Methods of sequencing 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 preparing a sequencing library, 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;
extending the joined primers to produce a first copy of the target sequence linked to a second copy of the target sequence, wherein the linked first and second copies are from sense and anti-sense strands, respectively, of the target sequence and have the same sequence; and
amplifying the linked first and second copies to generate amplification products.
2. The method of claim 1 , further comprising sequencing the amplification products.
3. The method of claim 1 , wherein at least one of the exposing, extending, and amplifying steps are performed within an aqueous partition.
4. The method of claim 3 , wherein the aqueous partition uses one selected from the group consisting of emulsions, droplets, or wells.
5. The method of claim 1 , wherein the joined primers are attached to a solid support.
6. The method of claim 5 , where the solid support is selected from the group consisting of a microbead, a bead, a channel wall, a microchip, and a surface.
7. The method of claim 5 , wherein the amplifying step generates a group or cluster of amplicons bound to the solid support.
8. The method of claim 7 , further comprising performing a sequencing reaction on the group or cluster.
9. The method of claim 8 , wherein the sequencing reaction includes:
introducing a DNA polymerase and a fluorescently labeled nucleotide;
detecting incorporation of the fluorescently labeled nucleotide;
a cleavage step that removes a fluorescent label; and
repeating the introducing, detecting, and cleavage step with other fluorescently labeled nucleotides.
10. The method of claim 9 , further comprising monitoring the incorporation of the fluorescently labeled nucleotides.
11. The method of claim 10 , further comprising identifying an error introduced during the amplifying step when the sequencing reaction gives a signal indicating disagreement between two bases at corresponding positions in the linked first and second copies.
12. The method of claim 1 , wherein the adapters comprise universal priming sites.
13. The method of claim 12 , wherein the linked copies of the target sequence are linked together with an in-phase orientation that prevents binding between the linked copies of the target sequence.
14. The method of claim 1 , wherein the ligating further comprises ligating molecular barcodes onto the plurality of nucleic acid fragments.
15. The method of claim 14 , where the molecular barcodes are sense specific.
16. A method for making a sequencing library, the method comprising:
ligating adapters onto a plurality of nucleic acid fragments, wherein the fragments include a target sequence;
exposing the fragments to a complex of joined primers, wherein the complex contains at least two joined primers that are linked together;
extending the joined primers to produce linked copies of the target sequence wherein the linked copies include a copy from a sense strand of the target sequence and a copy from an antisense strand of the target sequence and have an identical sequence; and
amplifying the linked copies.
17. The method of claim 16 , wherein the amplifying step is performed using primers bound to a solid support.
18. The method of claim 17 , wherein the amplifying step generates a plurality of amplicons bound to the solid support.
19. The method of claim 18 , further comprising performing a sequencing reaction on the plurality of amplicons bound to the solid support.
20. The method of claim 19 , further comprising identifying an error introduced during the amplifying step when the sequencing reaction gives a signal indicating disagreement between two bases at corresponding positions within the plurality of amplicons.
21. The method of claim 19 , further comprising monitoring the incorporation of labeled nucleotides during a polymerization step of the sequencing reaction.
22. The method of claim 19 , wherein the sequencing reaction includes:
introducing a DNA polymerase and a fluorescently labeled nucleotide;
detecting incorporation of the fluorescently labeled nucleotide;
a cleavage step that removes a fluorescent label; and
repeating the introducing, detecting, and cleavage step with other fluorescently labeled nucleotides.
23. The method of claim 18 , wherein the amplifying step is performed within a droplet of an emulsion.