Database resources of the National Center for Biotechnology Information.

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 34 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Custom implementations of the BLAST program provide sequence-based searching of many specialized datasets. New resources released in the past year include a new PubMed interface and NCBI datasets. Additional resources that were updated in the past year include PMC, Bookshelf, Genome Data Viewer, SRA, ClinVar, dbSNP, dbVar, Pathogen Detection, BLAST, Primer-BLAST, IgBLAST, iCn3D and PubChem. All of these resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.

ClinVar: improvements to accessing data.

ClinVar is a freely available, public archive of human genetic variants and interpretations of their relationships to diseases and other conditions, maintained at the National Institutes of Health (NIH). Submitted interpretations of variants are aggregated and made available on the ClinVar website (https://www.ncbi.nlm.nih.gov/clinvar/), and as downloadable files via FTP and through programmatic tools such as NCBI's E-utilities. The default view on the ClinVar website, the Variation page, was recently redesigned. The new layout includes several new sections that make it easier to find submitted data as well as summary data such as all diseases and citations reported for the variant. The new design also better represents more complex data such as haplotypes and genotypes, as well as variants that are in ClinVar as part of a haplotype or genotype but have no interpretation for the single variant. ClinVar's variant-centric XML had its production release in April 2019. The ClinVar website and E-utilities both have been updated to support the VCV (variation in ClinVar) accession numbers found in the variant-centric XML file. ClinVar's search engine has been fine-tuned for improved retrieval of search results.

Database resources of the National Center for Biotechnology Information.

The National Center for Biotechnology Information (NCBI) provides a large suite of online resources for biological information and data, including the GenBank® nucleic acid sequence database and the PubMed database of citations and abstracts published in life science journals. The Entrez system provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Custom implementations of the BLAST program provide sequence-based searching of many specialized datasets. New resources released in the past year include a new PubMed interface, a sequence database search and a gene orthologs page. Additional resources that were updated in the past year include PMC, Bookshelf, My Bibliography, Assembly, RefSeq, viral genomes, the prokaryotic genome annotation pipeline, Genome Workbench, dbSNP, BLAST, Primer-BLAST, IgBLAST and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Consensus coding sequence (CCDS) database: a standardized set of human and mouse protein-coding regions supported by expert curation.

The Consensus Coding Sequence (CCDS) project provides a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assembly in genome annotations produced independently by NCBI and the Ensembl group at EMBL-EBI. This dataset is the product of an international collaboration that includes NCBI, Ensembl, HUGO Gene Nomenclature Committee, Mouse Genome Informatics and University of California, Santa Cruz. Identically annotated coding regions, which are generated using an automated pipeline and pass multiple quality assurance checks, are assigned a stable and tracked identifier (CCDS ID). Additionally, coordinated manual review by expert curators from the CCDS collaboration helps in maintaining the integrity and high quality of the dataset. The CCDS data are available through an interactive web page (https://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi) and an FTP site (ftp://ftp.ncbi.nlm.nih.gov/pub/CCDS/). In this paper, we outline the ongoing work, growth and stability of the CCDS dataset and provide updates on new collaboration members and new features added to the CCDS user interface. We also present expert curation scenarios, with specific examples highlighting the importance of an accurate reference genome assembly and the crucial role played by input from the research community.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.

RefSeq: an update on mammalian reference sequences.

The National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database is a collection of annotated genomic, transcript and protein sequence records derived from data in public sequence archives and from computation, curation and collaboration (http://www.ncbi.nlm.nih.gov/refseq/). We report here on growth of the mammalian and human subsets, changes to NCBI's eukaryotic annotation pipeline and modifications affecting transcript and protein records. Recent changes to NCBI's eukaryotic genome annotation pipeline provide higher throughput, and the addition of RNAseq data to the pipeline results in a significant expansion of the number of transcripts and novel exons annotated on mammalian RefSeq genomes. Recent annotation changes include reporting supporting evidence for transcript records, modification of exon feature annotation and the addition of a structured report of gene and sequence attributes of biological interest. We also describe a revised protein annotation policy for alternatively spliced transcripts with more divergent predicted proteins and we summarize the current status of the RefSeqGene project.

Current status and new features of the Consensus Coding Sequence database.

The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.

Mouse genome annotation by the RefSeq project.

Complete and accurate annotation of the mouse genome is critical to the advancement of research conducted on this important model organism. The National Center for Biotechnology Information (NCBI) develops and maintains many useful resources to assist the mouse research community. In particular, the reference sequence (RefSeq) database provides high-quality annotation of multiple mouse genome assemblies using a combinatorial approach that leverages computation, manual curation, and collaboration. Implementation of this conservative and rigorous approach, which focuses on representation of only full-length and non-redundant data, produces high-quality annotation products. RefSeq records explicitly link sequences to current knowledge in a timely manner, updating public records regularly and rapidly in response to nomenclature updates, addition of new relevant publications, collaborator discussion, and user feedback. Whole genome re-annotation is also conducted at least every 12-18 months, and often more frequently in response to assembly updates or availability of informative data. This article highlights key features and advantages of RefSeq genome annotation products and presents an overview of NCBI processes to generate these data. Further discussion of NCBI's resources highlights useful features and the best methods for accessing our data.

Exploring and retrieving sequence and metadata for species across the tree of life with NCBI Datasets.

To explore complex biological questions, it is often necessary to access various data types from public data repositories. As the volume and complexity of biological sequence data grow, public repositories face significant challenges in ensuring that the data is easily discoverable and usable by the biological research community. To address these challenges, the National Center for Biotechnology Information (NCBI) has created NCBI Datasets. This resource provides straightforward, comprehensive, and scalable access to biological sequences, annotations, and metadata for a wide range of taxa. Following the FAIR (Findable, Accessible, Interoperable, and Reusable) data management principles, NCBI Datasets offers user-friendly web interfaces, command-line tools, and documented APIs, empowering researchers to access NCBI data seamlessly. The data is delivered as packages of sequences and metadata, thus facilitating improved data retrieval, sharing, and usability in research. Moreover, this data delivery method fosters effective data attribution and promotes its further reuse. This paper outlines the current scope of data accessible through NCBI Datasets and explains various options for exploring and downloading the data.

Contributions of functional genomics and proteomics to the study of immune responses in the Pacific white leg shrimp Litopenaeus vannamei.

The need for better control of infectious diseases in shrimp aquaculture and the ecological importance of crustacea in marine ecosystems have prompted interest in the study of crustacean immune systems, particularly those of shrimp. As shrimp and other crustacea are poorly understood from the immunological point of view, functional genomic and proteomic approaches have been applied as a means of quickly obtaining molecular information regarding immune responses in these organisms. In this article, a series of results derived from transcriptomic and proteomic studies in shrimp (Litopenaeus vannamei) are discussed. Expressed Sequence Tag analysis, differential expression cloning through Suppression Subtractive Hybridization, expression profiling using microarrays, and proteomic studies using mass spectrometry, have provided a wealth of useful data and opportunities for new avenues of research. Examples of new research directions arising from these studies in shrimp include the molecular diversity of antimicrobial effectors, the role of double stranded RNA as an inducer of antiviral immunity, and the possible overlap between antibacterial and antiviral responses in the shrimp.

Anti-lipopolysaccharide factor in Litopenaeus vannamei (LvALF): a broad spectrum antimicrobial peptide essential for shrimp immunity against bacterial and fungal infection.

Antimicrobial peptides are an essential component of the innate immune system of most organisms. Expressed sequence tag analysis from various shrimp (Litopenaeus vannamei) tissues revealed transcripts corresponding to two distinct sequences (LvALF1 and LvALF2) with strong sequence similarity to anti-lipopolysaccharide factor (ALF), an antimicrobial peptide originally isolated from the horseshoe crab Limulus polyphemus. Full-length clones contained a 528bp transcript with a predicted open reading frame coding for 120 amino acids in LvALF1, and a 623bp transcript with a predicted open reading frame coding for 93 amino acids in LvALF2. A reverse genetic approach was implemented to study the in vivo role of LvALF1 in protecting shrimp from bacterial, fungal and viral infections. Injection of double-stranded RNA (dsRNA) corresponding to the LvALF1 message resulted in a significant reduction of LvALF1 mRNA transcript abundance as determined by qPCR. Following knockdown, shrimp were challenged with low pathogenic doses of Vibrio penaeicida, Fusarium oxysporum or white spot syndrome virus (WSSV) and the resulting mortality curves were compared with controls. A significant increase of mortality in the LvALF1 knockdown shrimp was observed in the V. penaeicida and F. oxysporum infections when compared to controls, showing that this gene has a role in protecting shrimp from both bacterial and fungal infections. In contrast, LvALF1 dsRNA activated the sequence-independent innate anti-viral immune response giving increased protection from WSSV infection.

Genomic structure and transcriptional regulation of the penaeidin gene family from Litopenaeus vannamei.

Penaeidins are a family of shrimp antimicrobial peptides that have a unique molecular structure consisting of a highly conserved leader peptide followed by an N-terminal proline-rich domain and a C-terminal cysteine-rich domain. Three distinct classes of penaeidins, named PEN2, PEN3, and PEN4, are expressed in the hemocytes of the Pacific white shrimp, Litopenaeus vannamei. Multiple isoforms, generated by substitutions and deletions within the proline and cysteine-rich domains, have been reported at the mRNA level for all three classes of penaeidins suggesting that this is a highly diverse gene family; however, the genetic mechanisms by which sequence variability in the penaeidin gene family is generated are unknown. The present study examines the genomic sources for both class and isoform diversity in the penaeidin family. We show that each penaeidin class is encoded by a unique gene and that isoform diversity is generated by polymorphism within each penaeidin gene locus. Furthermore, the genomic regions upstream of each penaeidin gene were partially characterized and found to drive transcription.

