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 nucleic acids of interest, the method comprising:
ligating adapters onto a plurality of nucleic acid fragments, wherein the fragments include a target sequence;
exposing the fragments to a pair of joined primers, wherein the exposing step occurs under conditions that permit annealing of both of the joined primers to a first fragment of the plurality fragments;
extending the joined primers to produce linked, identical first and second copies of the target sequence;
amplifying the linked, identical first and second copies to generate amplification products; and
sequencing the amplification products.
2. The method of claim 1 , wherein the joined primers are annealed and extended sequentially to make the first and second copies sequentially.
3. The method of claim 1 , wherein the adapters comprise universal priming sites.
4. The method of claim 1 , wherein the ligating step further comprises ligating molecular barcodes onto the plurality of nucleic acid fragments.
5. The method of claim 4 , where the molecular barcodes are sense specific.
6. The method of claim 1 , wherein the pair of joined primers comprise a first primer and a second primer joined via a linker.
7. The method of claim 6 , 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.
8. The method of claim 6 , further comprising using a second pair of joined primers to create antisense copies antisense to the first and second copies prior to the amplification step.
9. The method of claim 6 , wherein the linker comprises polyethylene glycol (PEG).
10. The method of claim 9 , 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.