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 of sequencing a nucleic acid, the method comprising:
joining two amplification primers with a linker;
amplifying a nucleic acid fragment with the amplification primers, thereby creating a complex comprising two copies of the fragment;
annealing the complex to a solid support comprising binding sites for the complex;
amplifying the complex to create a cluster of amplification products; and
sequencing the amplification products.
2. The method of claim 1 , wherein at least one of the amplification primers is a universal amplification primer.
3. The method of claim 1 , wherein the nucleic acid fragment comprises a universal priming site.
4. The method of claim 3 , further comprising ligating the universal priming site to a target nucleic acid to produce the nucleic acid fragment.
5. The method of claim 1 , further comprising amplifying a target nucleic acid with a primer comprising the universal priming site to produce the nucleic acid fragment.
6. 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, and a protein.
7. The method of claim 1 , wherein the linker is removed prior to amplifying the complex.
8. The method of claim 1 , wherein the complex forms the seed for a single cluster.
9. The method of claim 1 , wherein the linker comprises polyethylene glycol (PEG).
10. The method of claim 1 , wherein the linker comprises a modified polyethylene glycol (PEG) selected from the group consisting of DBCO-PEG4, PEG-11, and N-hydroxysuccinimide (NHS) modified PEG.
11. The method of claim 1 , wherein the linker is selected from the group consisting of an oligosaccharide, a lipid, a hydrocarbon, a polymer, an antibody, and a protein.
12. The method of claim 1 , wherein the linker comprises an inverted or modified base.