Compositions and methods for amplifying polynucleotides

1. A composition for amplifying a polynucleotide, the composition comprising:

a substrate comprising a first region and a second region;

a first plurality of capture primers coupled to the first region of the substrate;

a second plurality of capture primers coupled to the second region of the substrate, the capture primers of the second plurality of capture primers being longer than the capture primers of the first plurality of capture primers;

a first plurality of orthogonal capture primers coupled to the first region of the substrate; and

a second plurality of orthogonal capture primers coupled to the second region of the substrate, the orthogonal capture primers of the second plurality of orthogonal capture primers being shorter than the orthogonal capture primers of the first plurality of orthogonal capture primers.

2. The composition of claim 1 , further comprising a fluid comprising target polynucleotides, each of the target polynucleotides comprising a first adapter that is complementary to the orthogonal capture primers of the first and second pluralities of orthogonal capture primers, and a second adapter that is complementary to the capture primers of the first and second pluralities of capture primers.

3. The composition of claim 2 , wherein the first adapters of the target polynucleotides are shorter than the orthogonal capture primers of the first plurality of orthogonal capture primers, and wherein the second adapters of the target polynucleotides are shorter than the capture primers of the second plurality of capture primers.

4. The composition of claim 2 , wherein the first adapter of a first one of the target polynucleotides is hybridized to a first one of the orthogonal capture primers of the first plurality of orthogonal capture primers, or wherein the second adapter of the first one of the target polynucleotides is hybridized to a first one of the capture primers of the second plurality of capture primers.

5. The composition of claim 4 , wherein a duplex formed from the hybridization between the first adapter of the first one of the target polynucleotides and the first one of the orthogonal capture primers has a melting temperature (Tm) of greater than about 40° C.

6. The composition of claim 1 , further comprising a first amplicon covalently coupled to one of the orthogonal capture primers of the first plurality of orthogonal capture primers, the first amplicon having a second adapter completely hybridized to one of the capture primers of the second plurality of capture primers.

7. The composition of claim 1 , further comprising a second amplicon covalently coupled to one of the capture primers of the first plurality of capture primers, the second amplicon having a first adapter completely hybridized to one of the orthogonal capture primers of the second plurality of orthogonal capture primers.

8. The composition of claim 1 , further comprising a third amplicon covalently coupled to one of the orthogonal capture primers of the first plurality of orthogonal capture primers, the third amplicon having a second adapter that is unable to completely hybridize to any of the capture primers of the first plurality of capture primers.

9. The composition of claim 8 , wherein the inability of the second adapter to completely hybridize to any of the capture primers of the first plurality of capture primers inhibits amplification of the third amplicon.

10. The composition of claim 9 , wherein any partial duplex between the second adapter of the third amplicon and any of the capture primers of the first plurality of capture primers has a melting temperature (Tm) of less than about 20° C.

11. The composition of claim 1 , further comprising a fourth amplicon covalently coupled to one of the capture primers of the second plurality of capture primers, the fourth amplicon having a first adapter that is unable to completely hybridize to any of the orthogonal capture primers of the second plurality of orthogonal capture primers.

12. The composition of claim 11 , wherein the inability of the first adapter to completely hybridize to any of the orthogonal capture primers of the second plurality of orthogonal capture primers inhibits amplification of the fourth amplicon.

13. The composition of claim 1 , wherein the capture primers of the first plurality of capture primers are at least 5 bases shorter than the capture primers of the second plurality of capture primers, and wherein the orthogonal capture primers of the second plurality of orthogonal capture primers are at least 5 bases shorter than the orthogonal capture primers of the first plurality of orthogonal capture primers.

14. The composition of claim 13 , wherein the capture primers of the second plurality of capture primers are P5 capture primers, and wherein the orthogonal capture primers of the first plurality of orthogonal capture primers are P7 capture primers.

15. The composition of claim 14 , wherein the capture primers of the first plurality of capture primers are shortened P5 capture primers, and wherein the orthogonal capture primers of the second plurality of orthogonal capture primers are shortened P7 capture primers.

16. The composition of claim 13 , wherein the capture primers of the first plurality of capture primers are approximately the same length as the orthogonal capture primers of the second plurality of capture primers.

17. The composition of claim 13 , wherein the capture primers of the second plurality of capture primers are approximately the same length as the orthogonal capture primers of the first plurality of capture primers.

18. The composition of claim 1 , wherein the first region of the substrate is adjacent to the second region of the substrate.

19. The composition of claim 1 , wherein the first region of the substrate surrounds the second region of the substrate.

20. The composition of claim 1 , wherein the second region of the substrate surrounds the first region of the substrate.