Genotyping by next-generation sequencing

• US 10,704,091 B2
• Filed: 03/12/2018
• Issued: 07/07/2020
• Est. Priority Date: 01/13/2012
• Status: Active Grant

• Current Assignee: Data2Bio
• Original Assignee: Data2Bio
• Inventors: Schnable, Patrick S., Liu, Sanzhen, Wu, Wei
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/917,977
• Publication Number:
  US 20180201996A1
• Time in Patent Office: 848 Days
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• 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 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• 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

Abstract

Provided herein is technology relating to genotyping and particularly, but not exclusively, to methods for genotyping one or more organisms by genome sequencing.

Claims

1. A composition comprising:

a) a nucleic acid template comprising single-stranded ends, said nucleic acid template comprising;

i) a double-stranded restriction fragment;

ii) a first single-stranded oligonucleotide ligated to a 3′

end of a first strand of the double-stranded restriction fragment, said first single-stranded oligonucleotide comprising a sequence comprising a first primer binding site; and

ii) a second single-stranded oligonucleotide ligated to a 5′

end of said first strand of the double-stranded restriction fragment, said second single-stranded oligonucleotide comprising a sequence complementary to a second primer binding site;

b) a first primer capable of hybridizing to said first primer binding site; and

c) a second primer capable of hybridizing to said second primer binding site.

2. The composition of claim 1 wherein said first primer is hybridized to said first single-stranded oligonucleotide at said first primer binding site and said second primer is not hybridized to said nucleic acid template.

3. The composition of claim 1 further comprising a polymerase.

4. The composition of claim 1 further comprising an amplicon, wherein said amplicon comprises a sequence of said double-stranded restriction fragment.

5. The composition of claim 1 wherein said double-stranded restriction fragment comprises degenerate sticky ends.

6. The composition of claim 1 wherein said first primer binding site comprises at least 8 nucleotides and/or wherein said second primer binding site comprises at least 8 nucleotides.

7. The composition of claim 1 wherein said first primer is complementary to said first primer binding site and said second primer is complementary to said second primer binding site.

8. The composition of claim 1 wherein said double-stranded restriction fragment comprises a polymorphic locus.

9. The composition of claim 1 wherein said first single-stranded oligonucleotide and/or said second single-stranded oligonucleotide comprises a barcode.

10. The composition of claim 1 wherein said first primer comprises a phosphorothioate bond and/or said second primer comprises a phosphorothioate bond.

11. The composition of claim 1 further comprising an amplicon, wherein said amplicon comprises a sequence of said double-stranded restriction fragment and a barcode sequence.