A general approach for detecting expressed mutations in AML cells using single cell RNA-sequencing.

Virtually all tumors are genetically heterogeneous, containing mutationally-defined subclonal cell populations that often have distinct phenotypes. Single-cell RNA-sequencing has revealed that a variety of tumors are also transcriptionally heterogeneous, but the relationship between expression heterogeneity and subclonal architecture is unclear. Here, we address this question in the context of Acute Myeloid Leukemia (AML) by integrating whole genome sequencing with single-cell RNA-sequencing (using the 10x Genomics Chromium Single Cell 5' Gene Expression workflow). Applying this approach to five cryopreserved AML samples, we identify hundreds to thousands of cells containing tumor-specific mutations in each case, and use the results to distinguish AML cells (including normal-karyotype AML cells) from normal cells, identify expression signatures associated with subclonal mutations, and find cell surface markers that could be used to purify subclones for further study. This integrative approach for connecting genotype to phenotype is broadly applicable to any sample that is phenotypically and genetically heterogeneous.

A variant by any name: quantifying annotation discordance across tools and clinical databases.

BACKGROUND: Clinical genomic testing is dependent on the robust identification and reporting of variant-level information in relation to disease. With the shift to high-throughput sequencing, a major challenge for clinical diagnostics is the cross-identification of variants called on their genomic position to resources that rely on transcript- or protein-based descriptions. METHODS: We evaluated the accuracy of three tools (SnpEff, Variant Effect Predictor, and Variation Reporter) that generate transcript and protein-based variant nomenclature from genomic coordinates according to guidelines by the Human Genome Variation Society (HGVS). Our evaluation was based on transcript-controlled comparisons to a manually curated set of 126 test variants of various types drawn from data sources, each with HGVS-compliant transcript and protein descriptors. We further evaluated the concordance between annotations generated by Snpeff and Variant Effect Predictor and those in major germline and cancer databases: ClinVar and COSMIC, respectively. RESULTS: We find that there is substantial discordance between the annotation tools and databases in the description of insertions and/or deletions. Using our ground truth set of variants, constructed specifically to identify challenging events, accuracy was between 80 and 90% for coding and 50 and 70% for protein changes for 114 to 126 variants. Exact concordance for SNV syntax was over 99.5% between ClinVar and Variant Effect Predictor and SnpEff, but less than 90% for non-SNV variants. For COSMIC, exact concordance for coding and protein SNVs was between 65 and 88% and less than 15% for insertions. Across the tools and datasets, there was a wide range of different but equivalent expressions describing protein variants. CONCLUSIONS: Our results reveal significant inconsistency in variant representation across tools and databases. While some of these syntax differences may be clear to a clinician, they can confound variant matching, an important step in variant classification. These results highlight the urgent need for the adoption and adherence to uniform standards in variant annotation, with consistent reporting on the genomic reference, to enable accurate and efficient data-driven clinical care.

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.

DbVar and DGVa: public archives for genomic structural variation.

Much has changed in the last two years at DGVa (http://www.ebi.ac.uk/dgva) and dbVar (http://www.ncbi.nlm.nih.gov/dbvar). We are now processing direct submissions rather than only curating data from the literature and our joint study catalog includes data from over 100 studies in 11 organisms. Studies from human dominate with data from control and case populations, tumor samples as well as three large curated studies derived from multiple sources. During the processing of these data, we have made improvements to our data model, submission process and data representation. Additionally, we have made significant improvements in providing access to these data via web and FTP interfaces.

Online resources for genomic structural variation.

