D108 Nucleic Acids Research, 2008, Vol. 36, Database issue BACKGROUND A consequence of the escalating pace of genomic sequencing has been the requirement for novel methodol- ogy and large-scale eﬀorts to interpret and annotate sequence function. Initial eﬀorts to achieve this were primarily focused on identifying protein-coding genes, RNA genes and repetitive DNA, since the rules governing their presence are generally tractable. However, less annotated, due to their small size and variability, gene regulatory sequences are widely regarded to be at least as important to our understanding of biological systems. To aid in their identiﬁcation, computational techniques such as phylogenetic footprinting, transcription factor (TF)- binding matrices, and motif clustering have been devel- oped (1–3). Unfortunately, the predictive ability of such methods has been diﬃcult to assess without large, well- described and comprehensive collections of biologically validated regulatory sequences (3). Sets of cis-regulatory sequences have been annotated by curation from the primary literature and several databases have been developed to collect and disseminate these sets (4–11). However, these databases are often species- or process- speciﬁc, and do not provide suﬃcient details about the experiments or conditions under which function was demonstrated, and in some cases require payment for access. Data access is generally limited to web-based search pages without any option for the programmatic interaction essential to most bioinformatics studies. Finally, they are typically ‘closed systems’ in that they do not allow continued addition or annotation by the research community and as such are not maintainable over the long term without vast resources. We have developed the Open Regulatory Annotation database (ORegAnno) to overcome these challenges and support research in regulatory biology (12). ORegAnno provides standardized technologies for the long-term, community- driven, open-access curation of cis-regulatory data. Here we provide an update of developments on the ORegAnno database and progress in the ﬁeld of open regulatory annotation. OVERVIEW ORegAnno (http://www.oreganno.org) is a database and literature curation system for community-based annota- tion of experimentally proven DNA regulatory regions, transcription factor binding sites (TFBS) and regulatory variants. A ‘publication queue’ allows papers of interest to be added to the system for future curation. Thus both regulatory papers and their regulatory sequences are managed in the system. ORegAnno is based on open- source technology and is comprised of a MySQL database with a Java-based web application that indexes new annotations using the Lucene search engine (http:// lucene.apache.org/) and provides programmatic access to the underlying data using Hibernate (http://www. hibernate.org/) and SOAP Web Services. Figure 1 outlines the annotation process and information ﬂow. Users in the gene regulation community can enter or ‘check out’ papers from the publication queue for detailed manual curation, using a series of annotation pages. A typical record entry consists of species, sequence type, sequence (plus suﬃcient ﬂanking sequence for genome alignment), target gene, binding factor, experimental outcome and one or more detailed lines of experimental evidence demonstrating function of the sequence. Records are cross-referenced to Ensembl or Entrez Gene identiﬁers, PubMed and dbSNP (for regulatory polymorphisms). Before committing a record to the database, ORegAnno performs a number of error checks (e.g. that the sequence has not been entered previously) and asks the user to verify its contents. A BLAST-based mapping agent then assigns genome coordinates to each sequence, allowing it to be viewed as a track in the Ensembl or UCSC genome browsers. Once ﬁnished with a paper, a user will then ‘close’ it in the queue and assign an annotation result (success, neutral or failure). Existing records can be updated, commented and scored (positive if veriﬁed as correct; negative if a problem is identiﬁed) by any registered user or deprecated and replaced by a ‘Validator’ user. The complete database or any subset can be searched or downloaded in a number of formats or accessed programmatically. RECENT DEVELOPMENTS New entries Since ORegAnno was ﬁrst released, the collection has grown by 10-fold from 2691 to 30 145 records. This total includes 15 738 regulatory regions, 14 229 TFBSs and 178 regulatory variants (polymorphisms and haplotypes) from 19 species (Table 1). A total of 29 433 records have been mapped to one of 14 species representing a mapping success rate of 98%. New additions were incorporated from external datasets including a large set of human promoters (13), the REDﬂy resource (9), HBB and Erythroid modules (14,15), the Vista Enhancer dataset (11), ChIP–chip sites for CTCF(16) and multiple yeast TFs(17,18) and ChIP-Seq sites for STAT1 (19) and REST (20). Apart from the 11 external datasets currently in ORegAnno, extensive manual curation of the literature has produced an additional 1293 original sequence records. A large number of annotations were entered during the RegCreative Jamboree (http://www.dmbr. ugent.be/bioit/contents/regcreative/) at which 130 scienti- ﬁc articles were examined in depth with 96 papers meeting the criteria for annotation and resulting in 501 new regulatory sequence records. In total, 922 publications have been curated by 45 contributing users (from >300 registered users). The complete set of records contain regulatory sequences for over 3853 genes and 465 TFs, describe 41 856 experimental sources of evidence referen- cing 31 diﬀerent cell types and are further annotated by 49 807 user-comments. The majority of records (98.9%) had positive experimental outcomes (i.e. the experiments demonstrated the sequence to be functional) but a small set of negative or neutral results have also been catalogued.