Play all audios:
ABSTRACT The human genome is arguably the most complete mammalian reference assembly1,2,3, yet more than 160 euchromatic gaps remain4,5,6 and aspects of its structural variation remain
poorly understood ten years after its completion7,8,9. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule,
real-time DNA sequencing10. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats,
often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including
inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5
kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results
suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read
sequencing technology. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution
Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Buy this article * Purchase on SpringerLink * Instant access to full
article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs *
Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS A DRAFT HUMAN PANGENOME REFERENCE Article Open access 10 May 2023 BEYOND ASSEMBLY: THE INCREASING FLEXIBILITY OF
SINGLE-MOLECULE SEQUENCING TECHNOLOGY Article 09 May 2023 HIGHLY ACCURATE LONG-READ HIFI SEQUENCING DATA FOR FIVE COMPLEX GENOMES Article Open access 17 November 2020 ACCESSION CODES PRIMARY
ACCESSIONS SEQUENCE READ ARCHIVE * SRP040522 * SRP044331 * SRX533609 DATA DEPOSITS All underlying SMRT WGS read data have been released within the NCBI Sequence Read Archive (SRA) under
accession SRX533609 and may also be accessed as part of all the SMRT data sets (NCBI SRA accession SRP040522). Illumina WGS data for CHM1 are available in the NCBI SRA under accession
SRP044331 as well as finished BAC and fosmid clone inserts using SMRT sequence data (GenBank accessions in Supplementary Table 35). For the purpose of mapping and annotation, we developed a
patched GRCh37 reference genome including a track hub for upload into the UCSC Genome Browser. A complete list of all inaccessible regions of the human genome and a database of
heterochromatic and subtelomeric sequence reads that could not be assembled are available at (http://eichlerlab.gs.washington.edu/publications/chm1-structural-variation). REFERENCES * The
1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. _Nature_ 491, 56–65 (2012) * The International HapMap Project Consortium. The International
HapMap Project. _Nature_ 426, 789–796 (2003) * International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. _Nature_ 431, 931–945 (2004) *
Kurahashi, H. et al. Molecular cloning of a translocation breakpoint hotspot in 22q11. _Genome Res._ 17, 461–469 (2007) Article CAS Google Scholar * Genovese, G. et al. Using population
admixture to help complete maps of the human genome. _Nature Genet._ 45, 406–414 (2013) Article CAS Google Scholar * Bovee, D. et al. Closing gaps in the human genome with fosmid
resources generated from multiple individuals. _Nature Genet._ 40, 96–101 (2008) Article CAS Google Scholar * Mills, R. E. et al. Mapping copy number variation by population-scale genome
sequencing. _Nature_ 470, 59–65 (2011) Article CAS Google Scholar * Kidd, J. M. et al. A human genome structural variation sequencing resource reveals insights into mutational mechanisms.
_Cell_ 143, 837–847 (2010) Article CAS Google Scholar * Eichler, E. E., Clark, R. A. & She, X. An assessment of the sequence gaps: unfinished business in a finished human genome.
_Nature Rev. Genet._ 5, 345–354 (2004) Article CAS Google Scholar * Eid, J. et al. Real-time DNA sequencing from single polymerase molecules. _Science_ 323, 133–138 (2009) Article ADS
CAS Google Scholar * Chaisson, M. J. & Tesler, G. Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory. _BMC
Bioinformatics_ 13, 238 (2012) Article CAS Google Scholar * Lee, H. & Schatz, M. C. Genomic dark matter: the reliability of short read mapping illustrated by the genome mappability
score. _Bioinformatics_ 28, 2097–2105 (2012) Article CAS Google Scholar * Myers, E. W. et al. A whole-genome assembly of _Drosophila_. _Science_ 287, 2196–2204 (2000) Article ADS CAS
Google Scholar * Chin, C. S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. _Nature Methods_ 10, 563–569 (2013) Article CAS Google Scholar *
Huddleston, J. et al. Reconstructing complex regions of genomes using long-read sequencing technology. _Genome Res._ 24, 688–696 (2014) Article CAS Google Scholar * Kimelman, A. et al. A
vast collection of microbial genes that are toxic to bacteria. _Genome Res._ 22, 802–809 (2012) Article CAS Google Scholar * Lander, E. S. et al. Initial sequencing and analysis of the
human genome. _Nature_ 409, 860–921 (2001) Article ADS CAS Google Scholar * Venter, J. C. et al. The sequence of the human genome. _Science_ 291, 1304–1351 (2001) Article ADS CAS
Google Scholar * Conrad, D. F. et al. Origins and functional impact of copy number variation in the human genome. _Nature_ 464, 704–712 (2010) Article CAS Google Scholar * Kong, A. et
al. A high-resolution recombination map of the human genome. _Nature Genet._ 31, 241–247 (2002) Article CAS Google Scholar * Stewart, C. et al. A comprehensive map of mobile element
insertion polymorphisms in humans. _PLoS Genet._ 7, e1002236 (2011) Article CAS Google Scholar * Steinberg, K. M. et al. Single haplotype assembly of the human genome from a hydatidiform
