The lack of biosynthetic pathways for several amino acids and purines suggests that this organism depends on other organisms, either in close association or in symbiosis, for critical nutrients. However, size fractionation experiments using natural populations have so far not provided evidence of a symbiotic association with another microorganism. The UCYN-A cyanobacterium is a paradox in evolution and adaptation to the marine environment, and is an example of the tight metabolic coupling between microorganisms in oligotrophic oceanic microbial communities.

Whole-Genome Comparison Reveals Novel Genetic Elements That Characterize the Genome of Industrial Strains of Saccharomyces cerevisiae

Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.

Genomic analyses of the microsporidian Nosema ceranae, an emergent pathogen of honey bees

Recent steep declines in honey bee health have severely impacted the beekeeping industry, presenting new risks for agricultural commodities that depend on insect pollination. Honey bee declines could reflect increased pressures from parasites and pathogens. The incidence of the microsporidian pathogen Nosema ceranae has increased significantly in the past decade. Here we present a draft assembly (7.86 MB) of the N. ceranae genome derived from pyrosequence data, including initial gene models and genomic comparisons with other members of this highly derived fungal lineage. N. ceranae has a strongly AT-biased genome (74% A+T) and a diversity of repetitive elements, complicating the assembly. Of 2,614 predicted protein-coding sequences, we conservatively estimate that 1,366 have homologs in the microsporidian Encephalitozoon cuniculi, the most closely related published genome sequence. We identify genes conserved among microsporidia that lack clear homology outside this group, which are of special interest as potential virulence factors in this group of obligate parasites. A substantial fraction of the diminutive N. ceranae proteome consists of novel and transposable-element proteins. For a majority of well-supported gene models, a conserved sense-strand motif can be found within 15 bases upstream of the start codon; a previously uncharacterized version of this motif is also present in E. cuniculi. These comparisons provide insight into the architecture, regulation, and evolution of microsporidian genomes, and will drive investigations into honey bee–Nosema interactions.

The Cassava Genome: Current Progress, Future Directions

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance.

Detection of low-frequency pretherapy chemokine (CXC motif) receptor 4 (CXCR4)-using HIV-1 with ultra-deep pyrosequencing.

Objective:Identification of low-frequency variants is of clinical importance in the identification of preexisting drug resistance. Using ‘ultra-deep’ sequencing, we address the detection of potential resistance to the chemokine (C–C motif) receptor 5 (CCR5) antagonist, maraviroc, due to the pretreatment presence of low levels of chemokine (CXC motif) receptor 4 (CXCR4)-using virus. Methods:We present a novel protocol for the phenotyping of HIV based on ‘454’ pyrosequence data and apply this to two large data sets comprised of 104 628 (before treatment, day 1) and 191 637 (after treatment, day 11) reads from the envelope region. We study resistance in the context of the evolutionary history of the intrapatient viral population. Variation was also investigated both within and outside the V3 region, the region associated with the receptor switch. Results:CXCR4-using virus can be detected at low frequency prior to maraviroc treatment (∼0.5%) and at high frequency after failure of monotherapy (∼81%). Inferring an evolutionary tree from the 1674 unique reads that span the V3 region confirms that the CXCR4-using population emerged from low-frequency CXCR4-using variants present before treatment. Changes in the frequency of amino acid residues used at individual sites were found in regions outside the V3 region, indicative of other potential sites associated with receptor usage. Conclusion:We have provided a high-resolution snapshot of intrapatient viral variation, prior and after treatment with maraviroc, and detected preexisting CXCR4-using variants present at an extremely low frequency. The evolutionary analysis demonstrates the extent of diversity present at a single time point within an infected individual and the rapid effect of drug pressure on the structure of a viral population.

Assessing the feasibility of GS FLX Pyrosequencing for sequencing the Atlantic salmon genome

BackgroundWith a whole genome duplication event and wealth of biological data, salmonids are excellent model organisms for studying evolutionary processes, fates of duplicated genes and genetic and physiological processes associated with complex behavioral phenotypes. It is surprising therefore, that no salmonid genome has been sequenced. Atlantic salmon (Salmo salar) is a good representative salmonid for sequencing given its importance in aquaculture and the genomic resources available. However, the size and complexity of the genome combined with the lack of a sequenced reference genome from a closely related fish makes assembly challenging. Given the cost and time limitations of Sanger sequencing as well as recent improvements to next generation sequencing technologies, we examined the feasibility of using the Genome Sequencer (GS) FLX pyrosequencing system to obtain the sequence of a salmonid genome. Eight pooled BACs belonging to a minimum tiling path covering ~1 Mb of the Atlantic salmon genome were sequenced by GS FLX shotgun and Long Paired End sequencing and compared with a ninth BAC sequenced by Sanger sequencing of a shotgun library.ResultsAn initial assembly using only GS FLX shotgun sequences (average read length 248.5 bp) with ~30× coverage allowed gene identification, but was incomplete even when 126 Sanger-generated BAC-end sequences (~0.09× coverage) were incorporated. The addition of paired end sequencing reads (additional ~26× coverage) produced a final assembly comprising 175 contigs assembled into four scaffolds with 171 gaps. Sanger sequencing of the ninth BAC (~10.5× coverage) produced nine contigs and two scaffolds. The number of scaffolds produced by the GS FLX assembly was comparable to Sanger-generated sequencing; however, the number of gaps was much higher in the GS FLX assembly.ConclusionThese results represent the first use of GS FLX paired end reads for de novo sequence assembly. Our data demonstrated that this improved the GS FLX assemblies; however, with respect to de novo sequencing of complex genomes, the GS FLX technology is limited to gene mining and establishing a set of ordered sequence contigs. Currently, for a salmonid reference sequence, it appears that a substantial portion of sequencing should be done using Sanger technology.

Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development.

The Actinomycetales bacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 bioconvert a diverse range of organic substrates through lipid biosynthesis into large quantities of energy-rich triacylglycerols (TAGs). To describe the genetic basis of the Rhodococcus oleaginous metabolism, we sequenced and performed comparative analysis of the 9.27 Mb R. opacus PD630 genome. Metabolic-reconstruction assigned 2017 enzymatic reactions to the 8632 R. opacus PD630 genes we identified. Of these, 261 genes were implicated in the R. opacus PD630 TAGs cycle by metabolic reconstruction and gene family analysis. Rhodococcus synthesizes uncommon straight-chain odd-carbon fatty acids in high abundance and stores them as TAGs. We have identified these to be pentadecanoic, heptadecanoic, and cis-heptadecenoic acids. To identify bioconversion pathways, we screened R. opacus PD630, R. jostii RHA1, Ralstonia eutropha H16, and C. glutamicum 13032 for growth on 190 compounds. The results of the catabolic screen, phylogenetic analysis of the TAGs cycle enzymes, and metabolic product characterizations were integrated into a working model of prokaryotic oleaginy.

De Novo Next Generation Sequencing of Plant Genomes

The genome sequencing of all major food and bioenergy crops is of critical importance in the race to improve crop production to meet the future food and energy security needs of the world. Next generation sequencing technologies have brought about great improvements in sequencing throughput and cost, but do not yet allow for de novo sequencing of large repetitive genomes as found in most crop plants. We present a strategy that combines cutting edge next generation sequencing with “old school” genomics resources and allows rapid cost-effective sequencing of plant genomes.