Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome

Korbel, Jan O.; Urban, Alexander; Affourtit, Jason P.; Godwin, Brian; Grubert, Fabian; Simons, Jan Fredrik; Kim, Philip M.; Palejev, Dean; Carriero, Nicholas; Du, Lei; Taillon, Bruce E.; Chen, Zhoutao; Tanzer, Andrea; Saunders, A. C. Eugenia; Chi, Jianxiang; Yang, Fengtang; Carter, Nigel P.; Hurles, Matthew E.; Weissman, Sherman M.; Harkins, Timothy T.; Gerstein, Mark; Egholm, Michael; Snyder, M

doi:10.1126/science.1149504

Cited by 1,017 publications

(1,032 citation statements)

References 22 publications

Supporting

Mentioning

992

Contrasting

Unclassified

Order By: Relevance

“…Six micrograms of intact genomic DNA was sheared hydrodynamically (Hydroshear, Genomic Solutions) and purified with AMPure TM SPRI beads into DNA fragments B3 kbp in length. After methylation of EcoRI restriction sites, a biotinylated hairpin adaptor was ligated to the ends of the P. pastoris DNA fragments, followed by EcoRI digestion with a subsequent circularization 37 . The restriction of the circularized DNA fragments with MmeI, the subsequent ligation of paired-end adaptors and the amplification of the remaining DNA fragments resulted in a doublestranded paired-end library 130 bp in length.…”

Section: Methodsmentioning

confidence: 99%

Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

View full text Add to dashboard Cite

The methylotrophic yeast Pichia pastoris is widely used for the production of proteins and as a model organism for studying peroxisomal biogenesis and methanol assimilation. P. pastoris strains capable of human-type N-glycosylation are now available, which increases the utility of this organism for biopharmaceutical production. Despite its biotechnological importance, relatively few genetic tools or engineered strains have been generated for P. pastoris. To facilitate progress in these areas, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris. We also provide manually curated annotation for its 5,313 protein-coding genes.The methylotrophic yeast Pichia pastoris is by far the most commonly used yeast species in the production of recombinant proteins 1 and is employed in laboratories around the world to produce proteins for basic research and medical applications. It is also an important model organism for the investigation of peroxisomal proliferation and methanol assimilation. The P. pastoris expression technology has been commercially available for many years. P. pastoris grows to high cell density, provides tightly controlled methanol-inducible transgene expression and efficiently secretes heterologous proteins in defined media. Several P. pastoris-produced biopharmaceuticals that are either not glycosylated (such as human serum albumin 2 ) or for which glycosylation is needed only for proper folding (such as several vaccines 3 ) are already on the market. An important recent breakthrough has been the development of P. pastoris strains with humantype N-glycosylation [4][5][6] . Humanized glycosylation will further increase the importance of P. pastoris for biopharmaceutical production; indeed, proteins produced with this system are moving into clinical development 7 . Moreover, monoclonal antibodies can be made at gramper-liter scale in the humanized glycosylation-homogenous strains 8 .For further strain engineering, a better understanding of all aspects of the yeast's protein production machinery is needed, and a number of studies relating to P. pastoris's secretory system and engineered promoters have been forthcoming 9,10 . To facilitate the investigation of P. pastoris and other methylotrophic yeasts, we present the 9.43 Mbp genomic sequence of the GS115 strain of P. pastoris.

show abstract

Section: Methodsmentioning

confidence: 99%

Genome sequence of the recombinant protein production host Pichia pastoris

et al. 2009

View full text Add to dashboard Cite

show abstract

“…The orientation and the distance of the reads within a pair are known in the donor, and when the corresponding mappings on the reference do not agree with this pattern, structural variations can be called similar to the split read approach. This paired-end mapping technique, introduced by Korbel et al [30], will be explained in detail in the following section.…”

Section: Introductionmentioning

confidence: 99%

Unraveling overlapping deletions by agglomerative clustering

Wittler

2013

BMC Genomics

View full text Add to dashboard Cite

BackgroundStructural variations in human genomes, such as deletions, play an important role in cancer development. Next-Generation Sequencing technologies have been central in providing ways to detect such variations. Methods like paired-end mapping allow to simultaneously analyze data from several samples in order to, e.g., distinguish tumor from patient specific variations. However, it has been shown that, especially in this setting, there is a need to explicitly take overlapping deletions into consideration. Existing tools have only minor capabilities to call overlapping deletions, unable to unravel complex signals to obtain consistent predictions.ResultWe present a first approach specifically designed to cluster short-read paired-end data into possibly overlapping deletion predictions. The method does not make any assumptions on the composition of the data, such as the number of samples, heterogeneity, polyploidy, etc. Taking paired ends mapped to a reference genome as input, it iteratively merges mappings to clusters based on a similarity score that takes both the putative location and size of a deletion into account.ConclusionWe demonstrate that agglomerative clustering is suitable to predict deletions. Analyzing real data from three samples of a cancer patient, we found putatively overlapping deletions and observed that, as a side-effect, erroneous mappings are mostly identified as singleton clusters. An evaluation on simulated data shows, compared to other methods which can output overlapping clusters, high accuracy in separating overlapping from single deletions.

show abstract

“…105 In the study, libraries of 3-kb fragments for two female samples from the International HapMap Project were prepared and sequenced by Roche 454 sequencing, and they found 1297 structural variations, including 122 inversions. Using the same approach, hundreds of inversions were also uncovered by whole genome resequencing studies; for example, 91 and 415 inversions were detected in the African NA18507 genome and Korean SJK genome, respectively.…”

Section: Copy Neutral Variations-inversions and Translocationsmentioning

confidence: 99%

The discovery of human genetic variations and their use as disease markers: past, present and future

Loy

Salim

et al. 2010

J Hum Genet

View full text Add to dashboard Cite

The field of human genetic variations has progressed rapidly over the past few years. It has added much information and deepened our knowledge and understanding of the diversity of genetic variations in the human genome. This significant progress has been driven mainly by the developments of microarray and next generation sequencing technologies. The array-based methods have been widely used for large-scale copy number variation (CNV) detection in the human genome. The arrival of next generation sequencing technologies, which enabled the completion of several whole genome resequencing studies, has also resulted in a massive discovery of genetic variations. These studies have identified several hundred thousand short indels and a total of thousands of CNVs and other structural variations in the human genome. The discovery of these 'newer' types of genetic variations, indels, CNVs and copy neutral variations (inversions and translocations) has also widened the scope of genetic markers in human genetic and disease gene mapping studies. The aim of this review article is to summarize the latest developments in the discovery of human genetic variations and address the issue of inadequate coverage of genetic variations in the current genome-wide association studies, which mainly focuses on common SNPs. Finally, we also discuss the future directions in the field and their impacts on next generation genome-wide association studies.

show abstract

Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome

Cited by 1,017 publications

References 22 publications

Genome sequence of the recombinant protein production host Pichia pastoris

Genome sequence of the recombinant protein production host Pichia pastoris

Unraveling overlapping deletions by agglomerative clustering

The discovery of human genetic variations and their use as disease markers: past, present and future

Contact Info

Product

Resources

About