Compositions and methods for improving sample identification in indexed nucleic acid libraries

1. A composition comprising:

a first plurality of adapter-target-adapter molecules comprising double-stranded target fragments isolated from a first source,

wherein the adapter comprises a first sample-specific universal adapter,

wherein the first sample-specific universal adapter comprises

(i) a region of double stranded nucleic acid, and

(ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,

wherein the first sample-specific universal adapter further comprises a first set of sample-specific tag sequences that differentiates the first plurality of adapter-target-adapter molecules from adapter-target-adapter molecules originating from a different source, the first set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and

an exonuclease, wherein the exonuclease does not comprise a DNA polymerase activity.

2. The composition of claim 1 , further comprising first sample-specific universal adapters not attached to a target fragment.

3. The composition of claim 1 , wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity that is biased for double stranded DNA that comprises a 5′ phosphate at the 5′ end of the region of double stranded nucleic acid.

4. The composition of claim 3 , wherein the exonuclease is lambda exonuclease.

5. The composition of claim 1 , wherein the exonuclease comprises a 3′ to 5′ DNA exonuclease activity that is biased for double stranded DNA that is blunt ended and/or has a recessed 3′ terminus.

6. The composition of claim 1 , the composition further comprising a second plurality of adapter-target-adapter molecules comprising double-stranded target fragments isolated from a second source,

wherein the adapter comprises a second sample-specific universal adapter that comprises a second set of sample-specific tag sequences that differentiates the first and second pluralities of adapter-target-adapter molecules.

7. The composition of claim 6 , wherein 3′ ends of the first and second pluralities of adapter-target-adapter molecules, or a combination thereof, are blocked.

8. The composition of claim 2 , wherein 3′ ends of the first sample-specific universal adapters not attached to a target fragment are blocked.

9. The composition of claim 1 , further comprising terminal deoxynucleotidyl transferase, a ddNTP, a DNA polymerase, or a combination thereof.

10. A method, comprising:

providing a first solution of a plurality of double-stranded target fragments isolated from a first source;

ligating a first sample-specific universal adapter to both ends of the double-stranded target fragments from the first source to form a first plurality of adapter-target-adapter molecules,

wherein each of the first plurality of adapter-target-adapter molecules comprises a target fragment flanked by the first sample-specific universal adapter,

wherein the first sample-specific universal adapter comprises (i) a region of double stranded nucleic acid, and (ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,

wherein the first sample-specific universal adapter further comprises a first set of sample-specific tag sequences that differentiates the first plurality of adapter-target- adapter molecules from adapter-target-adapter molecules originating from a different source, the first set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and

wherein the ligating covalently attaches the region of double stranded nucleic acid of the first sample-specific universal adapter to each end of the double-stranded target fragments from the first source; and

contacting the solution with an exonuclease, wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity that is biased for double stranded DNA, wherein the exonuclease does not comprise a DNA polymerase activity,

wherein the exonuclease selectively degrades first sample-specific universal adapters present in the first solution not ligated to a target fragment.

11. The method of claim 10 , wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity that is biased for double stranded DNA that comprises a 5′ phosphate at the 5′ end of the region of double stranded nucleic acid.

12. The method of claim 11 , wherein the exonuclease is lambda exonuclease.

13. The method of claim 10 , further comprising:

providing a second solution of a plurality of double-stranded target fragments isolated from a second source;

ligating a second sample-specific universal adapter to both ends of the double-stranded target fragments from the second source to form a second plurality of adapter-target-adapter molecules,

wherein each of the second plurality of adapter-target-adapter molecules comprises a target fragment from the second source flanked by the second sample-specific universal adapter,

wherein the second sample-specific universal adapter comprises (i) a region of double stranded nucleic acid, and (ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,

wherein the second sample-specific universal adapter further comprises a second set of sample-specific tag sequences that differentiates the second plurality of adapter-target-adapter molecules from adapter-target-adapter molecules originating from a different source, the second set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and

wherein the ligating covalently attaches the region of double stranded nucleic acid of the second sample-specific universal adapter to each end of the double-stranded target fragments from the second source; and

contacting the solution with an exonuclease, wherein the exonuclease comprises a 5′ to 3′ DNA exonuclease activity that is biased for double stranded DNA,

wherein the exonuclease selectively degrades second sample-specific universal adapters present in the second solution not ligated to a target fragment.

14. The method of claim 13 , further comprising blocking 3 ′ ends of the first and second pluralities of adapter-target-adapter molecules.

15. The method of claim 14 , wherein the blocking comprises enzymatically incorporating a dideoxynucleotide onto the 3′ ends of the first and second pluralities of adapter-target-adapter molecules and the 3′ ends of the first and second sample-specific universal adapters that are not attached to a target fragment.

16. The method of claim 13 , further comprising:

providing a surface comprising a plurality of amplification sites,

wherein the amplification sites comprise at least two populations of attached single stranded nuclei acids having a free 3′ end, and

contacting the surface comprising amplification sites with a mixture of the first and second pluralities of adapter-target-adapter molecules under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual adapter-target-adapter molecule.

17. A method, comprising:

providing a first solution of a plurality of double-stranded target fragments isolated from a first source;

ligating a first sample-specific universal adapter to both ends of the double-stranded target fragments from the first source to form a first plurality of adapter-target-adapter molecules,

wherein each of the first plurality of adapter-target-adapter molecules comprises a target fragment flanked by the first sample-specific universal adapter, wherein the first sample-specific universal adapter comprises (i) a region of double stranded nucleic acid, and (ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,

wherein the first sample-specific universal adapter further comprises a first set of sample-specific tag sequences that differentiates the first plurality of adapter-target-adapter molecules from adapter-target-adapter molecules originating from a different source, the first set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and

wherein the ligating covalently attaches the region of double stranded nucleic acid of the first sample-specific universal adapter to each end of the double-stranded target fragments from the first source; and

contacting the solution with an exonuclease, wherein the exonuclease comprises a 3′ to 5′ exonuclease activity that is biased for double stranded DNA having a blunt or recessed 3′ terminus, wherein the exonuclease does not comprise a DNA polymerase activity,

wherein the ligating also forms a plurality of incomplete products comprising adapter-target molecules, and wherein the exonuclease selectively degrades adapter-target molecules and first sample-specific universal adapters present in the first solution not ligated to a target fragment.

18. The method of claim 17 , wherein the exonuclease is exonuclease III.

19. The method of claim 17 , wherein the region of double stranded nucleic acid distal to the region of single-stranded non-complementary nucleic acid strands terminates as a blunt end structure.

20. The method of claim 19 , wherein the double-stranded target fragments comprise blunt end structures.

21. The method of claim 17 , wherein the region of double stranded nucleic acid distal to the region of single-stranded non-complementary nucleic acid strands terminates as a 3′ overhang structure.

22. The method of claim 17 , further comprising:

providing a second solution of a plurality of double-stranded target fragments isolated from a second source;

ligating a second sample-specific universal adapter to both ends of the double-stranded target fragments from the second source to form a second plurality of adapter-target-adapter molecules,

wherein each of the second plurality of adapter-target-adapter molecules comprises a target fragment from the second source flanked by the second sample-specific universal adapter,

wherein the second sample-specific universal adapter comprises (i) a region of double stranded nucleic acid, and (ii) a region of single-stranded non-complementary nucleic acid strands comprising at least one universal primer binding site,

wherein the second sample-specific universal adapter further comprises a second set of sample-specific tag sequences that differentiates the second plurality of adapter-target-adapter molecules from adapter-target-adapter molecules originating from a different source, the second set of sample-specific tag sequences present in the single stranded non-complementary nucleic acid strands, and

wherein the ligating covalently attaches the region of double stranded nucleic acid of the second sample-specific universal adapter to each end of the double-stranded target fragments from the second source; and

contacting the solution with an exonuclease, wherein the exonuclease comprises a 3′ to 5′ exonuclease activity that is biased for double stranded DNA having a blunt or 3′ recessed terminus, wherein the exonuclease does not comprise a DNA polymerase activity, wherein the ligating also forms a plurality of incomplete products, wherein the incomplete products comprise adapter-target molecules, and wherein the exonuclease selectively degrades adapter-target molecules and second sample-specific universal adapters present in the second solution not ligated to a target fragment.

23. The method of claim 22 , further comprising blocking 3′ ends of the first and second pluralities of adapter-target-adapter molecules.

24. The method of claim 23 , wherein the blocking comprises enzymatically incorporating a dideoxynucleotide onto the 3′ ends of the first and second pluralities of adapter-target-adapter molecules and the 3′ ends of the first and second sample-specific universal adapters that are not attached to a target fragment.

25. The method of claim 22 , further comprising:

providing a surface comprising a plurality of amplification sites, wherein the amplification sites comprise at least two populations of attached single stranded nuclei acids having a free 3′ end, and

contacting the surface comprising amplification sites with a mixture of the first and second pluralities of adapter-target-adapter molecules under conditions suitable to produce a plurality of amplification sites that each comprise a clonal population of amplicons from an individual adapter-target-adapter molecule.