mole. _Genome Res_ (in press) * Parsons, J. D. Miropeats: graphical DNA sequence comparisons. _Comput. Appl. Biosci._ 11, 615–619 (1995) CAS PubMed Google Scholar * Jurka, J., Klonowski,
P., Dagman, V. & Pelton, P. CENSOR–a program for identification and elimination of repetitive elements from DNA sequences. _Comput. Chem._ 20, 119–121 (1996) Article CAS Google Scholar
* Smit, A. F. A., Hubley, R. & Green, P. RepeatMasker Open-3.0 http://www.repeatmasker.org (1996–2010) * Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment
libraries by high-density in vitro transposition. _Genome Biol._ 11, R119 (2010) Article CAS Google Scholar * Wu, T. & Watanabe GMAP: a genomic mapping and alignment program for mRNA
and EST sequences. _Bioinformatics_ 21, 1859–1875 (2005) Article CAS Google Scholar Download references ACKNOWLEDGEMENTS We thank D. Alexander, D. Church and A. Klammer for discussions,
K. Mohajeri and L. Harshman for technical assistance and T. Brown for assistance in manuscript preparation. This work was supported, in part, by US National Institutes of Health (NIH) grant
HG002385 and HG007497 to E.E.E. M.Y.D. is supported by the US National Institute of Neurological Disorders and Stroke (award K99NS083627). E.E.E. is an investigator of the Howard Hughes
Medical Institute. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Genome Sciences, University of Washington School of Medicine, Seattle, 98195, Washington, USA Mark J. P.
Chaisson, John Huddleston, Megan Y. Dennis, Peter H. Sudmant, Maika Malig, Fereydoun Hormozdiari, Richard Sandstrom, John A. Stamatoyannopoulos & Evan E. Eichler * Howard Hughes Medical
Institute, University of Washington, Seattle, 98195, Washington, USA John Huddleston & Evan E. Eichler * Dipartimento di Biologia, Università degli Studi di Bari ‘Aldo Moro’, Bari 70125,
Italy, Francesca Antonacci * Department of Pathology, University of Pittsburgh, Pittsburgh, 15261, Pennsylvania, USA Urvashi Surti * Pacific Biosciences of California, Inc., Menlo Park,
94025, California, USA Matthew Boitano, Jane M. Landolin, Michael W. Hunkapiller & Jonas Korlach Authors * Mark J. P. Chaisson View author publications You can also search for this
author inPubMed Google Scholar * John Huddleston View author publications You can also search for this author inPubMed Google Scholar * Megan Y. Dennis View author publications You can also
search for this author inPubMed Google Scholar * Peter H. Sudmant View author publications You can also search for this author inPubMed Google Scholar * Maika Malig View author publications
You can also search for this author inPubMed Google Scholar * Fereydoun Hormozdiari View author publications You can also search for this author inPubMed Google Scholar * Francesca Antonacci
View author publications You can also search for this author inPubMed Google Scholar * Urvashi Surti View author publications You can also search for this author inPubMed Google Scholar *
Richard Sandstrom View author publications You can also search for this author inPubMed Google Scholar * Matthew Boitano View author publications You can also search for this author inPubMed
Google Scholar * Jane M. Landolin View author publications You can also search for this author inPubMed Google Scholar * John A. Stamatoyannopoulos View author publications You can also
search for this author inPubMed Google Scholar * Michael W. Hunkapiller View author publications You can also search for this author inPubMed Google Scholar * Jonas Korlach View author
publications You can also search for this author inPubMed Google Scholar * Evan E. Eichler View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS
E.E.E., M.J.P.C., M.Y.D., J.H. and J.K. designed experiments; M.M. prepared DNA; M.M. and M.B. prepared libraries and generated sequence data; P.H.S., J.H. and M.Y.D. identified clones for
sequencing; J.H., P.H.S., M.Y.D., F.H. and M.J.P.C. performed bioinformatics analyses; M.Y.D., F.A. and M.M. performed targeted sequencing of clones; M.J.P.C. designed algorithms and
pipelines for mapping SMRT sequence data and detection of structural variants; M.W.H., U.S., R.S. and J.A.S. provided access to critical resources; J.M.L. deposited SMRT sequence data into
SRA; M.J.P.C., J.H. and E.E.E. wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Evan E. Eichler. ETHICS DECLARATIONS COMPETING INTERESTS M.B., J.L., M.W.H. and J.K. are employees
of Pacific Biosciences, Inc., a company commercializing DNA sequencing technologies; E.E.E. is on the scientific advisory board (SAB) of DNAnexus, Inc. and was formerly an SAB member of
Pacific Biosciences, Inc. (2009–2013) and SynapDx Corp. (2011–2013); and M.J.P.C. was a former employee for Pacific Biosciences, Inc. EXTENDED DATA FIGURES AND TABLES EXTENDED DATA FIGURE 1
SEQUENCE CONTENT OF GAP CLOSURES. A–C, Gap closures are enriched for simple repeats compared to equivalently sized regions randomly sampled from GRCh37; examples of the organization of these
regions are shown using Miropeats for chromosome 4 (GRCh37, chr4:59724333–59804333) (A), chromosome 11 (GRCh37, chr11:87673378–87753378) (B), and chromosome X (GRCh37,
chrX:143492324–143572324) (C). Dotplots show the architecture of the degenerate STRs with the core motif highlighted below. Shared sequence motifs between blocks are indicated by colour.
