Arrayed biomolecules and their use in sequencing

• US 7,232,656 B2
• Filed: 06/30/2003
• Issued: 06/19/2007
• Est. Priority Date: 07/30/1998
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Ltd. (Illumina Incorporated)
• Inventors: Balasubramanian, Shankar, Klenerman, David, Barnes, Colin, Osborne, Mark Allen
• Primary Examiner(s): Whisenant; Ethan
• Application Number:
  US10/610,305
• Publication Number:
  US 20040091903A1
• Time in Patent Office: 1,450 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/0054 Means for coding or tagging the apparatus or the reagents
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 : Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00707 : separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/501 : Ligase
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• CPC Class Codes: C12Q 2565/507 : characterised by the density of the capture oligonucleotide
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

The invention is directed to a method for analysing genome wide variation in an individual. The method comprises randomly fragmenting the individual'"'"'s genome and generating sequence reads of multiple bases on all fragments of the individual'"'"'s genome, aligning the sequence reads generated with a known genomic reference sequence, and analysing variations between the sequence reads derived from the individual'"'"'s genome and the known genomic reference sequence.

Claims

1. A method for analysing genome wide variation in an individual comprising:

i) randomly fragmenting a genome of said individual;

ii) generating sequence reads of multiple bases on all fragments of said genome;

iii) aligning the sequence reads with a known genomic reference sequence; and

iv) analysing variations between the sequence reads derived from the genome of the individual and the known genomic reference sequence.

2. A method according to claim 1 further comprising identifying regions of variation across the whole of the genome of said individual with respect to said reference sequence to provide a genetic signature for said individual.

3. A method according to claim 1, wherein said fragments of genomic DNA are immobilised onto the surface of a solid support to form an array of polynucleotide molecules capable of interrogation and which molecules can be individually resolved by optical microscopy.

4. A method according to claim 1, wherein each molecule is immobilised by covalent attachment to the surface of said solid support other than at that part of each molecule that can be interrogated.

5. A method according to claim 4, wherein the sequences of said fragments are established by contacting the array with each of the bases A, T G and C under conditions that permit the polymerase reaction to proceed and thereby form sequences complementary to those in the array, and determining the incorporation of a base at each of selected positions in the complementary sequences.

6. A method according to claim 5, wherein the determination of the incorporation of said base is repeated between 10 and 20 times to generate a partial complementary sequence of between 10–

20 nucleotides.

8. A method of generating a genetic signature for an individual, which method comprises:

i) immobilising fragments of the genome of said individual onto the surface of a solid support to form an array of polynucleotide molecules capable of interrogation, wherein the array allows the molecules to be individually resolved by optical microscopy, and wherein each molecule is immobilised by covalent bonding to the surface, other than at that part of each molecule that can be interrogated;

ii) contacting the fragments with a series of nucleotide primers comprising sequences capable of hybridising to regions on said fragments specific for each of the regions of variation identified according to claim 2, in the presence of bases A, T, G and C under conditions that permit the polymerase reaction to proceed;

iii) determining the successive incorporation of a base at each of selected positions to produce sequences of a defined length complementary to the immobilised fragments;

iv) identifying the sequence of those fragments that have undergone the polymerase reaction to produce a genetic signature for said individual.

7. A method for analysing genome wide variation in an individual comprising:

i) randomly fragmenting a genome of said individual;

ii) arraying the fragments such that different fragments can be individually resolved by optical microscopy;

iii) generating sequence reads of multiple bases on all fragments of said genome;

iv) aligning said sequence reads with a known genomic reference sequence; and

v) analysing variations between the sequence reads derived from the individual sample and the known genomic reference sequence.

9. A method according to claim 7 or 8, wherein in step iii) the incorporation of said base is repeated between 10 and 20 times to produce a complementary sequence of between 10 and 20 nucleotides.