System and method for improved processing of nucleic acids for production of sequencable libraries

• US 20110003701A1
• Filed: 02/23/2009
• Published: 01/06/2011
• Est. Priority Date: 02/27/2008
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Albert, Thomas J., Simons, Jan F., Riches, David Roderick, Hutchison, Stephen Kyle, Braverman, Michael S., Jeddeloh, Jeffrey, Ferreri, Gianni Calogero, Egholm, Michael, Palmer, Melinda D., Ronan, Michael Todd, Godwin, Brian C., Kitzman, Jacob
• Application Number:
  US12/380,139
• Publication Number:
  US 20110003701A1
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

An embodiment of an adaptor element for efficient target processing is described that comprises a semi-complementary double stranded nucleic acid adaptor comprising a non-complementary region and a complementary region, where the non-complementary region comprises a first amplification primer site and a second amplification primer site and the complementary region comprises a sequencing primer site and one or more inosine species.

Claims

1. An adaptor element for efficient target processing, comprising:

a semi-complementary double stranded nucleic acid adaptor comprising a non-complementary region and a complementary region, wherein the non-complementary region comprises a first amplification primer site and a second amplification primer site and the complementary region comprises a sequencing primer site and one or more inosine species.

2. The adaptor element of claim 1, wherein:

the non-complementary region comprises a detectable moiety.

3. The adaptor element of claim 2, wherein:

the detectable moiety comprises a fluorescent label.

4. The adaptor element of claim 3, wherein:

the fluorescent label is selected from the group consisting of Cy3, Cy5, carboxyfluorescein (FAM), Alexafluor, Rhodamine green, Texas Red, R-Phycoerytherin, semiconductor nanocrytals.

5. The adaptor element of claim 1, wherein:

the complementary region comprises a blunt end.

6. The adaptor element of claim 5, wherein:

The blunt end is ligatable to a blunt end of a target nucleic acid.

7. The adaptor element of claim 1, wherein:

the complementary region comprises a sticky end.

8. The adaptor element of claim 7, wherein:

the sticky end comprises a single base overhang.

9. The adaptor element of claim 8, wherein:

the single base overhang comprises a T nucleotide species.

10. The adaptor element of claim 7, wherein:

the sticky end comprises an overhang comprising a plurality a bases.

11. The adaptor element of claim 1, wherein:

the complementary region comprises a multiplex identifier element.

12. The adaptor element of claim 11, wherein:

the multiplex identifier element comprises 11 sequence postions.

13. The adaptor element of claim 12, wherein:

the multiplex identifier element is selected from the group consisting of SEQ ID NO 1-SEQ ID NO 133.

14. The adaptor element of claim 11, wherein:

the multiplex identifier element comprises a design that enables detection of up to two sequencing errors and correction of one of the sequencing errors.

15. The adaptor element of claim 1, wherein:

the inosine species are positionally located in a single strand.

16. The adaptor element of claim 15, wherein:

the inosine species are positionally located at least four sequence positions from the end of the strand.

17. The adaptor element of claim 15, wherein:

at least two of the inosine species are positionally located no closer than four sequence positions from each other.

18. The adaptor element of claim 1, wherein:

the complementary region comprises one or more phosphorothioate species.

19. The adaptor element of claim 18, wherein:

the non-complementary region comprises one or more phosphorothioate species.

20. The adaptor element of claim 19, wherein:

the phosphorothioate species are positionally located in an end region of the complementary and non-complementary regions.

21. The adaptor element of claim 18, wherein:

the phosphorothioate species protect the end regions from exonuclease digestion.

22. A kit comprising:

the semi-complementary double stranded nucleic acid adaptor of claim 1.

23. A method for efficient target processing, comprising:

ligating a species of a double stranded nucleic acid adaptor to each end of a linear double stranded nucleic acid molecule to produce an adapted double stranded nucleic acid molecule, wherein the species of the double stranded nucleic acid adaptor comprises a complementary region amenable for ligation to the linear double stranded nucleic acid molecule and a non-complementary region that inhibits ligation;

dissociating the adapted double stranded nucleic acid molecule to produce a first strand and a second strand each comprising a first amplification primer site and a sequencing primer site at a first end and a second amplification site at a second end; and

individually amplifying the first and second strands to produce a first clonal population comprising copies of the first strand and a second clonal population comprising copies of the second strand.

24. The method of claim 23, further comprising:

sequencing the first clonal population to produce a sequence composition of the first strand.

25. The method of claim 24, further comprising:

associating the sequence composition with a sample of origin, wherein the sequence composition comprises a sequence from a multiplex identifier element included in the double stranded nucleic acid adaptor.

26. The method of claim 25, wherein:

the multiplex identifier element comprises 11 sequence postions.

27. The method of claim 26, wherein:

the multiplex identifier element is selected from the group consisting of SEQ ID NO 1-SEQ ID NO 133.

28. The method of claim 25, wherein:

the step of associating comprises detection of up to two errors in the sequence from the multiplex identifier element and correction of up to one of the sequencing errors.

29. The method of claim 23, further comprising:

prior to the step of dissociating, determining a quantity of the adapted double stranded nucleic acid, wherein the double stranded nucleic acid adaptor comprises a fluorescent moiety.

30. The method of claim 29, further comprising:

the fluorescent moiety emits light in response to an excitation light and is measured by a detector, wherein a level of the measured emitted light is associated with the quantity.

31. The method of claim 29, further comprising:

the fluorescent moiety is selected from the group consisting of Cy3, Cy5, carboxyfluorescein (FAM), Alexafluor, Rhodamine green, Texas Red, R-Phycoerytherin, semiconductor nanocrytals.

32. The method of claim 23

the complementary region comprises one or more inosine species.

33. The method of claim 32, wherein:

the inosine species are positionally located in a single strand.

34. The element of claim 33, wherein:

the inosine species are positionally located at least six sequence positions from the end of the strand.

35. The element of claim 33, wherein:

at least two of the inosine species are positionally located no closer than four sequence positions from each other.

36. The element of claim 33, wherein:

the inosine species inhibit the formation of hairpin structures of the first strand and the second strand.

37. The element of claim 33, wherein:

the inosine species improve amplification efficiency of the first strand and the second strand.

38. A method for multiplex target processing and enrichment, comprising:

ligating a species of a double stranded nucleic acid adaptor to each end of a plurality of linear double stranded nucleic acid molecules from a plurality of samples to produce a pool of adapted double stranded nucleic acid molecules, wherein the species of the double stranded nucleic acid adaptor comprises a sample specific identifier element;

dissociating a plurality of members from the pool adapted double stranded nucleic acid molecules to produce a first strand and a second strand from each of the dissociated members to produce a population of single stranded molecules;

hybridizing a plurality of members of the population of single stranded molecules to a substrate bound capture probe, wherein the population of single stranded molecules comprises at least one member that does not hybridize to a substrate bound capture probe;

eluting the hybridized members from the substrate bound capture probe to produce an enriched population of single stranded molecules;

amplifying a plurality of members of the enriched population of single stranded molecules to produce a clonal population from each amplified member;

individually sequencing the clonal populations to produce sequence data for each amplified member that comprises a sequence composition for the multiplex identifier element;

associating the sequence data with one of the samples using the sample specific identifier.