EXTENDED DATA FIGURE 2 VARIANT DETECTION PIPELINE. At every variant locus, we collected the full-length reads that overlap the locus, performed _de novo_ assembly using the Celera assembler,
and called a consensus using Quiver after remapping reads used in the assembly as well as reads flanking the assembly (yellow reads) to increase consensus quality at the boundaries of the
assembly. BLASR is used to align the assembly consensus sequences to the reference, and insertions and deletions in the alignments are output as variants. Reads spanning a deletion event
within a single alignment are shown as bars connected by a solid line, and double hard-stop reads spanning a larger deletion event and split into two separate alignments of the same read are
shown as a dotted line. EXTENDED DATA FIGURE 3 GENOME DISTRIBUTION OF CLOSED GAPS AND INSERTIONS. Chromosome ideogram heatmap depicts the normalized density of inserted CHM1 base pairs per
5-Mb bin with a strong bias noted near the end of most chromosomes. Locations of structural variants and closed gaps are given by coloured diamonds to the left of each chromosome: closed gap
sequences (red), inversions (green), and complex events (blue). EXTENDED DATA FIGURE 4 CONFIRMATION OF COMPLEX INSERTIONS IN ADDITIONAL GENOMES. Top, genotypes of polymorphic complex
regions using read depth of unique _k_-mers (blue: present; white: absent). Bottom, extended examples of complex insertion events: alignment to chimpanzee panTro4 reference (dark blue);
existing human reference hg19 (light teal); inserted sequence (dark teal). The bottom rows show repeat annotations, with darker hues for repeats overlapping the inserted region. EXTENDED
DATA FIGURE 5 INVERSION VALIDATION BY BAC-INSERT SEQUENCING. Inversions detected by alignment of single long reads were validated by sequencing clones from the CHM1 BAC library (CHORI17), in
which end mappings to GRCh37 spanned the putative inversions. Inversions were validated by aligning the corresponding BAC sequences to GRCh37 with Miropeats. Shared sequence between the
BACs and GRCh37 is shown in black; inversion events are indicated in red. EXTENDED DATA FIGURE 6 CHM1 CLONE-BASED ASSEMBLY OF THE HUMAN 10Q11 GENOMIC REGION. A, The clone-based assembly is
composed primarily of BACs from the CH17 library as shown in the tiling path below the internal repeat structure of the region. Coloured arrows indicate large segmental duplications with
homologous sequences connected by coloured lines (Miropeats). Genes annotated from alignment of RefSeq messenger RNA sequences with GMAP27 are shown. B, Miropeats comparisons of the 10q11
clone-based assembly against the corresponding sequence from GRCh37, with gaps shown in red, highlight the degree to which the reference was misassembled. SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION This file contains Supplementary Methods, Text and Data, Supplementary Figures 1-29, Supplementary Tables 1-35 and additional references. Tables shown in this file
represent views of the full tables given in the Supplementary Tables file. (PDF 5107 kb) SUPPLEMENTARY TABLES This file contains the full table values for the Supplementary Tables 1-35 (see
separate Supplementary information file). (XLSX 442 kb) POWERPOINT SLIDES POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 RIGHTS AND PERMISSIONS Reprints
and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Chaisson, M., Huddleston, J., Dennis, M. _et al._ Resolving the complexity of the human genome using single-molecule sequencing. _Nature_
517, 608–611 (2015). https://doi.org/10.1038/nature13907 Download citation * Received: 03 July 2014 * Accepted: 30 September 2014 * Published: 10 November 2014 * Issue Date: 29 January 2015
* DOI: https://doi.org/10.1038/nature13907 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not
currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative