Universal blocking oligo system and improved hybridization capture methods for multiplexed capture reactions

1. A composition for use in massive parallel nucleic acid sequencing comprising:

a) a library of nucleic acids comprising a plurality of library inserts wherein each nucleic acid of the library comprises (i) at least one library insert obtained from one of four or more samples, (ii) a first non-native nucleic acid, and (iii) a second non-native nucleic acid, wherein the first non-native nucleic acid and the second non-native nucleic acid are located on opposing sides of the at least one library insert, and the first non-native nucleic acid comprises a first distinguishable nucleic acid barcode and the second non-native nucleic acid comprises a second distinguishable nucleic acid barcode, wherein the first and second distinguishable nucleic acid barcodes are unique to the one of the four or more samples; and

b) four universal blocking (U-block) nucleic acids, wherein (i) a first U-block nucleic acid and a second U-block nucleic acid are configured to hybridize to the first non-native nucleic acid on opposing sides of the first distinguishable nucleic acid barcode, (ii) a third U-block nucleic acid and a fourth U-block nucleic acid are configured to hybridize to the second non-native nucleic acid on opposing sides of the second distinguishable nucleic acid barcode, and (iii) each of the U-block nucleic acids does not substantially hybridize to a portion of the first or second distinguishable nucleic acid barcode.

2. The composition of claim 1 , wherein the library of nucleic acids comprises at least eight distinguishable nucleic acid barcodes, and optionally, wherein each of the at least eight distinguishable nucleic acid barcodes is present on a different nucleic acid of the library.

3. The composition of claim 1 , wherein the first and second U-block nucleic acids are substantially complementary to a portion of the first non-native nucleic acid, and the third and fourth U-block nucleic acids are substantially complementary to a portion of the second non-native nucleic acid.

4. The composition of claim 1 , comprising no more than four U-block nucleic acids.

5. The composition of claim 1 , comprising one or more capture nucleic acids, wherein,

(i) the capture nucleic acids comprise a member of a binding pair; and

(ii) each of the capture nucleic acids is configured to specifically hybridize to a subset of nucleic acids of the library.

6. The composition of claim 1 , wherein the library of nucleic acids comprises ten or more distinguishable nucleic acid barcodes.

7. The composition of claim 1 , wherein the first and second non-native nucleic acids comprise adapter nucleic acids.

8. The composition of claim 1 , wherein each of the four U-block nucleic acids comprises a length of 10 to 40 nucleotides.

9. The composition of claim 1 , wherein each of the four U-block nucleic acids comprises locked nucleic acids.

10. The composition of claim 1 , wherein each of the four U-block nucleic acids comprises bridged nucleic acids.

11. The composition of claim 1 , wherein each of the four U-block nucleic acids has a melting temperature of at least 65° C.

12. The composition of claim 1 , wherein the four U-block nucleic acids do not substantially hybridize to a distinguishable nucleic acid barcode.

13. The composition of claim 1 , wherein the four U-block nucleic acids comprise a chain terminator.

14. A method of analyzing a nucleic acid library comprising:

a) obtaining a library of nucleic acids comprising a plurality of library inserts wherein each nucleic acid of the library comprises (i) at least one library insert obtained from one of four or more samples, (ii) a first non-native nucleic acid, and (iii) a second non-native nucleic acid, wherein the first non-native nucleic acid and the second non-native nucleic acid are located on opposing sides of the at least one library insert, and the first non-native nucleic acid comprises a first distinguishable nucleic acid barcode and the second non-native nucleic acid comprises a second distinguishable nucleic acid barcode, wherein the first and second distinguishable nucleic acid barcodes are unique to the one of the four or more samples;

b) contacting the library of nucleic acids with four universal blocking (U-block) nucleic acids, wherein (i) a first U-block nucleic acid and a second U-block nucleic acid are configured to hybridize to the first non-native nucleic acid on opposing sides of the first distinguishable nucleic acid barcode, (ii) a third U-block nucleic acid and a fourth U-block nucleic acid are configured to hybridize to the second non-native nucleic acid on opposing sides of the second distinguishable nucleic acid barcode, and (iii) each of the U-block nucleic acids does not substantially hybridize to a portion of the first or second distinguishable nucleic acid barcode;

c) contacting the library of nucleic acids with one or more capture nucleic acids, each comprising a first member of a binding pair, wherein the one or more capture nucleic acids are configured to specifically hybridize to a subset of the nucleic acids of the library;

d) capturing the capture nucleic acids, thereby providing captured nucleic acids comprising the subset of nucleic acids of the library;

e) contacting the captured nucleic acids with a set of primers under amplification condition, thereby providing amplicons; and

f) analyzing the amplicons.

15. The method of claim 14 , wherein the library of nucleic acids comprises ten or more distinguishable nucleic acid barcodes.

16. The method of claim 14 , wherein each of the four U-block nucleic acids comprises a length of 10 to 40 nucleotides.

17. The method of claim 14 , wherein each of the four U-block nucleic acids comprises locked nucleic acids.

18. The method of claim 14 , wherein each of the four U-block nucleic acids comprises bridged nucleic acids.

19. The method of claim 14 , wherein each of the four U-block nucleic acids has a melting temperature of at least 65° C.