Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library regeneration

• US 9,650,628 B2
• Filed: 01/25/2013
• Issued: 05/16/2017
• Est. Priority Date: 01/26/2012
• Status: Active Grant

• Current Assignee: Tecan Genomics
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith, Amorese, Doug, Armour, Chris
• Primary Examiner(s): Bertagna, Angela M
• Application Number:
  US13/750,768
• Publication Number:
  US 20130231253A1
• Time in Patent Office: 1,572 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• 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
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present invention provides methods, compositions and kits for targeted nucleic acid sequence enrichment in a nucleic acid sample and for high efficiency nucleic acid library generation for next generation sequencing (NGS). Specifically, the methods, compositions and kits provided herein are useful for the production and capture of amplification-ready, target-specific and strand-specific regions of interest from nucleic acid samples containing complex DNA.

Claims

1. A method for enriching for a nucleic acid sequence of interest in a sample comprising nucleic acids, the method comprising:

(a) fragmenting nucleic acids in the sample comprising the nucleic acids, thereby generating nucleic acid fragments, wherein the nucleic acid fragments comprise the nucleic acid sequence of interest;

(b) ligating a first adaptor sequence to a 5′

end of the nucleic acid fragments, thereby generating nucleic acid fragments ligated to the first adaptor sequence;

(c) purifying the nucleic acid fragments ligated to the first adaptor sequence;

(d) annealing one or more oligonucleotides in solution to the nucleic acid sequence of interest in the purified nucleic acid fragments ligated to the first adaptor sequence, wherein the one or more oligonucleotides in solution comprise a 3′

portion with at least 10 bases that are complementary to the nucleic acid sequence of interest and a 5′

tail, wherein the 5′

tail comprises a second adaptor sequence non-complementary to the sequence of interest;

(e) extending the one or more oligonucleotides annealed to the nucleic acid sequence of interest in the nucleic acid fragments ligated to the first adaptor sequence with a polymerase in a reaction mixture, wherein the reaction mixture does not comprise a primer that anneals to sequence complementary to the first adaptor sequence, thereby generating one or more oligonucleotide extension products comprising sequence complementary to the ligated first adaptor sequence at a first end, sequence complementary to the nucleic acid sequence of interest, and the second adaptor sequence at a second end;

(f) amplifying the one or more oligonucleotide extension products using a first primer that anneals to the complement of the first adaptor sequence and a second primer that anneals to a complement of the second adaptor sequence to enrich for the nucleic acid sequence of interest, wherein products of the amplifying comprise a 3′

end with sequence complementary to a sequence on a surface, thereby providing an enriched nucleic acid sequence of interest;

(g) annealing a strand of the products of the amplifying to the sequence on the surface using the 3′

end with sequence complementary to the sequence on the surface; and

(h) sequencing the enriched nucleic acid sequence of interest on a massively parallel sequencing platform.

2. The method of claim 1, wherein the nucleic acid sequence of interest comprises genomic DNA, RNA, or cDNA.

3. The method of claim 1, wherein the first adaptor sequence and the second adaptor sequence are distinct from each other.

4. The method of claim 1, wherein the first adaptor sequence and/or the second adaptor sequence comprise barcode sequence.

5. The method of claim 1, wherein the polymerase is a DNA polymerase.

6. The method of claim 5, wherein the DNA polymerase is a DNA polymerase without 3′

-exonuclease activity.

7. The method of claim 1, wherein the one or more oligonucleotides comprise more than one oligonucleotide, and wherein each of the more than one oligonucleotides comprises a 3′

portion that is complementary to a different nucleic acid sequence of interest in the nucleic acid fragments and a 5′

portion comprising the second adaptor sequence.

8. The method of claim 1, wherein the nucleic acid fragments are double-stranded.

9. The method of claim 8, further comprising denaturing the purified nucleic acid fragments ligated to the first adaptor sequence prior to step (d), thereby generating single-stranded nucleic acid fragments ligated to the first adaptor sequence.

10. The method of claim 1, wherein the first adaptor sequence comprises a restriction and/or cleavage site for a nucleic acid modifying enzyme.

11. The method of claim 1, wherein the 3′

portion of the one or more oligonucleotides comprises a random sequence.

12. The method of claim 1, wherein the first adaptor sequence is in a first strand of a partial duplex adaptor.

13. The method of claim 12, wherein the first strand is a long strand of the partial duplex adaptor.

14. The method of claim 12, wherein the partial duplex adaptor comprises a second strand, wherein the second strand is a short strand.

15. The method of claim 14, wherein the short strand is ligated to a 3′

end of the nucleic acid fragments.

