The Human Pangenome Project: a global resource to map genomic diversity.

The human reference genome is the most widely used resource in human genetics and is due for a major update. Its current structure is a linear composite of merged haplotypes from more than 20 people, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent global human genomic variation. A high-quality reference with global representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. The Human Pangenome Reference Consortium aims to create a more sophisticated and complete human reference genome with a graph-based, telomere-to-telomere representation of global genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete diploid genomes. With attention to ethical frameworks, the human pangenome reference will contain a more accurate and diverse representation of global genomic variation, improve gene-disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome, and serve as the ultimate genetic resource for future biomedical research and precision medicine.

The NCBI Comparative Genome Viewer (CGV) is an interactive visualization tool for the analysis of whole-genome eukaryotic alignments.

We report a new visualization tool for analysis of whole-genome assembly-assembly alignments, the Comparative Genome Viewer (CGV) (https://ncbi.nlm.nih.gov/genome/cgv/). CGV visualizes pairwise same-species and cross-species alignments provided by National Center for Biotechnology Information (NCBI) using assembly alignment algorithms developed by us and others. Researchers can examine large structural differences spanning chromosomes, such as inversions or translocations. Users can also navigate to regions of interest, where they can detect and analyze smaller-scale deletions and rearrangements within specific chromosome or gene regions. RefSeq or user-provided gene annotation is displayed where available. CGV currently provides approximately 800 alignments from over 350 animal, plant, and fungal species. CGV and related NCBI viewers are undergoing active development to further meet needs of the research community in comparative genome visualization.

Telomere-to-telomere assembly of a complete human X chromosome.

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.

Database resources of the National Center for Biotechnology Information.

The National Center for Biotechnology Information (NCBI) provides online information resources for biology, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for most of these databases. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, SciENcv, the NIH Comparative Genomics Resource (CGR), NCBI Virus, SRA, RefSeq, foreign contamination screening tools, Taxonomy, iCn3D, ClinVar, GTR, MedGen, dbSNP, ALFA, ClinicalTrials.gov, Pathogen Detection, antimicrobial resistance resources, and PubChem. These resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information in 2023.

The National Center for Biotechnology Information (NCBI) provides online information resources for biology, including the GenBank® nucleic acid sequence database and the PubMed® database of citations and abstracts published in life science journals. NCBI provides search and retrieval operations for most of these data from 35 distinct databases. The E-utilities serve as the programming interface for most of these databases. New resources include the Comparative Genome Resource (CGR) and the BLAST ClusteredNR database. Resources receiving significant updates in the past year include PubMed, PMC, Bookshelf, IgBLAST, GDV, RefSeq, NCBI Virus, GenBank type assemblies, iCn3D, ClinVar, GTR, dbGaP, ALFA, ClinicalTrials.gov, Pathogen Detection, antimicrobial resistance resources, and PubChem. These resources can be accessed through the NCBI home page at https://www.ncbi.nlm.nih.gov.

Accessing NCBI data using the NCBI Sequence Viewer and Genome Data Viewer (GDV).

The National Center for Biotechnology Information (NCBI) is an archive providing free access to a wide range and large volume of biological sequence data and literature. Staff scientists at NCBI analyze user-submitted data in the archive, producing gene and SNP annotation and generating sequence alignment tools. NCBI's flagship genome browser, Genome Data Viewer (GDV), displays our in-house RefSeq annotation; is integrated with other NCBI resources such as Gene, dbGaP, and BLAST; and provides a platform for customized analysis and visualization. Here, we describe how members of the biomedical research community can use GDV and the related NCBI Sequence Viewer (SV) to access, analyze, and disseminate NCBI and custom biomedical sequence data. In addition, we report how users can add SV to their own web pages to create a custom graphical sequence display without the need for infrastructure investments or back-end deployments.

The NIH Comparative Genomics Resource: addressing the promises and challenges of comparative genomics on human health.

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.

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.

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 38 distinct databases. The E-utilities serve as the programming interface for the Entrez system. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. New resources released in the past year include PubMed Labs and a new sequence database search. Resources that were updated in the past year include PubMed, PMC, Bookshelf, genome data viewer, Assembly, prokaryotic genomes, Genome, BioProject, dbSNP, dbVar, BLAST databases, igBLAST, iCn3D and PubChem. All of these resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

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 dbGaP data browser: a new tool for browsing dbGaP controlled-access genomic data.

The database of Genotypes and Phenotypes (dbGaP) Data Browser (https://www.ncbi.nlm.nih.gov/gap/ddb/) was developed in response to requests from the scientific community for a resource that enable view-only access to summary-level information and individual-level genotype and sequence data associated with phenotypic features maintained in the controlled-access tier of dbGaP. Until now, the dbGaP controlled-access environment required investigators to submit a data access request, wait for Data Access Committee review, download each data set and locally examine them for potentially relevant information. Existing unrestricted-access genomic data browsing resources (e.g. http://evs.gs.washington.edu/EVS/, http://exac.broadinstitute.org/) provide only summary statistics or aggregate allele frequencies. The dbGaP Data Browser serves as a third solution, providing researchers with view-only access to a compilation of individual-level data from general research use (GRU) studies through a simplified controlled-access process. The National Institutes of Health (NIH) will continue to improve the Browser in response to user feedback and believes that this tool may decrease unnecessary download requests, while still facilitating responsible genomic data-sharing.

