Tamara Goldfarb

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.

Abstract Comprehensive genome annotation is essential to understand the impact of clinically relevant variants. However, the absence of a standard for clinical reporting and browser display complicates the process of consistent interpretation and reporting. To address these challenges, Ensembl/GENCODE 1 and RefSeq 2 launched a joint initiative, the Matched Annotation from NCBI and EMBL-EBI (MANE) collaboration, to converge on human gene and transcript annotation and to jointly define a high-value set of transcripts and corresponding proteins. Here, we describe the MANE transcript sets for use as universal standards for variant reporting and browser display. The MANE Select set identifies a representative transcript for each human protein-coding gene, whereas the MANE Plus Clinical set provides additional transcripts at loci where the Select transcripts alone are not sufficient to report all currently known clinical variants. Each MANE transcript represents an exact match between the exonic sequences of an Ensembl/GENCODE transcript and its counterpart in RefSeq such that the identifiers can be used synonymously. We have now released MANE Select transcripts for 97% of human protein-coding genes, including all American College of Medical Genetics and Genomics Secondary Findings list v3.0 (ref. 3 ) genes. MANE transcripts are accessible from major genome browsers and key resources. Widespread adoption of these transcript sets will increase the consistency of reporting, facilitate the exchange of data regardless of the annotation source and help to streamline clinical interpretation.

Reference sequences and annotations serve as the foundation for many lines of research today, from organism and sequence identification to providing a core description of the genes, transcripts and proteins found in an organism's genome. Interpretation of data including transcriptomics, proteomics, sequence variation and comparative analyses based on reference gene annotations informs our understanding of gene function and possible disease mechanisms, leading to new biomedical discoveries. The Reference Sequence (RefSeq) resource created at the National Center for Biotechnology Information (NCBI) leverages both automatic processes and expert curation to create a robust set of reference sequences of genomic, transcript and protein data spanning the tree of life. RefSeq continues to refine its annotation and quality control processes and utilize better quality genomes resulting from advances in sequencing technologies as well as RNA-Seq data to produce high-quality annotated genomes, ortholog predictions across more organisms and other products that are easily accessible through multiple NCBI resources. This report summarizes the current status of the eukaryotic, prokaryotic and viral RefSeq resources, with a focus on eukaryotic annotation, the increase in taxonomic representation and the effect it will have on comparative genomics. The RefSeq resource is publicly accessible at https://www.ncbi.nlm.nih.gov/refseq.

Recombination between moderately divergent DNA sequences is impaired compared with identical sequences. In yeast, an HO endonuclease-induced double-strand break can be repaired by single-strand annealing (SSA) between flanking homologous sequences. A 3% sequence divergence between 205-bp sequences flanking the double-strand break caused a 6-fold reduction in repair compared with identical sequences. This reduction in heteroduplex rejection was suppressed in a mismatch repair-defective msh6 Delta strain and partially suppressed in an msh2 separation-of-function mutant. In mlh1 Delta strains, heteroduplex rejection was greater than in msh6 Delta strains but less than in wild type. Deleting PMS1, MLH2,or MLH3 had no effect on heteroduplex rejection, but a pms1 Delta mlh2 Delta mlh3 Delta triple mutant resembled mlh1 Delta. However, correction of the mismatches within heteroduplex SSA intermediates required PMS1 and MLH1 to the same extent as MSH2 and MSH6. An SSA competition assay in which either diverged or identical repeats can be used for repair showed that heteroduplex DNA is likely to be unwound rather than degraded. This conclusion is supported by the finding that deleting the SGS1 helicase also suppressed heteroduplex rejection.