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.
At a glance
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Extended data figures and tables
Extended Data Figures
- Extended Data Figure 1: Sequence content of gap closures. (261 KB)
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. (212 KB)
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. (369 KB)
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. (769 KB)
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. (289 KB)
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. (428 KB)
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 (4.9 MB)
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.
- Supplementary Tables (442 KB)
This file contains the full table values for the Supplementary Tables 1-35 (see separate Supplementary information file).