The role of crustins in Litopenaeus vannamei in response to infection with shrimp pathogens: An in vivo approach.

Crustin antimicrobial peptides, identified in crustaceans, are hypothesized to have both antimicrobial and protease inhibitor activity based on their primary structure and in vitro assays. In this study, a reverse genetic approach was utilized to test the hypothesis that crustins are antimicrobial in vivo in response to bacterial and fungal challenge. Injection of double-stranded RNA specific to a 120-bp region of LvABP1, one of the most prominent crustin isoforms, yielded a significant reduction in the expression of both crustin mRNA and protein within the hemocytes. To test the role of crustins in the shrimp immune response, RNAi was first used to suppress crustin expression and animals were subsequently injected with low pathogenic doses of either Vibrio penaeicida or Fusarium oxysporum. A significant increase in mortality in crustin-depleted animals was observed in animals infected with V. penaeicida as compared to controls, whereas no significant change in shrimp mortality was observed following infection with F. oxysporum.

Lack of evidence for Litopenaeus vannamei Toll receptor (lToll) involvement in activation of sequence-independent antiviral immunity in shrimp.

Injection of non-specific dsRNA initiates a broad-spectrum innate antiviral immune response in the Pacific white shrimp, Litopenaeus vannamei, however, the receptor involved in recognition of this by-product of viral infections remains unknown. In vertebrates, dsRNA sensing is mediated by a class of Toll-like receptors (TLRs) and results in activation of the interferon system. Because a TLR (lToll) was recently characterized in L. vannamei, we investigated its potential role in dsRNA recognition. We showed that injection of non-specific RNA duplexes did not modify lToll gene expression. A reverse genetic approach was therefore implemented to study its role in vivo. Silencing of lToll did not impair the ability of non-specific dsRNA to trigger protection from white spot syndrome virus and did not increase the shrimp susceptibility to viral infection, when compared to controls. In contrast, gene-specific dsRNA injected to specifically silence lToll expression activated an antiviral response. These data strongly suggest that shrimp lToll plays no role in dsRNA-induced antiviral immunity.

Analysis of multiple tissue-specific cDNA libraries from the Pacific whiteleg shrimp, Litopenaeus vannamei.

Multiple small-scale transcriptome studies have been undertaken for various members of the Penaeidae. Penaeid shrimp are important both as members of diverse ecosystems around the world and for their importance as commercial commodities. Of the many shrimps, the most important from this family is the Pacific whiteleg shrimp, Litopenaeus vannamei, as it is the primary shrimp used in worldwide aquaculture. The sequencing and analysis of 13 656 expressed sequence tags (ESTs) from this species is presented. ESTs were derived from multiple tissue-specific cDNA libraries with an emphasis being placed on those tissues with predicted immune function. Assembly of the sequences into non-overlapping clusters yielded 7466 putative unigenes (1981 contigs and 5485 singletons). Multiple approaches were taken to assign putative function to each transcript; sequence homology searches using BLASTX (Basic Local Alignment Search Tool: Translated query versus protein database) of the National Center for Biotechnology Information's (NCBI) GenBank Database and Gene Ontology annotation, and still a significant portion of the shrimp ESTs (62%) had no homology with known proteins in the public databases. The sequence and complete annotation of all ESTs is available at www.marinegenomics.org, a publicly accessible database. In addition to providing the basic resources for microarray construction, transcript profiling, and novel gene discovery, this study constitutes the largest combined analysis of ESTs from any shrimp species and is a prelude to an even larger effort aimed at identifying and depleting highly redundant genes from shrimp cDNA libraries toward the goal of sequencing 100 000 shrimp ESTs.

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis.

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.

Diverse lectin repertoires in tunicates mediate broad recognition and effector innate immune responses.

It is widely recognized that humoral and phagocyte-associated lectins constitute critical components of innate immunity in vertebrates and invertebrates. Their functions include not only self/non-self recognition but also engaging associated effector mechanisms, such as complement-mediated opsonization and killing of potential pathogens. One of the unresolved questions concerns the diversity in recognition capacity of the lectin repertoire, particularly in those organisms lacking adaptive immunity. In this paper, we discuss evidence suggesting that lectin repertoire in invertebrates and protochordates is highly diversified, and includes most of the lectin classes described so far in vertebrate species, as well as associated effector pathways.