End Modification to Prevent Over-Representation of Fragments

• US 20090176662A1
• Filed: 02/07/2007
• Published: 07/09/2009
• Est. Priority Date: 02/08/2006
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Rigatti, Roberto, Bignell, Helen Rachel
• Granted Patent: US 9,012,184 B2
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides and also the use of the 5′ and 3′ modified library of templates in methods of solid-phase nucleic acid amplification. In particular, the invention relates to a method of preparing a 5′ and 3′ modified library of template polynucleotides which have common sequences at their 5′ ends and at their 3′ ends, wherein over-representation of “end” sequences of the primary polynucleotide molecules from whence the 5′ and 3′ modified library is generated is greatly reduced or prevented.

Claims

1. A method of generating a 5′

and 3′

modified library of template polynucleotide molecules from one or more primary polynucleotide molecules, wherein said primary polynucleotide molecules are modified primary polynucleotide molecules comprising a modification at or near each 5′

-terminus that prevents ligation to their 5′

-termini;

said method comprising the step of;

a) Fragmenting the modified primary polynucleotide molecules to produce target polynucleotide duplexes, wherein the target polynucleotide duplexes comprise modified target polynucleotide duplexes comprising the modification at or near a 5′

terminus and unmodified target polynucleotide duplexes comprising two ligatable termini;

b) ligating adapter polynucleotides to the two ligatable termini of the unmodified target polynucleotide duplexes to form one or more adapter-target-adapter constructs;

c) carrying out an amplification reaction, wherein a primer oligonucleotide is annealed to both 5′

-terminal adapter portions of each of the adapter-target-adapter constructs and extended by sequential addition of nucleotides to form extension products complementary to each strand of each of the adapter-target constructs, wherein the extension products have common sequences at their 5′

ends and common sequences at their 3′

ends and collectively provide a 5′ and

3′

modified library of template polynucleotide molecules;

wherein said method prevents the over-representation of the sequences at either end of the primary polynucleotide molecules in the 5′ and

3′

modified library of template polynucleotide molecules.

14. The method of claim 1, wherein at least one of the primer oligonucleotides used for amplification extends beyond the 5′

-bases of the adapter.

15. The method of claim 1, wherein the adapter contains a double stranded region and at least one single stranded region.

16. The method according to claim 1, further comprising the steps of:

(d) preparing clusters from the 5′ and

3′

modified library of template polynucleotide molecules; and

(e) sequencing the clusters by sequencing by synthesis.

17. A 5′

and 3′

modified library of template polynucleotide molecules prepared by the method of claim 1.

18. An array comprising the 5′

and 3′

modified library of template polynucleotide molecules of claim 17.

19. (canceled)

20. A method of preventing over-representation of the ends of a primary polynucleotide sequence in a library of fragments generated from said primary polynucleotide sequence, the method comprising modifying the ends of the primary polynucleotide sequence, wherein the modifying prevents the fragments originating from the ends of the primary polynucleotide from ligating to an adapter.

2. The method of claim 20, wherein said modification is at or near the 5′

terminus of the primary polynucleotide molecules and is introduced using an amplification reaction.

3. The method of claim 2, wherein said amplification reaction comprises one or more modified amplification primers.

4. The method of claim 3, wherein said modified amplification primers comprise a 5′

-amino or 5′

-biotin modification.

5. The method of claim 3, wherein said modified amplification primers comprise a modification that prevents nucleotide polymerase mediated copying of the full length of the primer.

6. The method of claim 5, wherein said modified amplification primers comprise an abasic site.

7. The method of claim 20, wherein said primary polynucleotides are double stranded, and said modification at or near the 5′

terminus of the primary polynucleotides is introduced using an enzyme.

8. The method according to claim 7, wherein said modification is a modified deoxynucleoside triphosphate introduced using a nucleotide polymerase.

9. The method according to claim 8, wherein said modification is a dideoxynucleoside triphosphate introduced using a terminal transferase.

10. The method according to claim 20, wherein the fragments of the primary polynucleotide sequences are generated by sonication or nebulization.

11. The method of claim 20, wherein the primary polynucleotide sequences are DNA molecules and the primary polynucleotide sequences are modified to produce modified primary polynucleotide molecules.

12. The method of claim 11, wherein the modified primary polynucleotide molecules are generated by polymerase chain reaction.

13. The method of claims 12, wherein the modified primary polynucleotide molecules are at least 5000 base pairs in length.