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High throughput genome sequencing on DNA arrays

a genome sequencing and high throughput technology, applied in the field of high throughput genome sequencing on dna arrays, can solve the problems of reducing the packing efficiency of target targets and placing a limit on overall sequencing efficiency

Inactive Publication Date: 2009-05-07
COMPLETE GENOMICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of these new approaches are restricted to determining a few tens of nucleotides before signals become significantly degraded, thereby placing a limit on overall sequencing efficiency.
Another limitation of traditional high-throughput sequencing techniques is that random positioning of DNA targets over an array surface, which is used in many sequencing methods, reduces the packing efficiency of those targets from what is possible by attaching DNA at predefined sites such as in a grid.

Method used

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  • High throughput genome sequencing on DNA arrays
  • High throughput genome sequencing on DNA arrays
  • High throughput genome sequencing on DNA arrays

Examples

Experimental program
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Effect test

example 1

RCR Based Formation and Attachment of DNBs

[0305]Two synthetic targets were co-amplified. About one million molecules were captured on the glass surface, and then probed for one of the targets. After imaging and photo-bleaching the first probe, the second target was probed. Successive hybridization with amplicon specific probes showed that each spot on the array corresponded uniquely to either one of the two amplicon sequences. It was also confirmed that the probe could be removed through heating to 70° C. and then re-hybridized to produce equally strong signals.

example 2

Validation of Circle Formation and Amplification

[0306]The circle formation and amplification process was validated using E. coli DNA (FIG. 6). A universal adaptor, which also served as the binding site for capture probes and RCR primer, was ligated to the 5′ end of the target molecule using a universal template DNA containing degenerate bases for binding to all genomic sequences. The 3′ end of the target molecule was modified by addition of a poly-dA tail using terminal transferase. The modified target was then circularized using a bridging template complementary to the adaptor and to the oligo-dA tail.

example 3

Validation of Ligation with Condensed Concatemers

[0307]The ability for probe ligation to occur with the condensed concatemers was tested. Reactions were carried out at 20° C. for 10 min using ligase, followed by a brief wash of the chamber to remove excess probes. The ligation of a 6-mer and a labeled 5-mer produced signal levels comparable to that of an 1-mer. Software modules, including image analysis of random arrays, were tested on simulated data for whole genome sequence reconstruction.

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Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 11 / 679,124, filed Feb. 26, 2007 which claims priority to provisional application Ser. No. 60 / 776,415, filed Feb. 24, 2006 and Ser. No. 60 / 821,960 filed Aug. 10, 2006.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This application has been partially funded by the Federal Government through Grant No. 1 U01 A1057315-01 of the National Institute of Health.BACKGROUND OF THE INVENTION[0003]Large-scale sequence analysis of genomic DNA is central to understanding a wide range of biological phenomena related to states of health and disease both in humans and in many economically important plants and animals, e.g. Collins et al (2003), Nature, 422: 835-847; Service, Science, 311: 1544-1546 (2006); Hirschhorn et al (2005), Nature Reviews Genetics, 6: 95-108; National Cancer Institute, Report of Working Group on Biomedical Technology, “Recommendation for a Human Cancer Genome Proj...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/04
CPCC12N15/64C12N15/66C12Q1/6874Y10T436/143333C12Q2521/313C12Q2525/191C12Q2525/151C12Q2525/131C12Q2565/518C12Q2533/107C12Q2531/125
Inventor DRMANAC, RADOJE T.CALLOW, MATTHEWDRMANAC, SNEZANA
Owner COMPLETE GENOMICS INC
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