functional analyses, including investigations of plastid tran- scriptomes and proteomes are also progressing rapidly (24). Several bioinformatic resources provide information on organelle genomes, and tools speciﬁc for these genomes have been developed (25). The standard repository for full genome sequences, the GenBank, EMBL and DDBJ nucleot- ide sequence databases, currently includes 44 complete plastid genomes sequenced since 1986. The NCBI GenBank genome section lists entire organelle genome sequences submitted to the database and reviewed by NCBI staff (26). GOBASE (27) also maintains a list of sequenced organelle genomes. A stand- ardized nomenclature for plastid-encoded protein genes is available through the UniProt database (http://www.expasy. org/txt/plastid.txt). A web-based annotation tool, DOGMA, provides a graphical user interface to annotate draft and ﬁn- ished organelle genomes based on sequence similarity searches and RNA secondary structure prediction (28). The program GRAPPA has been used for phylogenetic analysis of chloroplast gene order changes (29). A plastid gene order database was developed with uniform gene names for 32 plas- tid genomes (30). In addition, 500 primers are now available for targeted PCR ampliﬁcation of sequences from chloroplast genomes (http://bfw.ac.at/200/1859.html). A prerequisite of future research is the accessibility of well- annotated, easy-to-use sequence data. However, several major limiting factors exist including ﬂat ﬁle presentation of annot- ated organelle genomes, lack of standard data structure for relational databases, and non-uniform annotation quality. Errors in the annotation typically persist in the standard data- bases (e.g. the gene rpl2 is annotated as rp12 in the Oryza sativa chloroplast DNA, a human error). As a heritage of early annotations, gene name variants, unidentiﬁed ycfs and ORFs, and unannotated genes are present in some genomes. Given the ubiquity of phylogenetic studies based on plastid gene sequences, the ﬂat ﬁle format makes search and data retrieval cumbersome. RNA editing, a post-transcriptional process that alters spe- ciﬁc RNA bases prior to translation, is common in the chloro- plast genomes of some land plants (31). RNA editing can result in the creation of start codons and removal of stop codons, as well as making radical amino acid substitutions that would not be predicted based on the DNA sequence alone. Accurate genome annotation and inference of protein sequences often cannot be accomplished without knowledge of RNA editing sites (e.g. in the chloroplast DNA of Antho- ceros formosae, Adiantum capillus-veneris and Zea mays). The pace of new data generation and large-scale analyses demand a better integration of resources for chloroplast gen- ome research. ChloroplastDB is a relational database with a user-friendly interface and tools to aid the analysis of chloro- plast genome sequences. DATA MANAGEMENT AND ORGANIZATION ChloroplastDB was designed using a MySQL database struc- ture (Figure 1). The tables in the relational database store data related to the genes, nucleotide sequences and annotated pro- tein sequences for coding sequences (CDS). The databases contain fully sequenced plastid genomes obtained from the NCBI RefSeq section (http://www.ncbi.nlm.nih.gov/ Nucleic Acids Research, 2006, Vol. 34, Database issue D693 genomes/static/euk_o.html). All genes, including protein- coding genes, tRNA, rRNA, hypothetical ORFs (ycf, ORF) were parsed and incorporated into the database. The standard process for extracting and storing data was carried out as follows: (i) A GenBank XML file containing a plastid genome sequence is downloaded. The XML format ensures better integrity of parsed data than GenBank flat files. (ii) Using in-house XML parsers written in Perl, the XML data is extracted, filtered through quality control steps and for- matted properly. The cleaned data are stored in the data- base in a form conducive to efficient data transactions. (iii) Using the coordinates from the features (CDS, tRNA, rRNA, intron), the corresponding nucleotide sequence is extracted from the genome and stored in the database. (iv) In a few instances when a parsed sequence lacks appropriate annotation, the GenBank records are updated with expert annotations after automatic processing of the XML file. (v) Three BLAST databases are created: one for the whole genome sequence from all organisms, a second for the annotated protein sequences of all organisms in the data- base, and a third for the generated nucleotide CDS from each organism. When new sequences are added to the database, the proteins are sorted into potentially orthologous sets or ‘tribes’ using tribeMCL (32). First, a sequence similarity proﬁle is obtained by all-against-all BLAST on the protein sequences at a thresh- old of 1E-3. The BLAST output is fed to tribeMCL, which then generates a list of tribes representing protein fam- ilies. This output is parsed and the tribes are updated in the database. The quality control procedure is crucial in maintaining the integrity and accuracy of the extracted data. There appear to be some irregularities with the GenBank annotations. The genomic region spanning one gene and the gene features (CDS, tRNA, rRNA) share the same gene name. In case of overlapping and nested genes, the annotation for the second (or nested) gene could be attributed to the ﬁrst gene, resulting in confusions. Also, there are instances where the gene names are not included in the feature description. We have avoided the problem by using the coordinates for each ‘gene’ feature as the primary reference, and after that, the coordinates of other features are checked and assigned new gene names. For example, rps12 is a trans-spliced gene containing three exons in the angiosperm chloroplast genome. The ﬁrst exon is loc- ated 30 kb upstream of the second exon, and on the opposite coding strand. The initial parsed record for the gene contained many other genes nested in the intron region. After the ﬁltering step, those nested genes were dissociated from the name ‘rps12’ and assigned to their appropriate names. If no gene name was found, the feature was deemed to be an ‘ophan’ and assigned a local name (starting with ‘lcl_anno’). When the GenBank annotation included RNA edited sites, both the location and type of edits were extracted from the record. The information was used to generate an edited pseu- dosequence that was stored with a list of edited sites. Just 38 genes with 541 annotated, experimental veriﬁed sites from A.formosae and Physcomitrella patens are included in the current GenBank annotations. RNA editing has been reported