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...

A small proportion of men with infertility have Y-chromosome microdeletions, but the size and position of the deletions correlate poorly with the severity of spermatogenic failure, and a deletion does not preclude the presence of viable sperm and possible conception.

Cytogenetic and molecular deletion analyses of azoospermic and oligozoospermic males have suggested the existence of AZoospermia Factor(s) (AZF) residing in deletion intervals 5 and 6 of the human Y-chromosome and coinciding with three functional regions associated with spermatogenic failure. Nonpolymorphic microdeletions in AZF are associated with a broad spectrum of testicular phenotypes. Unfortunately, Sequence Tagged Sites (STSs) employed in screening protocols range broadly in number and mapsite and may be polymorphic. To thoroughly analyze the AZF region(s) and any correlations that may be drawn between genotype and phenotype, we describe the design of nine multiplex PCR reactions derived from analysis of 136 loci. Each multiplex contains 4-8 STS primer pairs, amplifying a total of 48 Y-linked STSs. Each multiplex consists of one positive control: either SMCX or MIC2. We screened four populations of males with these STSs. Population I consisted of 278 patients diagnosed as having significant male factor infertility: either azoospermia, severe oligozoospermia associated with hypogonadism and spermatogenic arrest or normal sperm counts associated with abnormal sperm morphology. Population II consisted of 200 unselected infertile patients. Population III consisted of 36 patients who had previously been shown to have aneuploidy, cytological deletions or translocations involving the Y-chromosome or normal karyotypes associated with severe phenotype abnormalities. Population IV consisted of 920 fertile (control) males. The deletion rates in populations I, II and III were 20.5%, 7% and 58.3%, respectively. A total of 92 patients with deletions were detected. The deletion rate in population IV was 0.87% involving 8 fertile individuals and 4 STSs which were avoided in multiplex panel construction. The ability of the nine multiplexes to detect pathology associated microdeletions is equal to or greater than screening protocols used in other studies. Furthermore, the data suggest a fourth AZF region between AZFb and AZFc, which we have termed AZFd. Patients with microdeletions restricted to AZFd may present with mild oligozoospermia or even normal sperm counts associated with abnormal sperm morphology. Though a definitive genotype/phenotype correlation does not exist, large deletions spanning multiple AZF regions or microdeletions restricted to AZFa usually result in patients with Sertoli Cell Only (SCO) or severe oligozoospermia, whereas microdeletions restricted to AZFb or AZFc can result in patients with phenotypes which range from SCO to moderate oligozoospermia. The panel of nine multiplexed reactions, the Y-deletion Detection System (YDDS), provides a fast, efficient and accurate method of assessing the integrity of the Y-chromosome. To date, this study provides the most extensive screening of a proven fertile male population in tandem with 514 infertile males, derived from three different patient selection protocols.