Resolving the complexity of the human genome using single-molecule sequencing

Abstract

The human genome is arguably the most complete mammalian reference assembly1–3 yet more than 160 euchromatic gaps remain4–6 and aspects of its structural variation remain poorly understood ten years after its completion7–9. In order to identify missing sequence and genetic variation, we sequenced and analyzed a haploid human genome (CHM1) using single-molecule, real-time (SMRT) DNA sequencing10. We closed or extended 55% of the remaining interstitial gaps in the human GRCh37 reference genome—78% of which carried long runs of degenerate short tandem repeats (STRs) often multiple kilobases in length embedded within GC-rich genomic regions. We resolved the complete sequence of 26,079 euchromatic structural variants at the basepair 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 kbp in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long STRs. 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.

De novo assembly and phasing of a Korean human genome

“…We also extended into 72 of the 85 remaining gaps with the addition of 663 kb of sequence into 4.1 Mb. These locations, previously intractable using only short reads, commonly contained simple tandem repeats, as reported previously 6,9 . One example (Fig.…”

“…We were able to validate 271 out of 276 SVs with BAC contigs generated by SMRT sequencing (Supplementary Table 12). Compared to previous studies 6,[8][9][10][11] , a total of 11,927 variants were previously unreported, which account for approximately 47% (3,465) and 76% (7,710) of all deletions and insertions, respectively ( Fig. 2a and Extended Data Fig.…”

An Incomplete Understanding of Human Genetic Variation

“…Sensitivity also varies as a function of size, with both ends of the SV spectrum adversely affected. Comparisons with SVs resolved using long-read sequencing technologies [e.g., single-molecule, real-time (SMRT) or Pacific Biosciences sequencing technology] suggest that the majority (.80%) of insertions and deletions between 50 bp and 1 kbp in length are missed using short-read sequencing technologies (Figure 1) (Chaisson et al 2015a), irrespective of frequency. These results argue that most widely used sequencing technologies are insufficient, because short reads fail to detect and accurately genotype a large fraction of SVs.…”

“…Detailed targeted sequencing of regions of the human genome suggests that indels should occur at approximately one-tenth of the frequency of SNVs (Bhangale et al 2005), suggesting that the current catalog may be missing at least 30-40% of all indels. Detection of indels associated with short tandem repeat (STR) sequences is particularly challenging and specialized methods have been developed to discover and accurately genotype these from next-generation sequencing datasets (Karakoc et al 2012;Narzisi et al 2014;Willems et al 2014;Chaisson et al 2015a). Sensitivity for indel variant discovery is generally much lower than for SNVs.…”

“…Several lines of evidence suggest that this missing variation will be critical to interpreting the "missing heritability" of human disease. First, the genes and regions associated with this variation are hotspots of recurrent mutation directly or indirectly associated with human disease and the emergence of novel genes associated with the evolution of human phenotypes (Chaisson et al 2015a;Florio et al 2015). The HLA locus is perhaps the most well-cited example of this (Raymond et al 2005) Second, SVs have been estimated to be enriched 50-fold for expression quantitative trait loci (eQTL) when compared to SNVs (relative to the number of events tested) (Sudmant et al 2015b).…”