Single haplotype assembly of the human genome from a hydatidiform mole.

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human 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 improve assembly, even the longest available reads do not resolve all regions. 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 and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly.

Clone DB: an integrated NCBI resource for clone-associated data.

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.

Rapid and sensitive detection of genome contamination at scale with FCS-GX.

Assembled genome sequences are being generated at an exponential rate. Here we present FCS-GX, part of NCBI's Foreign Contamination Screen (FCS) tool suite, optimized to identify and remove contaminant sequences in new genomes. FCS-GX screens most genomes in 0.1-10 min. Testing FCS-GX on artificially fragmented genomes demonstrates high sensitivity and specificity for diverse contaminant species. We used FCS-GX to screen 1.6 million GenBank assemblies and identified 36.8 Gbp of contamination, comprising 0.16% of total bases, with half from 161 assemblies. We updated assemblies in NCBI RefSeq to reduce detected contamination to 0.01% of bases. FCS-GX is available at https://github.com/ncbi/fcs/ or https://doi.org/10.5281/zenodo.10651084 .

Interactive visualization of whole eukaryote genome alignments using NCBI's Comparative Genome Viewer (CGV).

We report a new visualization tool for analysis of whole genome assembly-assembly alignments, the Comparative Genome Viewer (CGV) (https://ncbi.nlm.nih.gov/genome/cgv/). CGV visualizes pairwise same-species and cross-species alignments provided by NCBI using assembly alignment algorithms developed by us and others. Researchers can examine the alignments between the two assemblies using two alternate views: a chromosome ideogram-based view or a 2D genome dotplot. Whole genome alignment views expose large structural differences spanning chromosomes, such as inversions or translocations. Users can also navigate to regions of interest, where they can detect and analyze smaller-scale deletions and rearrangements within specific chromosome or gene regions. RefSeq or user-provided gene annotation is displayed in the ideogram view where available. CGV currently provides approximately 700 alignments from over 300 animal, plant, and fungal species. CGV and related NCBI viewers are undergoing active development to further meet needs of the research community in comparative genome visualization.

Rapid and sensitive detection of genome contamination at scale with FCS-GX.

Assembled genome sequences are being generated at an exponential rate. Here we present FCS-GX, part of NCBI's Foreign Contamination Screen (FCS) tool suite, optimized to identify and remove contaminant sequences in new genomes. FCS-GX screens most genomes in 0.1-10 minutes. Testing FCS-GX on artificially fragmented genomes demonstrates sensitivity >95% for diverse contaminant species and specificity >99.93%. We used FCS-GX to screen 1.6 million GenBank assemblies and identified 36.8 Gbp of contamination (0.16% of total bases), with half from 161 assemblies. We updated assemblies in NCBI RefSeq to reduce detected contamination to 0.01% of bases. FCS-GX is available at https://github.com/ncbi/fcs/.

Evaluation of Lou et al.: Proposed benefits of a population-specific genome reference.

One snapshot of the peer review process for "Haplo-type-resolved de novo assembly of a Tujia genome suggests the necessity for high-quality population-specific genome references" (Lou et al., 2022).

Personalized genome assembly for accurate cancer somatic mutation discovery using tumor-normal paired reference samples.

BACKGROUND: The use of a personalized haplotype-specific genome assembly, rather than an unrelated, mosaic genome like GRCh38, as a reference for detecting the full spectrum of somatic events from cancers has long been advocated but has never been explored in tumor-normal paired samples. Here, we provide the first demonstrated use of de novo assembled personalized genome as a reference for cancer mutation detection and quantifying the effects of the reference genomes on the accuracy of somatic mutation detection. RESULTS: We generate de novo assemblies of the first tumor-normal paired genomes, both nuclear and mitochondrial, derived from the same individual with triple negative breast cancer. The personalized genome was chromosomal scale, haplotype phased, and annotated. We demonstrate that it provides individual specific haplotypes for complex regions and medically relevant genes. We illustrate that the personalized genome reference not only improves read alignments for both short-read and long-read sequencing data but also ameliorates the detection accuracy of somatic SNVs and SVs. We identify the equivalent somatic mutation calls between two genome references and uncover novel somatic mutations only when personalized genome assembly is used as a reference. CONCLUSIONS: Our findings demonstrate that use of a personalized genome with individual-specific haplotypes is essential for accurate detection of the full spectrum of somatic mutations in the paired tumor-normal samples. The unique resource and methodology established in this study will be beneficial to the development of precision oncology medicine not only for breast cancer, but also for other cancers.