Methods for sequencing polynucleotides

• US 10,329,613 B2
• Filed: 04/10/2015
• Issued: 06/25/2019
• Est. Priority Date: 08/27/2010
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Rigatti, Roberto, Gormley, Niall Anthony, Boutell, Jonathan Mark
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US14/683,580
• Publication Number:
  US 20150225787A1
• Time in Patent Office: 1,537 Days
• Field of Search: 435 61, 435 611, 435 612, 435 911, 435 912, 435 9151, 435183, 4352831, 4352871, 4352872, 436 94, 436501, 536 231, 536 243, 536 2433, 536 253
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/119 Double strand sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Provided herein is a method for sequencing a polynucleotide molecules. The method includes the steps of providing a plurality of polynucleotide molecules attached to a surface, wherein a first portion of each polynucleotide molecule is attached to a first location of the surface and a second portion of each polynucleotide molecule is attached to a second location of the surface, the relative proximity of the first and second locations being correlated with the probability that the first and second portions are paired, separating the first and second portions of the polynucleotide molecules on the surface, determining the sequences of the first and second portions of the polynucleotide molecules and comparing the relative proximities and the sequences to determine which first and second portions are paired and to determine the sequence of the target polynucleotide molecules.

Claims

1. A method for determining a probability of whether the sequence of a first portion of one of a plurality of double-stranded polynucleotide molecules and the sequence of a second portion of the one of the plurality of double-stranded polynucleotide molecules are located on the same target polynucleotide molecule, comprising the steps of:

a) providing a plurality of double-stranded polynucleotide molecules having a known length;

b) attaching the plurality of double-stranded polynucleotide molecules to a surface, wherein a first portion on a first strand of each of the plurality of double-stranded polynucleotide molecules is attached to a first location of the surface and a second portion on a second strand of each of the plurality of double-stranded polynucleotide molecules is attached to a second location of the surface, wherein the first portion and the second portion are located at the opposite ends of two complementary strands of each of the plurality of double-stranded polynucleotide molecules;

c) separating the first portion on the first strand of each of the plurality of double-stranded polynucleotide molecules from the second portion on the second strand of each of the plurality of double-stranded polynucleotide molecules by denaturing the two complementary strands of each of the plurality of double-stranded polynucleotide molecules, thereby generating a plurality of denatured single polynucleotide strands, each of the plurality of denatured single polynucleotide strands is immobilized on the first location or the second location of the surface;

d) after step c), amplifying the first portion of each of the plurality of denatured single polynucleotide strands immobilized on the first location and amplifying the second portion of each of the plurality of denatured single polynucleotide strands immobilized on the second location , thereby producing a plurality of nucleic acid clusters on the surface;

e) after step d), identifying the locations of the plurality of nucleic acid clusters on the surface using an imaging device, and determining the distance from one cluster of the plurality of nucleic acid clusters to another cluster of the plurality of nucleic acid clusters on the surface;

f) after step e), determining the sequences of the first portion and the second portion of the plurality of nucleic acid clusters; and

g) comparing the location of each of the plurality of nucleic acid clusters on the surface, wherein the distance from one of the plurality of nucleic acid clusters to another of the plurality of nucleic acid clusters determines the probability of whether the sequence of the first portion of the one of the plurality of double-stranded polynucleotide molecules and the sequence of the second portion of the one of the plurality of double-stranded polynucleotide molecules are located on the same target polynucleotide molecule.

2. The method of claim 1, wherein the known length is a known average length.

3. The method of claim 1, wherein the first portion and second portion are noncontiguous.

4. The method of claim 1, wherein step (f) is carried out by sequencing by ligation, sequencing by synthesis or sequencing by hybridization.

5. The method of claim 1, further comprising comparing the sequences of the first portion and second portion to a reference sequence.

6. The method of claim 1, wherein each of the plurality of double-stranded polynucleotide molecules comprises indexing tags.

7. The method of claim 6, wherein the first portion and second portion of each of the plurality of double-stranded polynucleotide molecules comprise the same tag from the indexing tags.

8. The method of claim 7, wherein the first portion and second portion of each of the plurality of double-stranded polynucleotide molecules retain said same tag after step (c).

9. The method of claim 1, wherein the plurality of double-stranded polynucleotide molecules are produced by a plurality of fragments which is generated by fragmenting one or more target polynucleotide molecules, and wherein the first portion and the second portion in each of the plurality of double-stranded polynucleotide molecules are from the same fragment of the plurality of fragments.