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Methods of targeted sequencing

  • US 10,072,287 B2
  • Filed: 09/10/2010
  • Issued: 09/11/2018
  • Est. Priority Date: 09/10/2009
  • Status: Active Grant
First Claim
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1. A method of sequencing comprising:

  • (a) hybridizing a plurality of target polynucleotide fragments from a sample to a plurality of capture probes and a plurality of first solution probes, wherein each capture probe of the plurality of capture probes is attached to a solid support and at least one capture probe from the plurality of capture probes comprises sequence complementary to a first region of known sequence on one or more of the plurality of target polynucleotide fragments, and each first solution probe of the plurality of first solution probes are in solution and comprise a cleavage site at a 3′

    end and a first cap on the 3′

    end, and wherein at least one first solution probe of the plurality of first solution probes comprises sequence complementary to a second region of known sequence on the one or more of the plurality of target polynucleotide fragments, wherein the hybridizing comprises forming one or more hybridization complexes, wherein each of the one or more hybridization complexes comprises;

    (i) a capture probe from the plurality of capture probes;

    (ii) a target polynucleotide fragment from the plurality of target polynucleotide fragments; and

    (iii) a first solution probe from the plurality of first solution probes, wherein the capture probe hybridizes to the first region of the known sequence on the target polynucleotide fragment and the first solution probe hybridizes to the second region of the known sequence on the target polynucleotide fragment, whereby hybridization of the capture probe to the first region and the first solution probe to the second region of the target polynucleotide fragment is configured to permit ligation of the capture probe and the first solution probe;

    (b) ligating the capture probe and the first solution probe in the one or more hybridization complexes, thereby forming one or more target-capture probe-first solution probe hybridization complexes comprising the target polynucleotide fragment and ligated capture and first solution probes;

    (c) adding a second cap, wherein the second cap is attached to any of the plurality of capture probes which are not ligated to any of the plurality of first solution probes;

    (d) selectively removing the first cap from the one or more target-capture probe-first solution probe hybridization complexes by cleaving the cleavage site at the 3′

    end of the first solution probe in the one or more target-capture probe-first solution probe hybridization complexes(e) hybridizing a second solution probe to the one or more target-capture probe-first solution probe hybridization complexes from (d), wherein the second solution probe comprises sequence complementary to a third region of the known sequence on the target polynucleotide fragment, whereby the hybridizing of the second solution probe to the one or more target-capture probe-first solution probe hybridization complexes is configured to permit ligation of the second solution probe and the first solution probe;

    (f) ligating the second solution probe to the first solution probe, thereby forming one or more target-capture probe-first-second solution probe hybridization complexes, comprising ligated capture probe, first solution probe, and second solution probe;

    (g) hybridizing a third solution probe to the one or more target probe hybridization complexes from (f) wherein the third solution probe comprises sequence complementary to a fourth region of known sequence on the target polynucleotide fragment, and wherein the third solution probe comprises a universal primer sequence, whereby the hybridizing of the third solution probe to the target-probe hybridization complexes from (f) is configured to permit-ligation of the third solution probe and the second solution probe;

    (h) ligating the third solution probe to the second solution probe, thereby generating one or more target-capture probe-first-second-third solution probe hybridization complexes comprising ligated capture probe, first solution probe, second solution probe, and third solution probe;

    (i) removing the target polynucleotide fragment from the one or more target-capture probe-first-second-third solution probe hybridization complexes, thereby generating one or more single stranded complexes comprising capture probe-first solution probe-second solution probe-third solution probe polynucleotides comprising the universal primer sequence at the 3′

    end; and

    (j) determining the sequence of the one or more single-stranded complexes generated in (i) by hybridizing a primer directed against the universal primer sequence and performing a sequencing reaction.

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