Methods for sequencing polynucleotides

• US 9,029,103 B2
• Filed: 08/26/2011
• Issued: 05/12/2015
• 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
• Application Number:
  US13/219,422
• Publication Number:
  US 20120053063A1
• Time in Patent Office: 1,355 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• 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 and the sequence of a second portion 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 end of each of the polynucleotide molecules is attached to a first location of the surface and a second portion on a second end of each of the polynucleotide molecules is attached to a second location of the surface, wherein the first portion and second portion are located at the opposite ends of the double-stranded polynucleotide molecules;

c) cleaving each of the double-stranded polynucleotide molecules at a position between the first portion and second portion, thereby separating the first portion and second portion of each of the plurality of double-stranded polynucleotide molecules on the surface;

d) amplifying the first portion and second portion of each of the double-stranded polynucleotide molecules after step c) at the first location and second location, thereby producing a plurality of nucleic acid clusters on the surface;

e) 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) determining the sequences of the first portion and the second portion of each of the double-stranded polynucleotide molecules in the plurality of nucleic acid clusters after step e); and

g) comparing the locations of the plurality of nucleic acid clusters on the surface, wherein the distance from said one cluster to said another cluster determines the probability of whether the sequence of the first portion and the sequence of the second portion are located on the same target polynucleotide molecule.

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

3. The method of claim 1, wherein the cleaving step is carried out by cleaving a cleavable site between the first portion and the second portion.

4. The method of claim 3, wherein the cleavable site comprises a modified nucleotide or is a restriction enzyme site.

5. The method of claim 1, wherein the cleaving step is carried out by contacting each of the double-stranded molecules with a transposase to insert a transposon at a position between the first portion and second portion, thereby separating the first portion and second portion.

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

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

8. The method of claim 1, wherein each of the polynucleotide molecules comprises indexing tags.

9. The method of claim 8, wherein the first portion and second portion of each of the polynucleotide molecules comprise the same tag from the indexing tags.

10. The method of claim 9, wherein the first portion and second portion retain said same tag after step (c).