Genomic structural variation (SV) can be thought of on a continuum from a single base pair insertion/deletion (INDEL) to large megabase-scale rearrangements involving insertions, deletions, duplications, inversions, or translocations of whole chromosomes or chromosome arms. These variants can occur in coding or noncoding DNA, they can be inherited or arise sporadically in the germline or somatic cells. Many of these events are segregating in the population and can be considered common alleles while others are new alleles and thus rare events. All species studied to date harbor structural variants and these may be benign, contributing to phenotypes such as sensory perception and immunity, or pathogenic resulting in genomic disorders including DiGeorge/velocardiofacial, Smith-Margenis, Williams-Beuren, and Prader-Willi syndromes. As structural variants are identified, validated, and their significance, origin, and prevalence are elucidated, it is of critical importance that these data be collected and collated in a way that can be easily accessed and analyzed. This chapter describes current structural variation online resources (see Fig. 1 and Table 1), highlights the challenges in capturing, storing, and displaying SV data, and discusses how dbVar and DGVa, the genomic structural variation databases developed at NCBI and EBI, respectively, were designed to address these issues.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central (PMC), Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Primer-BLAST, COBALT, Electronic PCR, OrfFinder, Splign, ProSplign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, dbVar, Epigenomics, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART), IBIS, Biosystems, Peptidome, OMSSA, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. 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.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, Reference Sequence, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Trace Archive, Sequence Read Archive, Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool, Biosystems, Peptidome, Protein Clusters and the PubChem suite of small molecule databases. Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All 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.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through the NCBI web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs), Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART) and the PubChem suite of small molecule databases. Augmenting many of the web applications is custom implementation of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data available through NCBI's web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link, Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace, Assembly, and Short Read Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Database of Genotype and Phenotype, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting the web applications are custom implementations of the BLAST program optimized to search specialized data sets. 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.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link(BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace and Assembly Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs), Viral Genotyping Tools, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus (GEO), Entrez Probe, GENSAT, Online Mendelian Inheritance in Man (OMIM), Online Mendelian Inheritance in Animals (OMIA), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), the Conserved Domain Architecture Retrieval Tool (CDART) and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. 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.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, the Entrez Programming Utilities, MyNCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Retroviral Genotyping Tools, HIV-1, Human Protein Interaction Database, SAGEmap, Gene Expression Omnibus, Entrez Probe, GENSAT, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology Information.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data retrieval systems and computational resources for the analysis of data in GenBank and other biological data made available through NCBI's website. NCBI resources include Entrez, Entrez Programming Utilities, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs), Retroviral Genotyping Tools, HIV-1/Human Protein Interaction Database, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD) and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized datasets. All of the resources can be accessed through the NCBI home page at http://www.ncbi.nlm.nih.gov.

Analysis of segmental duplications and genome assembly in the mouse.

Limited comparative studies suggest that the human genome is particularly enriched for recent segmental duplications. The extent of segmental duplications in other mammalian genomes is unknown and confounded by methodological differences in genome assembly. Here, we present a detailed analysis of recent duplication content within the mouse genome using a whole-genome assembly comparison method and a novel assembly independent method, designed to take advantage of the reduced allelic variation of the C57BL/6J strain. We conservatively estimate that approximately 57% of all highly identical segmental duplications (>or=90%) were misassembled or collapsed within the working draft WGS assembly. The WGS approach often leaves duplications fragmented and unassigned to a chromosome when compared with the clone-ordered-based approach. Our preliminary analysis suggests that 1.7%-2.0% of the mouse genome is part of recent large segmental duplications (about half of what is observed for the human genome). We have constructed a mouse segmental duplication database to aid in the characterization of these regions and their integration into the final mouse genome assembly. This work suggests significant biological differences in the architecture of recent segmental duplications between human and mouse. In addition, our unique method provides the means for improving whole-genome shotgun sequence assembly of mouse and future mammalian genomes.

Database resources of the National Center for Biotechnology Information: update.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's website. NCBI resources include Entrez, PubMed, PubMed Central, LocusLink, the NCBI Taxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR, OrfFinder, Spidey, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SARS Coronavirus Resource, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD) and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

Database resources of the National Center for Biotechnology.

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources for the data in GenBank and other biological data made available through NCBI's Web site. NCBI resources include Entrez, PubMed, PubMed Central (PMC), LocusLink, the NCBITaxonomy Browser, BLAST, BLAST Link (BLink), Electronic PCR (e-PCR), Open Reading Frame (ORF) Finder, References Sequence (RefSeq), UniGene, HomoloGene, ProtEST, Database of Single Nucleotide Polymorphisms (dbSNP), Human/Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes and related tools, the Map Viewer, Model Maker (MM), Evidence Viewer (EV), Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB), the Conserved Domain Database (CDD), and the Conserved Domain Architecture Retrieval Tool (CDART). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

Initial sequencing and comparative analysis of the mouse genome.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

Database resources of the National Center for Biotechnology Information: 2002 update.

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, Human inverted exclamation markVMouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at http://www.ncbi.nlm.nih.gov.