The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

The human genome reference assembly is crucial for aligning and analyzing sequence data, and for genome annotation, among other roles. However, the models and analysis assumptions that underlie the current assembly need revising to fully represent human sequence diversity. Improved analysis tools and updated data reporting formats are also required.

Comparative genomics is the comparison of genetic information within and across organisms to understand the evolution, structure, and function of genes, proteins, and non-coding regions (Sivashankari and Shanmughavel, Bioinformation 1:376-8, 2007). Advances in sequencing technology and assembly algorithms have resulted in the ability to sequence large genomes and provided a wealth of data that are being used in comparative genomic analyses. Comparative analysis can be leveraged to systematically explore and evaluate the biological relationships and evolution between species, aid in understanding the structure and function of genes, and gain a better understanding of disease and potential drug targets. As our knowledge of genetics expands, comparative genomics can help identify emerging model organisms among a broader span of the tree of life, positively impacting human health. This impact includes, but is not limited to, zoonotic disease research, therapeutics development, microbiome research, xenotransplantation, oncology, and toxicology. Despite advancements in comparative genomics, new challenges have arisen around the quantity, quality assurance, annotation, and interoperability of genomic data and metadata. New tools and approaches are required to meet these challenges and fulfill the needs of researchers. This paper focuses on how the National Institutes of Health (NIH) Comparative Genomics Resource (CGR) can address both the opportunities for comparative genomics to further impact human health and confront an increasingly complex set of challenges facing researchers.

The human reference genome assembly plays a central role in nearly all aspects of today’s basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009 and reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that while the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.

The National Center for Biotechnology Information (NCBI) Clone DB (http://www.ncbi.nlm.nih.gov/clone/) is an integrated resource providing information about and facilitating access to clones, which serve as valuable research reagents in many fields, including genome sequencing and variation analysis. Clone DB represents an expansion and replacement of the former NCBI Clone Registry and has records for genomic and cell-based libraries and clones representing more than 100 different eukaryotic taxa. Records provide details of library construction, associated sequences, map positions and information about resource distribution. Clone DB is indexed in the NCBI Entrez system and can be queried by fields that include organism, clone name, gene name and sequence identifier. Whenever possible, genomic clones are mapped to reference assemblies and their map positions provided in clone records. Clones mapping to specific genomic regions can also be searched for using the NCBI Clone Finder tool, which accepts queries based on sequence coordinates or features such as gene or transcript names. Clone DB makes reports of library, clone and placement data on its FTP site available for download. With Clone DB, users now have available to them a centralized resource that provides them with the tools they will need to make use of these important research reagents.

An accurate and complete reference human genome sequence assembly is essential for accurately interpreting individual genomes and associating sequence variation with disease phenotypes. While the current reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can help overcome these problems, even the longest available reads do not resolve all regions of the human genome. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones, an optical map, and 100X whole genome shotgun (WGS) sequence coverage using short (lllumina) read pairs. We used the WGS sequence and the GRCh37 reference assembly to create a sequence assembly of the CHM1 genome. We subsequently incorporated 382 finished CHORI-17 BAC clone sequences to generate a second draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene and repeat content show this assembly to be of excellent quality and contiguity, and comparisons to ClinVar and the NHGRI GWAS catalog show that the CHM1 genome does not harbor an excess of deleterious alleles. However, comparison to assembly-independent resources, such as BAC clone end sequences and long reads generated by a different sequencing technology (PacBio), indicate misassembled regions. The great majority of these regions is enriched for structural variation and segmental duplication, and can be resolved in the future by sequencing BAC clone tiling paths. This publicly available first generation assembly will be integrated into the Genome Reference Consortium (GRC) curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly.