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Characterization of missing human genome sequences and copy-number polymorphic insertions.

ABSTRACT: The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18-37% of these new insertions are copy-number polymorphic, including loci that show extensive population stratification among Europeans, Asians and Africans. Complete sequencing of 156 of these insertions identified new exons and conserved noncoding sequences not yet represented in the reference genome. We developed a method to accurately genotype these new insertions by mapping next-generation sequencing datasets to the breakpoint, thereby providing a means to characterize copy-number status for regions previously inaccessible to single-nucleotide polymorphism microarrays.

SUBMITTER: Kidd JM

PROVIDER: S-EPMC2875995 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Characterization of missing human genome sequences and copy-number polymorphic insertions.

Kidd Jeffrey M JM Sampas Nick N Antonacci Francesca F Graves Tina T Fulton Robert R Hayden Hillary S HS Alkan Can C Malig Maika M Ventura Mario M Giannuzzi Giuliana G Kallicki Joelle J Anderson Paige P Tsalenko Anya A Yamada N Alice NA Tsang Peter P Kaul Rajinder R Wilson Richard K RK Bruhn Laurakay L Eichler Evan E EE

Nature methods 20100501 5

The extent of human genomic structural variation suggests that there must be portions of the genome yet to be discovered, annotated and characterized at the sequence level. We present a resource and analysis of 2,363 new insertion sequences corresponding to 720 genomic loci. We found that a substantial fraction of these sequences are either missing, fragmented or misassigned when compared to recent de novo sequence assemblies from short-read next-generation sequence data. We determined that 18-3 ...[more]

PMID: 20440878

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Project description:Recent large-scale genomic studies within human populations have identified numerous genomic regions as copy number variant (CNV). As these CNV regions often overlap coding regions of the genome, large lists of potentially copy number polymorphic genes have been produced that are candidates for disease association. Most of the current data regarding normal genic variation, however, has been generated using BAC or SNP microarrays, which lack precision especially with respect to exons. To address this, we assessed 2,790 candidate CNV genes defined from available studies in nine well-characterized HapMap individuals by designing a customized oligonucleotide microarray targeted specifically to exons. Using exon array comparative genomic hybridization (aCGH), we detected 255 (9%) of the candidates as true CNVs including 134 with evidence of variation over the entire gene. Individuals differed in copy number from the control by an average of 100 gene loci. Both partial- and whole-gene CNVs were strongly associated with segmental duplications (55 and 71%, respectively) as well as regions of positive selection. We confirmed 37% of the whole-gene CNVs using the fosmid end sequence pair (ESP) structural variation map for these same individuals. If we modify the end sequence pair mapping strategy to include low-sequence identity ESPs (98-99.5%) and ESPs with an everted orientation, we can capture 82% of the missed genes leading to more complete ascertainment of structural variation within duplicated genes. Our results indicate that segmental duplications are the source of the majority of full-length copy number polymorphic genes, most of the variant genes are organized as tandem duplications, and a significant fraction of these genes will represent paralogs with levels of sequence diversity beyond thresholds of allelic variation. In addition, these data provide a targeted set of CNV genes enriched for regions likely to be associated with human phenotypic differences due to copy number changes and present a source of copy number responsive oligonucleotide probes for future association studies.

Project description:The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.

Dataset Information

Characterization of missing human genome sequences and copy-number polymorphic insertions.

Publications

Characterization of missing human genome sequences and copy-number polymorphic insertions.

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