Next-generation sequencing libraries

• US 10,036,013 B2
• Filed: 08/19/2014
• Issued: 07/31/2018
• Est. Priority Date: 08/19/2013
• Status: Active Grant

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: Abbott Molecular Incorporated (Abbott Laboratories Incorporated)
• Inventors: Kim, Dae Hyun
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/463,498
• Publication Number:
  US 20150051116A1
• Time in Patent Office: 1,442 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• 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 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Provided herein is technology relating to next-generation sequencing and particularly, but not exclusively, to methods and compositions for preparing a next-generation sequencing library comprising short overlapping DNA fragments and using the library to sequence one or more target nucleic acids.

Claims

1. A method of generating a sequencing library, the method comprising:

a) amplifying a target nucleotide sequence using a primer comprising a target specific sequence and a universal sequence A to provide an amplicon, wherein the amplicon may be single stranded or double stranded;

b) ligating a first adaptor oligonucleotide comprising a universal sequence B to the amplicon to form an adaptor-amplicon; and

c) generating a ladder fragment library for use as a sequencing library, wherein said ladder fragment library comprises a plurality of nucleic acid fragments terminated by a 3′

-O-propargyl nucleotide.

2. The method of claim 1 wherein the primer further comprises a barcode nucleotide sequence associated with the target nucleic acid.

3. The method of claim 2 wherein the barcode nucleotide sequence comprises 1 to 20 nucleotides.

4. The method of claim 1 wherein the first adaptor oligonucleotide is ligated to the 3′

end of the amplicon.

5. The method of claim 1 further comprising circularizing the adaptor-amplicon to form a circular template.

6. The method of claim 1 further comprising ligating a second adaptor oligonucleotide comprising a universal sequence C to the 3′

ends of the fragments of the ladder fragment library to generate a sequencing library.

7. The method of claim 6 comprising use of a click chemistry reaction to ligate the second adaptor oligonucleotide comprising a universal sequence C to the 3′

ends of the fragments of the ladder fragment library to generate the sequencing library.

8. The method of claim 1 wherein the first adaptor oligonucleotide comprises 10 to 80 nucleotides.

9. The method of claim 1, wherein the nucleotide sequences of the fragments of the ladder fragment library correspond to overlapping nucleotide subsequences within the target nucleotide sequence and the nucleotide sequences of the fragments have 3′

ends corresponding to different nucleotides of the target nucleotide sequence.

10. The method of claim 1 wherein each nucleotide sequence of each fragment of the ladder fragment library comprises 15 to 1,000 nucleotides.

11. The method of claim 1 wherein generating said ladder fragment library comprises using a primer complementary to said universal sequence A.