16. The method of claim 1, wherein the first adaptor sequence is in a ligation strand of a first adaptor, wherein the first adaptor comprises the ligation strand and a non-ligation strand, wherein the ligation strand ligates to a 5′

end of the nucleic acid fragments.

17. The method of claim 1, wherein the sequencing comprises bridge amplification.

18. The method of claim 1, wherein the sequencing comprises use of four labeled reversible terminators.

19. The method of claim 1, wherein the sequencing comprises semiconductor sequencing.

20. The method of claim 1, further comprising purifying the nucleic acid fragments prior to step (b).

21. The method of claim 1, further comprising purifying the products of the amplifying prior to step (g).

22. The method of claim 1, wherein the 3′

portion of the one or more oligonucleotides that hybridizes to the nucleic acid sequence of interest is synthesized precisely according to antisense sequence of the nucleic acid sequence of interest.

23. A method for enriching for a nucleic acid sequence of interest from a library, wherein the library comprises nucleic acid inserts comprising the nucleic acid sequence of interest, wherein the nucleic acid inserts have a first adaptor sequence attached on a first end and a second adaptor sequence attached on a second end, the method comprising:

(a) denaturing the nucleic acid inserts with the first adaptor sequence on the first end and the second adaptor sequence on the second end, thereby generating a library of single-stranded nucleic acid inserts with the first adaptor sequence on a first end and the second adaptor sequence on a second end;

(b) annealing one or more oligonucleotides in solution to the sequence of interest in the single-stranded nucleic acid inserts with the first adaptor sequence on the first end and the second adaptor sequence on the second end, wherein the one or more oligonucleotides in solution comprise a 3′

portion with at least 10 bases designed to be complementary to the nucleic acid sequence of interest present in the nucleic acid inserts and a 5′

tail, wherein the 5′

tail comprises a third adaptor sequence, wherein the third adaptor sequence is distinct from the first adaptor sequence and the second adaptor sequence, and the 5′

tail is non-complementary to the sequence of interest;

(c) extending the one or more oligonucleotides with a polymerase in a reaction mixture, wherein the reaction mixture does not comprise a primer that anneals to sequence complementary to the first adaptor sequence or the second adaptor sequence, thereby generating one or more oligonucleotide extension products comprising sequence complementary to the first adaptor sequence at a first end, sequence complementary to the nucleic acid sequence of interest, and the third adaptor sequence at a second end;

(d) amplifying the one or more oligonucleotide extension products using a first primer that anneals to the complement of the first adaptor sequence and a second primer that anneals to a complement of the third adaptor sequence to enrich for the nucleic acid sequence of interest, wherein products of the amplifying comprise a 3′

end with sequence complementary to a sequence on a surface, thereby providing an enriched nucleic acid sequence of interest;

(e) purifying the products of the amplifying;

(f) annealing a strand of the purified products of the amplifying to the sequence on the surface using the 3′

end with sequence complementary to the sequence on the surface; and

(g) sequencing the enriched nucleic acid sequence of interest on a massively parallel sequencing platform.

24. The method of claim 23, wherein the nucleic acid sequence of interest comprises genomic DNA, RNA or cDNA.

25. The method of claim 23, wherein the third adaptor sequence is the same among the one or more oligonucleotides.

26. The method of claim 23, wherein the first adaptor sequence and the second adaptor sequence are distinct from each other.

27. The method of claim 23, wherein the first adaptor sequence and the complement of the second adaptor sequence are the same.

28. The method of claim 23, wherein the first adaptor sequence, the second adaptor sequence and/or the third adaptor sequence comprise barcode sequence.

29. The method of claim 23, wherein the 3′

portion of each of the one or more oligonucleotides comprises a random sequence.

30. The method of claim 23, wherein the sequencing comprises bridge amplification.

31. The method of claim 23, wherein the sequencing comprises use of four labeled reversible terminators.

32. The method of claim 23, wherein the sequencing comprises semiconductor sequencing.

33. The method of claim 23, wherein neither the first adaptor sequence nor the second adaptor sequence comprises a 3′

block.

34. The method of claim 23, further comprising ligating the first adaptor sequence to the first end and the second adaptor sequence to the second end of the nucleic acid inserts prior to step (a), thereby generating the library of nucleic acid inserts with the first adaptor sequence on the first end and the second adaptor sequence on the second end.

35. The method of claim 34, further comprising purifying the nucleic acid inserts with the first adaptor sequence on the first end and the second adaptor sequence on the second end after the ligating.

36. The method of claim 23, wherein the 3′

portion of the one or more oligonucleotides that hybridizes to the nucleic acid sequence of interest is synthesized precisely according to antisense sequence of the nucleic acid sequence of interest.

37. The method of claim 23, wherein the polymerase is a DNA polymerase without 3′

-exonuclease activity.