EcoGene-RefSeq: EcoGene tools applied to the RefSeq prokaryotic genomes.

SUMMARY: EcoGene.org is a genome database and website dedicated to Escherichia coli K-12 substrain MG1655 that is revised daily using information derived from the biomedical literature and in-house analysis. EcoGene is a major source of annotation updates for the MG1655 Genbank record, one of only a few Genbank genome records that are updated by a community effort. The Reference Sequence (RefSeq) database, built by The National Center for Biotechnology Information, comprises a set of duplicate Genbank genome records that can be modified by the NCBI staff annotators. EcoGene-RefSeq is being developed as a stand-alone internet resource to facilitate the usage of EcoGene-based tools on any of the >2400 completed prokaryotic genome records that are currently available at the RefSeq database. AVAILABILITY: The web interface of EcoGene-RefSeq is available at http://www.ecogene.org/refseq. CONTACT: krudd@med.miami.edu or j.zhou1@miami.edu.

EcoGene 3.0.

EcoGene (http://ecogene.org) is a database and website devoted to continuously improving the structural and functional annotation of Escherichia coli K-12, one of the most well understood model organisms, represented by the MG1655(Seq) genome sequence and annotations. Major improvements to EcoGene in the past decade include (i) graphic presentations of genome map features; (ii) ability to design Boolean queries and Venn diagrams from EcoArray, EcoTopics or user-provided GeneSets; (iii) the genome-wide clone and deletion primer design tool, PrimerPairs; (iv) sequence searches using a customized EcoBLAST; (v) a Cross Reference table of synonymous gene and protein identifiers; (vi) proteome-wide indexing with GO terms; (vii) EcoTools access to >2000 complete bacterial genomes in EcoGene-RefSeq; (viii) establishment of a MySql relational database; and (ix) use of web content management systems. The biomedical literature is surveyed daily to provide citation and gene function updates. As of September 2012, the review of 37 397 abstracts and articles led to creation of 98 425 PubMed-Gene links and 5415 PubMed-Topic links. Annotation updates to Genbank U00096 are transmitted from EcoGene to NCBI. Experimental verifications include confirmation of a CTG start codon, pseudogene restoration and quality assurance of the Keio strain collection.

Bacterial genome reengineering.

The web application PrimerPair at ecogene.org generates large sets of paired DNA sequences surrounding- all protein and RNA genes of Escherichia coli K-12. Many DNA fragments, which these primers amplify, can be used to implement a genome reengineering strategy using complementary in vitro cloning and in vivo recombineering. The integration of a primer design tool with a model organism database increases the level of quality control. Computer-assisted design of gene primer pairs relies upon having highly accurate genomic DNA sequence information that exactly matches the DNA of the cells being used in the laboratory to ensure predictable DNA hybridizations. It is equally crucial to have confidence that the predicted start codons define the locations of genes accurately. Annotations in the EcoGene database are queried by PrimerPair to eliminate pseudogenes, IS elements, and other problematic genes before the design process starts. These projects progressively familiarize users with the EcoGene content, scope, and application interfaces that are useful for genome reengineering projects. The first protocol leads to the design of a pair of primer sequences that were used to clone and express a single gene. The N-terminal protein sequence was experimentally verified and the protein was detected in the periplasm. This is followed by instructions to design PCR primer pairs for cloning gene fragments encoding 50 periplasmic proteins without their signal peptides. The design process begins with the user simply designating one pair of forward and reverse primer endpoint positions relative to all start and stop codon positions. The gene name, genomic coordinates, and primer DNA sequences are reported to the user. When making chromosomal deletions, the integrity of the provisional primer design is checked to see whether it will generate any unwanted double deletions with adjacent genes. The bad designs are recalculated and replacement primers are provided alongside the requested primers. A list of all genes with overlaps includes those expressed from the translational coupling motifs 5'-UGAUG-3' and 5'-AUGA-3'. Rigid alignments of the 893 ribosome binding sites (RBSs) linked to the AUG codons of this coupled subset are assessed for information content using WebLogo 3.0. These specialized logos are missing the G at the prominent information peak position normally seen in the rigid alignment of all genes. This novel GHOLE motif was apparently masked by the normal RBSs in two previously published rigid alignments. We propose a model constraining the distance between the ATG and the RBS, obviating- the need for a flexible linker model to reveal a Shine-Dalgarno-like sequence.