Methods of sequencing with linked fragments
The invention generally relates to sequencing library preparation methods. In certain embodiments, two template nucleic acids are joined together by a linking molecule, such as a PEG derivative. The linked template nucleic acids is amplified, creating linked amplicons.
1. A method for sequencing a nucleic acid, the method comprising:
annealing to a solid support a complex comprising two nucleic acid templates joined by a linker, wherein the linker comprises a molecule that is not selected from the group consisting of guanine, cytosine, adenine, uracil or thymine;
amplifying the complex to generate amplification products; and
sequencing the amplification products.
2. The method of claim 1 , wherein the linker is selected from the group consisting of a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, an antibody, and a protein.
3. The method of claim 1 , wherein the linker comprises an inverted or modified base.
4. The method of claim 1 , wherein the linker is removed prior to the amplifying step.
5. The method of claim 1 , wherein the method further comprises introducing an adapter to the complex.
6. The method of claim 1 , wherein the method further comprises the step of introducing primers to the complex.
7. The method of claim 1 , wherein the amplifying step comprises bridge amplification.
8. The method of claim 1 , further comprising attaching the amplification products to the solid support.
9. The method of claim 8 , wherein the amplification products form a cluster on the solid support.
10. The method of claim 1 , wherein the sequencing step comprises sequencing-by-synthesis.
11. The method of claim 10 , wherein said sequencing-by-synthesis comprises addition of fluorescently-labeled nucleotides.
12. A method of sequencing a nucleic acid, the method comprising:
attaching at least two copies of a nucleic acid fragment, forming a unit;
joining at least two units with a linker to form a complex, wherein the linker comprises a molecule that is not selected from the group consisting of guanine, cytosine, adenine, uracil or thymine;
annealing the complex to a solid support;
amplifying the complex; and
sequencing the amplification products.
13. The method of claim 12 , wherein the linker is selected from the group consisting of a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, an antibody, and a protein.
14. The method of claim 12 , wherein the linker comprises an inverted or modified base.
15. The method of claim 12 , wherein the method further comprises introducing an adapter to the complex.