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Methods and compositions for efficient base calling in sequencing reactions

  • US 20090263802A1
  • Filed: 01/28/2009
  • Published: 10/22/2009
  • Est. Priority Date: 01/28/2008
  • Status: Active Grant
First Claim
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1. A method of identifying a first nucleotide at a detection position of a target sequence comprising:

  • a) providing a surface comprising a plurality of concatemers, each concatemer comprising a plurality of monomers, each monomer comprising;

    i) a first target domain of said target sequence;

    ii) a first detection position;

    iii) a first adapter adjacent to said first target domain, wherein said first adapter comprises a first anchor site;

    b) providing a first sequencing set of sequencing probes comprising;

    i) a first probe set comprising;

    1. a first unique label;

    2. a first probe domain complementary to said first target domain; and

    3. a first unique nucleotide at a first interrogation position;

    ii) a second probe set comprising;

    1. a second unique label;

    2. said first probe domain; and

    3. a second unique nucleotide at said first interrogation position;

    iii) a third probe set comprising;

    1. said first label and said second label;

    2. said first probe domain; and

    3. a third unique nucleotide at said first interrogation position;

    iv) a fourth probe set comprising;

    1. said first probe domain; and

    2. a fourth unique nucleotide at said first interrogation position;

    (c) hybridizing an anchor probe to said first anchor site;

    (d) applying said set of sequencing probes to said surface, wherein if a sequencing probe from said sequencing set has a unique nucleotide that is complementary to said first nucleotide, said sequencing probe hybridizes to said concatemer;

    (e) ligating said hybridized sequencing probe to said hybridized anchor probe to form ligation products;

    (f) determining the identity of said first nucleotide.

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