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Massively parallel continguity mapping

A technology of adjacency information and adapters, applied in the field of D, can solve problems such as the astonishingly high price of DNA sequencing

Active Publication Date: 2013-12-11
UNIV OF WASHINGTON CENT FOR COMMERICIALIZATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Despite rapid improvements in DNA sequencing technology platforms, the cost of DNA sequencing remains prohibitively expensive for some targets

Method used

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example

[0099]Several properties of in vitro transposition can be exploited to develop ultra-low-cost, massively parallel sequencing methods for capturing contiguity information at different scales. First, the modified Tn5 transposome attacked DNA in vitro with high efficiency and density in a reaction that catalyzed the insertion of consensus sequences, with or without breaks, depending on whether the synthetic transposon was continuous or discontinuous. Second, the pattern of transposome attack is relatively random with respect to sequence content. Third, degenerate subsequences plus consensus adapter sequences can be easily included in synthetic transposons. Fourth, in vitro transposition is inexpensive as a one-volume, aqueous-phase, enzymatic reaction. Examples 1-3 relate to the development of massively parallel methods using in vitro transposition to inform small, medium and large range contiguities, respectively. Example 4 concerns the development of a method for capturing co...

example 6

[0099]Several properties of in vitro transposition can be exploited to develop ultra-low-cost, massively parallel sequencing methods for capturing contiguity information at different scales. First, the modified Tn5 transposome attacked DNA in vitro with high efficiency and density in a reaction that catalyzed the insertion of consensus sequences, with or without breaks, depending on whether the synthetic transposon was continuous or discontinuous. Second, the pattern of transposome attack is relatively random with respect to sequence content. Third, degenerate subsequences plus consensus adapter sequences can be easily included in synthetic transposons. Fourth, in vitro transposition is inexpensive as a one-volume, aqueous-phase, enzymatic reaction. Examples 1-3 relate to the development of massively parallel methods using in vitro transposition to inform small, medium and large range contiguities, respectively. Example 4 concerns the development of a method for capturing co...

example 1

[0107] Example 1: Small-scale adjacency

[0108] 1.A. Symmetrically and Uniquely Mark Fragmentation Events

[0109] Genomic DNA breaks, whether by mechanical or enzymatic means, result in a complete loss of information about the pairing of molecules originating from either side of any individual "break". To preserve this information, methods were designed so that unique barcodes were bound to both ends of fragments derived from each disruption introduced by in vitro transposition ( Figure 4 ). Briefly, transposases can be used to catalyze the in vitro insertion of synthetic transposons containing nicking restriction endonucleases into very low amounts of genomic DNA, i.e., less than 5 haploid human genome equivalents. A degenerate single-stranded "bubble" flanking the site. and figure 1 In contrast to the method described in , the synthetic transposon is contiguous, containing a 19 bp ME sequence together with two endonuclease nicking sites flanked by 25 bp of degenera...

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Abstract

Contiguity information is important to achieving high-quality de novo assembly of mammalian genomes and the haplotype-resolved resequencing of human genomes. The methods described herein pursue cost-effective, massively parallel capture of contiguity information at different scales.

Description

[0001] priority statement [0002] This application claims priority to U.S. Provisional Patent Application No. 61 / 438,935, filed February 2, 2011, and U.S. Provisional Patent Application No. 61 / 473,083, filed April 7, 2011, both of which are The subject matter of is hereby incorporated by reference as if fully set forth herein. [0003] statement of government support [0004] This invention was made with Government support under Grant Numbers 3U54AI057141-06S1880509 and 1R01HG006283-01 awarded by the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] Over the past few years, massively parallel sequencing platforms have reduced the cost / base of DNA sequencing by orders of magnitude (Shendureh and Ji 2008). Almost all of the commercially available 'next generation' technologies rely on iterative cycles of biochemistry and imaging of dense arrays of sequencing features to generate relatively short reads, the 'c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B30/04
CPCC12N15/1093Y02P20/582
Inventor 杰·阿肖克·申杜雷杰罗德·约瑟夫·施瓦兹安德鲁·科林·阿迪卓立·李约瑟夫·布莱恩·海特雅各布·奥托·基茨曼阿卡什·库马尔
Owner UNIV OF WASHINGTON CENT FOR COMMERICIALIZATION
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