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

• US 10,036,012 B2
• Filed: 03/28/2017
• Issued: 07/31/2018
• Est. Priority Date: 01/26/2012
• Status: Active Grant

• Current Assignee: Tecan Genomics
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Amorese, Doug, Armour, Chris, Kurn, Nurith
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US15/471,785
• Publication Number:
  US 20170298345A1
• Time in Patent Office: 490 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 sequencing an enriched nucleic acid sequence of interest, the method comprising:

a) obtaining a nucleic acid fragment ligated to a partial duplex adaptor, wherein the nucleic acid fragment comprises a nucleic acid sequence of interest, wherein the partial duplex adaptor comprises a first adaptor sequence, and wherein the partial duplex adaptor comprises a first strand and a second strand, wherein the first strand is longer than the second strand;

b) annealing one or more oligonucleotides in solution to the nucleic acid sequence of interest in the nucleic acid fragment ligated to the partial duplex adaptor, wherein the one or more oligonucleotides comprise a 3′

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

tail portion comprising a second adaptor sequence that is non-complementary to the nucleic acid sequence of interest;

c) extending the one or more oligonucleotides annealed to the nucleic acid sequence of interest in the nucleic acid fragment ligated to the partial duplex adaptor with a polymerase, 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 second adaptor sequence at a second end;

d) amplifying the one or more oligonucleotide extension products using a first primer that anneals to a complement of the first adaptor sequence and a second primer that anneals at its 3′

end 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;

e) 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

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

2. The method of claim 1, wherein the nucleic acid fragment comprises genomic DNA.

3. The method of claim 1, wherein the nucleic acid fragment comprises cDNA.

4. The method of claim 1, wherein the nucleic acid fragment comprises RNA.

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

6. The method of claim 1, wherein the one or more oligonucleotides comprise more than one oligonucleotide, and wherein the second adaptor sequence is common to the more than one oligonucleotides.

7. The method of claim 1, wherein the first adaptor sequence, the second adaptor sequence, or both comprise barcode sequence.

8. The method of claim 7, wherein the barcode sequence is at least 8 nucleotides in length.

9. The method of claim 7, wherein the barcode sequence is 12 nucleotides in length.

10. The method of claim 1, wherein the obtaining comprises obtaining a plurality of nucleic acid fragments ligated to a partial duplex adaptor, wherein each partial duplex adaptor of each nucleic acid fragment of the plurality of nucleic acid fragments comprises a barcode, and wherein each barcode is unique.

11. The method of claim 1, wherein the nucleic acid fragment ligated to the partial duplex adaptor is double-stranded prior to step b).

12. The method of claim 11, further comprising denaturing the nucleic acid fragment ligated to the partial duplex adaptor prior to step b), thereby generating a single-stranded nucleic acid fragment comprising the nucleic acid sequence of interest ligated to the first adaptor sequence.

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

14. The method of claim 1, wherein the first strand of the partial duplex adaptor is ligated to a 5′

end of the nucleic acid fragment.

15. The method of claim 1, wherein the second strand of the partial duplex adaptor is ligated to a 3′

end of the nucleic acid fragment.

16. The method of claim 1, further comprising fragmenting a nucleic acid, thereby generating a nucleic acid fragment;

and purifying the nucleic acid fragment prior to obtaining the nucleic acid fragment ligated to the partial duplex adaptor in step a).

17. The method of claim 1, further comprising performing bridge amplification of the annealed strand after step e).

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 fragmenting a nucleic acid, thereby generating a nucleic acid fragment;

and performing end repair on the nucleic acid fragment prior to obtaining the nucleic acid fragment ligated to the partial duplex adaptor in step a).

21. The method of claim 1, wherein the nucleic acid fragment of the nucleic acid fragment ligated to the partial duplex adaptor in step a) is obtained by performing reverse transcription on an RNA fragment prior to obtaining the nucleic acid fragment ligated to the partial duplex adaptor in step a).

22. The method of claim 1, wherein each end of the nucleic acid fragment is ligated to a partial duplex adaptor.