'PACLIMS': A component LIM system for high-throughput functional genomic analysis

BACKGROUND: Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. RESULTS: The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. CONCLUSION: Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors

\u27PACLIMS\u27: a component LIM system for high-throughput functional genomic analysis

BACKGROUND: Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the approximately 11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. RESULTS: The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. CONCLUSION: Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

MAP V1.0: User Manual

p. The Los Alamos chromosome 16 clones were digested by multiple enzymes; Balding and Torney [91] used this fragment data to calculate the probability of overlap between every two clones. Such probabilities, along with the associated clone names, are the input to MAP. Complexity. The clone assembly problem (with only false negative overlaps and no false positive overlaps) has been proven to be NP-complete [Golumbic and Shamir 92]. As will be shown, there are false positive and false negative overlaps; therefore, there is no deterministic polynomial algorithm to solve this problem, unless P=NP. In order to aid the user in solving the problem, MAP combines interactive graphics, editing abilities, and nondeterministic and deterministic solutions. This non-deterministic processing uses shuffle and annealing [Churchill et al. 92]. The deterministic processing uses interval graphs [Booth and Leuker 76]. 1. Department of Computer Science, Rensselaer P

Magnaporthe grisea Infection Triggers RNA Variation and Antisense Transcript Expression in Rice1[W]

Rice blast disease, caused by the fungal pathogen Magnaporthe grisea, is an excellent model system to study plant-fungal interactions and host defense responses. In this study, comprehensive analysis of the rice (Oryza sativa) transcriptome after M. grisea infection was conducted using robust-long serial analysis of gene expression. A total of 83,382 distinct 21-bp robust-long serial analysis of gene expression tags were identified from 627,262 individual tags isolated from the resistant (R), susceptible (S), and control (C) libraries. Sequence analysis revealed that the tags in the R and S libraries had a significant reduced matching rate to the rice genomic and expressed sequences in comparison to the C library. The high level of one-nucleotide mismatches of the R and S library tags was due to nucleotide conversions. The A-to-G and U-to-C nucleotide conversions were the most predominant types, which were induced in the M. grisea-infected plants. Reverse transcription-polymerase chain reaction analysis showed that expression of the adenine deaminase and cytidine deaminase genes was highly induced after inoculation. In addition, many antisense transcripts were induced in infected plants and expression of four antisense transcripts was confirmed by strand-specific reverse transcription-polymerase chain reaction. These results demonstrate that there is a series of dynamic and complex transcript modifications and changes in the rice transcriptome at the M. grisea early infection stages

The oryza map alignment project (OMAP): a new resource for comparative genome studies within oryza

Rice (Oryza sativa L.) is the most important human food crop in the world. The agronomic importance of rice, its shared evolutionary history with major cereal crops, and small genome size have led to the generation of a high-quality finished genome sequence by the International Rice Genome Sequencing Project (2005). The highly accurate and public IRGSP sequence now serves as a unifying research platform for a complete functional characterization of the rice genome. Such an analysis will investigate the rice transcriptome, proteome, and metabolome, with the goal of understanding the biological function of all 35,000 to 40,000 rice genes and applying that information to improve rice production and quality. This comprehensive analysis will utilize a variety of techniques and resources from expression and genome tiling arrays to collections of tagged mutant populations developed in elite cultivars grown around the world. Comparative genomics between the cereal genomes and within the genus Oryza will also play a critical role in our understanding of the rice genome (Ahn et al. 1993; Ahn and Tanksley 1993; Bennetzen and Ma 2003; Han and Xue 2003; Huang and Kochert 1994; Jena et al. 1994; Ma and Bennetzen 2004). By comparing genome organization, genes, and intergenic regions between cereal species, one can identify regions of the genome that are highly conserved or rapidly evolving. Such regions are expected to yield key insights into genome evolution, speciation, and domestication. The study of conserved noncoding sequences (CNSs) between cereal genomes will also increase our ability to understand and isolate regulatory elements required for precise developmental and temporal gene expression (Kaplinsky et al. 2002). The genus Oryza is composed of two cultivated (O. sativa and O. glaberrima) and 21 wild species (Khush 1997; Vaughan et al. 2003). Based on recent phylogenetic data, Ge et al. (1999) proposed that Porteresia coarctata should be included in the genus as the 24th Oryza species. Cultivated rice is classified as an AA genome diploid and has 6 wild AA genome relatives. The remaining 15 wild species are classified into 9 other genome types that include both diploid and tetraploid species. Figure 15.1 shows a proposed phylogenetic tree of the genus Oryza as described by Ge et al. (1999) based on the analysis of two nuclear genes and one chloroplast gene. The wild rice species offer a largely untapped resource of agriculturally important genes that have the potential to solve many of the problems in rice production that we face today, such as yield, drought, and salt tolerance as well as disease and insect resistance. To better understand the wild species of rice and take advantage of the IRGSP genome sequence, we have embarked on an ambitious comparative genomics program entitled the Oryza Map Alignment Project (OMAP). The long-term objective of OMAP is to create a genome-level closed experimental system for the genus Oryza that can be used as a research platform to study evolution, development, genome organization, polyploidy, domestication, gene regulatory networks, and crop improvement. The specific objectives of OMAP are to (1) construct deep-coverage large-insert bacterial artificial chromosome (BAC) libraries from 11 wild and 1 cultivated African Oryza species (O. glaberrima); (2) fingerprint and end-sequence clones from all 12 BAC libraries; (3) construct physical maps for all 12 Oryza species and align them to the IRGSP genome sequence; and (4) perform a detailed reconstruction of rice chromosomes 1, 3, and 10 across all 12 Oryza species (Wing et al. 2005). This chapter presents our current progress for OMAP and some early glimpses into the results we are finding

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ∼ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI

A bacterial artificial chromosome library for Biomphalaria glabrata , intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata , members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata , a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni , was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 ´ coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI