High-resolution human genome structure by single-molecule analysis

Abstract

Variation in genome structure is an important source of human genetic polymorphism: It affects a large proportion of the genome and has a variety of phenotypic consequences relevant to health and disease. In spite of this, human genome structure variation is incompletely characterized due to a lack of approaches for discovering a broad range of structural variants in a global, comprehensive fashion. We addressed this gap with Optical Mapping, a highthroughput, high-resolution single-molecule system for studying genome structure. We used Optical Mapping to create genomewide restriction maps of a complete hydatidiform mole and three lymphoblast-derived cell lines, and we validated the approach by demonstrating a strong concordance with existing methods. We also describe thousands of new variants with sizes ranging from kb to Mb. structural variation | copy number variation | optical mapping | single-molecule genomics | genome assembly R ecent reports (1-11) have firmly established genome structural variation as an important and pervasive source of genetic polymorphism. Since the initial reports (1, 2) of widespread copy-number variation between the genomes of phenotypically normal individuals, investigators have applied hybridizationbased methods (3, 7, 9, 11), computational approaches (5, 6), clone paired-end sequencing (4, 10) and most recently a pairedend sequencing by synthesis approach (8) to the discovery and characterization of structural polymorphism. Others have described phenotypic consequences of these variants, including associations with myocardial infarction, neuroblastoma, autism, and schizophrenia (reviewed recently in ref. 12). Finally, their consistent association with segmental duplications and other classes of repeats (13) provides a mechanistic explanation for their origin (14) and points to a previously unappreciated role in evolution (15) as well as disease.Unfortunately, despite all efforts, a comprehensive picture of genome structure polymorphism has not yet emerged. Current genome-wide studies of structural variation manifest only modest concordance, possibly due to ascertainment biases arising from the techniques employed. For example, hybridization-based methods (2,3,7,9,11,16) are subject to nonspecific hybridization in repeat-rich regions, while clone-based strategies (4,8,10) are limited by maximum clone insert sizes and a wide clone size distribution relative to the events they are trying to detect. More recently, several entire human genomes were sequenced using high-throughput methods (17)(18)(19)(20), but the difficulty of interrogating repeat-rich regions is compounded by these systems' short read lengths.In an effort to overcome these challenges, we have applied Optical Mapping to the problem of discerning structural variation in normal human genomes. Optical Mapping (21-35) is a highthroughput system that combines single-molecule measurements with dedicated computational analysis to produce ordered restriction maps from individual molecules of genomic DNA: essentially, a singl...

Characterizing polymorphic inversions in human genomes by single-cell sequencing

“…For instance, the breakpoints of the inversions we mapped at Chr 16p12.3 showed >86% overlap with an inversion characterized in six independent studies using combinations of BAC clone sequencing, mate-pair sequencing, optical mapping, PCR, and FISH technologies (Supplemental Fig. S12; Tuzun et al 2005;Korbel et al 2007;Kidd et al 2008Kidd et al , 2010Pang et al 2010;Teague et al 2010). Of note, in some genomes from our population study, we observed multiple inversions with distinct genotypes at this locus (see Fig.…”

“…Techniques such as karyotyping, fluorescence in situ hybridization (FISH), and optical mapping allow visualization and genotyping of inversions at the single-cell and single-chromosome level. However, the low resolution of these approaches limits their application to mapping large microscopic events that disrupt visible patterns (typically at the megabase-scale), and their low throughput limits the number of cells or individuals that can be studied at a time (Youings et al 2004;Zody et al 2008;Antonacci et al 2009;Feuk 2010;Teague et al 2010).…”

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

“…In collaboration with various external groups, we identified and investigated reported path issues and associated assembly gaps using a combination of techniques, including optical maps (Teague et al 2010;Howe and Wood 2015), Strand-seq (Falconer et al 2012), admixture mapping (Genovese et al 2013a) and reevaluation of component sequences and overlaps (Mueller et al 2013). These analyses uncovered some substantial misassemblies in GRCh37 that spanned several megabases and many genes, including the regions at 1q21, 10q11, and a peri-centromeric inversion of Chromosome 9.…”

DNA confined in a two-dimensional strip geometry

“…Important biomolecules, such as, chromosomal DNAs, or proteins whose functionalities are crucially dependent on the exact sequence of the nucleotides or amino acids usually exist in highly compact conformations. By straightening these molecules on a two dimensional sheet [2][3][4] or inside a nanochannel [5][6][7][8][9][10] it is possible to obtain the structural details of these molecules. It is believed that a complete characterization of the DNA sequence for each individual and a proper understanding the role of genetic variations will lead to personalized medicine for diseases, such as, cancer [11].…”

“…Naturally quests for efficient but low cost techniques have attracted considerable attention. Along with optical maps [2,7], recently DNA melting characteristics inside a nanochannel have been studied showing further promises [9]. These recent experiments have generated renewed interests in theoretical and computational studies of confined polymers [13]- [24].…”