Journal Article

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

Boitano, Matthew, Landolin, Jane M., Stamatoyannopoulos, John A., Korlach, Jonas, Surti, Urvashi, Hormozdiari, Fereydoun, Eichler, Evan E., Antonacci, Francesca, Huddleston, John, Dennis, Megan Y., Malig, Maika, Sandstrom, Richard, Sudmant, Peter H., Chaisson, Mark J. P., Hunkapiller, Michael W.

2015

Overview

Single-molecule, real-time DNA sequencing is used to analyse a haploid human genome (CHM1), thus closing or extending more than half of the remaining 164 euchromatic gaps in the human genome; the complete sequences of euchromatic structural variants (including inversions, complex insertions and tandem repeats) are resolved at the base-pair level, suggesting that a greater complexity of the human genome can now be accessed. Deep-sequencing the human genome The human genome is considered sequenced, yet more than 160 euchromatic gaps remain and many aspects of its structural variation are poorly understood. Evan Eichler and colleagues sequenced and analysed a haploid human genome (CHM1) using single-molecule, real-time (SMRT) DNA sequencing and by doing so closed — or in some cases extended — more than half of the remaining gaps. They also resolved the complete sequence of numerous euchromatic structural variants at the base-pair level, revealing inversions, complex insertions and long tracts of tandem repeats, some of them previously unknown. Thanks to the longer-read sequencing technology applied here, the complexity of the human genome that stems from variation of longer and more complex repetitive DNA can now be largely resolved. The human genome is arguably the most complete mammalian reference assembly 1 , 2 , 3 , yet more than 160 euchromatic gaps remain 4 , 5 , 6 and aspects of its structural variation remain poorly understood ten years after its completion 7 , 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 sequencing 10 . 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 45 / 45/23 / 631/208/212/748 / 631/208/726/649/2157 / Chromosome Inversion - genetics / Chromosomes, Human, Pair 10 - genetics / Cloning, Molecular / Deoxyribonucleic acid / DNA / DNA sequencing / GC Rich Sequence - genetics / Genetic diversity / Genetic research / Genetic Variation - genetics / Genome, Human - genetics / Genomes / Genomics / Haploidy / Human genome / Humanities and Social Sciences / Humans / letter / Methods / Molecular biology / multidisciplinary / Mutagenesis, Insertional - genetics / Nucleotide sequencing / Reference Standards / Science / Sequence Analysis, DNA - methods / Tandem Repeat Sequences - genetics