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STRATEGIES FOR SEQUENCING COMPLEX GENOMES USING HIGH THROUGHPUT SEQUENCING TECHNOLOGIES

  • US 20090142758A1
  • Filed: 06/23/2006
  • Published: 06/04/2009
  • Est. Priority Date: 06/23/2005
  • Status: Abandoned Application
First Claim
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1. A method for determining a genome sequence comprising the steps of:

  • (a) providing a first subset of the genome by digesting the genome with at least one first restriction endonuclease to provide restriction fragments;

    (b) ligating at least one adaptor to the restriction fragments of the first subset to provide a first set of adaptor-ligated restriction fragments;

    (c) selectively amplifying the first set of adaptor-ligated restriction fragments using a first primer combination wherein at least a first primer contains a section that is complementary to the adaptor and to part of the recognition sequence of the restriction endonuclease and that further contains a first selected sequence at the 3′

    end of the primer sequence, wherein the first selected sequence comprises 1-10 selective nucleotides, to provide a first subset of amplified adaptor-ligated restriction fragments;

    (d) repeating step (c) with at least a second and/or further primer combinations wherein the primer contains a different second and/or further selected sequence at its 3′

    end that contains the same number of selective nucleotides, to provide for second and/or further subsets of amplified adaptor-ligated restriction fragments;

    (e) fragmenting each of the first, second and/or further subsets of amplified adaptor-ligated restriction fragments to generate first, second and/or further sequencing libraries, followed by optional pooling of the libraries;

    (f) determine (at least part of) the nucleotide sequence of (at least part of) the fragments contained in each of the first, second and/or further libraries;

    (g) aligning the sequence of the fragments in each of the first, second and/or further libraries to generate contigs of the amplified adaptor-ligated restriction fragments derived representing dispersed fractions of the genome;

    (h) repeating steps (a)-(g) for at least one second and/or further restriction endonucleases;

    (i) aligning the contigs obtained in step (g) and (h) for each of the second and/or further restriction endonucleases to provide for a sequence of the genome.

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