Paired end sequencing

• US 20090233291A1
• Filed: 01/28/2009
• Published: 09/17/2009
• Est. Priority Date: 06/06/2005
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Riches, David Roderick, Ferreri, Gianni Calogero, Chen, Zhoutao, Godwin, Brian Christopher
• Application Number:
  US12/322,119
• Publication Number:
  US 20090233291A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

An embodiment of a method for obtaining a DNA construct comprising two end regions of a target nucleic acid in an in vitro reaction is described that comprises the steps of: fragmenting a large nucleic acid molecule to produce a target nucleic acid molecule; ligating a recombination adaptor element to each end of the target nucleic acid molecule to produce an adapted target nucleic acid molecule; exposing the adapted target nucleic acid to a site specific recombinase to produce a circular nucleic acid product and a linear nucleic acid product from the adapted target nucleic acid, wherein the circular nucleic acid product comprises the target nucleic acid molecule; and fragmenting the circular nucleic acid product to produce a template nucleic acid molecule comprising a sequence region from each end of the target nucleic acid molecule.

Claims

1. A method for obtaining a DNA construct comprising two end regions of a target nucleic acid in an in vitro reaction comprising the steps of:

fragmenting a large nucleic acid molecule to produce a target nucleic acid molecule;

ligating a recombination adaptor element to each end of the target nucleic acid molecule to produce an adapted target nucleic acid molecule;

exposing the adapted target nucleic acid to a site specific recombinase to produce a circular nucleic acid product and a linear nucleic acid product from the adapted target nucleic acid, wherein the circular nucleic acid product comprises the target nucleic acid molecule; and

fragmenting the circular nucleic acid product to produce a template nucleic acid molecule comprising a sequence region from each end of the target nucleic acid molecule.

2. The method of claim 1, wherein:

after the step of exposing the adapted target nucleic acid to a site specific recombinase the method further comprises the step of removing the non-circular molecules.

3. The method of claim 2, wherein:

the non-circular molecules comprise the linear nucleic acid product and an adaptor dimer product, wherein the adaptor dimer product is generated from a ligation of two of the recombination adaptor elements to each other.

4. The method of claim 2, further comprising the steps of:

the non-circular molecules are removed using at least one exonuclease.

5. The method of claim 2, further comprising:

adding a plurality of circular carrier DNA molecules to the circular nucleic acid product;

fragmenting the circular nucleic acid product and the carrier DNA molecules to produce the template molecule and a plurality of linear carrier molecules;

determining the efficiency of the fragmentation from the template molecule and the linear carrier molecules;

amplifying the template molecule to produce a population comprising a plurality of substantially identical copies, wherein the linear carrier molecules are un-amplifiable; and

sequencing the population to produce sequence data comprising the sequence composition of the template nucleic acid.

6. The method of claim 5, wherein:

the circular carrier molecules comprise pUC19.

7. The method of claim 5, wherein:

the circular carrier molecules comprise damaged DNA wherein the damaged DNA is un-amplifiable.

8. The method of claim 7, wherein:

the damaged DNA includes a type of damage selected from the group consisting of UV damage, alkylation/methylation, X-ray damage, hydrolysis, and oxidative damage.

9. The method of claim 1, further comprising the steps of:

amplifying the template nucleic acid to produce a population comprising a plurality of substantially identical copies; and

sequencing the population to produce sequence data comprising the sequence composition of the template nucleic acid.

10. The method of claim 9, further comprising the steps of:

ligating a second set of adaptor elements to the template nucleic acid molecule, wherein the second set of adaptor elements comprise a first primer element and a second primer element and further wherein the step of amplifying employs the first primer element and the step of sequencing employs the second primer element.

11. The method of claim 9, wherein:

the sequence composition of the template nucleic acid comprises a sequence composition for each of the sequence regions from the ends of the target molecule.

12. The method of claim 1, wherein:

the recombination adaptor elements comprise a first recombination adaptor element and a second recombination adaptor element, wherein the first and second recombination adaptor elements both comprise a directional element.

13. The method of claim 12, wherein:

the circular nucleic acid product and the linear nucleic acid product are produced when the directional elements in the first and second recombination adaptor elements are in an identical directional relationship.

14. The method of claim 13, wherein:

the first and second recombination adaptor elements each comprise a blunt end that ligates to the target nucleic acid molecule in an orientation that promotes the identical directional relationship of the directional elements.

15. The method of claim 12, wherein:

the first and second recombination adaptor elements comprise an overhang end that inhibits formation of adaptor concatemers.

16. The method of claim 12, wherein:

the directional element comprises a lox sequence element.

17. The method of claim 12, wherein:

The first and second recombination adaptors elements comprise a palindromic sequence element flanking both ends of the directional element.

18. The method of claim 1, wherein:

the site specific recombinase comprises a Cre recombinase.

19. The method of claim 1, wherein:

the target nucleic acid molecule comprises a length selected from the group consisting of at least 3 Kb, at least 8 Kb, at least 10 Kb, at least 20 Kb, at least 50 Kb, and at least 100 Kb.

20. The method of claim 1, wherein:

the large nucleic acid molecule comprises genomic DNA.

21. The method of claim 1, wherein:

the circular nucleic acid product comprises a first hybrid recombination adaptor and the linear nucleic acid product comprises a second hybrid recombination adaptor, wherein the first and second hybrid recombination adaptors comprise elements from the ligated recombination adaptors.

22. The method of claim 21, wherein:

the template nucleic acid comprises the first hybrid recombination adaptor positioned between the end sequence regions.

23. The method of claim 22, wherein:

the template nucleic acid comprises at least one enrichment tag associated with the first hybrid recombination adaptor.

24. The method of claim 23, wherein:

the enrichment tag comprises a Biotin tag.

25. The method of claim 1, wherein:

the step of fragmenting the circular nucleic acid product comprises nebulization.

26. The method of claim 25, wherein:

the step of fragmenting the circular nucleic acid product further comprises a first break of the circular nucleic acid product using a type II restriction enzyme and a second break using the nebulization, wherein the type II restriction enzyme cuts at a restriction site in a hybrid adaptor region of the circular nucleic acid product and produces a short sequence region from the target nucleic acid and the nebulization produces a long sequence region from the target nucleic acid.

27. The method of claim 26, wherein:

the type II restriction enzyme comprises MmeI and the short sequence region comprises a 20 bp sequence length.

29. A kit for performing the method of claim 1, comprising:

a plurality of recombination adaptor elements; and

a site specific recombinase.

31. A kit for performing the method of claim 5, comprising:

a plurality of recombination adaptor elements;

a site specific recombinase;

an exonuclease; and

a circular carrier DNA.

32. The kit of claim 29, wherein:

the site specific recombinase comprises Cre recombinase and the circular carrier DNA comprises pUC19.

28. A method for obtaining a plurality of DNA constructs comprising two end regions of a target nucleic acid in an in vitro reaction comprising the steps of:

fragmenting a large nucleic acid molecule to produce a plurality of target nucleic acid molecules;

ligating a recombination adaptor element to each end of the target nucleic acid molecules to produce a plurality of adapted target nucleic acid molecules;

exposing the adapted target nucleic acid molecules to a site specific recombinase to produce a plurality of circular nucleic acid products and a plurality of linear nucleic acid products from the adapted target nucleic acid molecule, wherein the circular nucleic acid products comprise the target nucleic acid molecules; and

fragmenting the circular nucleic acid products to produce a plurality of template nucleic acid molecules comprising a sequence region from each end of the target nucleic acid molecules.

30. The kit of claim 28, wherein:

the site specific recombinase comprises Cre recombinase.