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.
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Sequence Read Archive
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).
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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.
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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.
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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)
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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
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DOI: https://doi.org/10.1038/nature13907
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