GENOTYPING BY NEXT-GENERATION SEQUENCING

• US 20180201996A1
• Filed: 03/12/2018
• Published: 07/19/2018
• Est. Priority Date: 01/13/2012
• Status: Active Grant

• Current Assignee: Data2Bio
• Original Assignee: Data2Bio
• Inventors: Schnable, Patrick S., Liu, Sanzhen, Wu, Wei
• Granted Patent: US 10,704,091 B2
• Time in Patent Office: 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

1-14. -14. (canceled)

15. A method for genotyping-by-sequencing a sample, the method comprising:

a) ligating nucleic acid fragments to a first single-stranded oligonucleotide comprising a first primer binding site and a second single-stranded oligonucleotide comprising a second primer binding site, wherein said ligating produces templates comprising single-stranded ends;

b) amplifying said templates using a first amplification primer that hybridizes to the first primer binding site or complement thereof and a second amplification primer that hybridizes to the second primer binding site or complement thereof to produce amplicons; and

c) genotyping the sample by acquiring sequence reads from the amplicons.

16. The method of claim 15 wherein the first and/or second single-stranded oligonucleotide comprises a barcode.

17. The method of claim 15 further comprising mapping a sequence read to a reference sequence.

18. The method of claim 15 wherein acquiring sequence reads comprises providing amplicons for sequencing and receiving nucleotide sequence data.

19. The method of claim 15 further comprising sequencing amplicons.

20. The method of claim 16 further comprising using a barcode to assign a genotype to a sample.

21. The method of claim 15 further comprising identifying a polymorphism in a sequence read.

22. The method of claim 15 wherein the first amplification primer comprises a first sequence that is complementary to the first primer binding site and a second sequence that is adjacent to the first sequence and comprises a base that is complementary to a base of the nucleic acid fragment.

23. The method of claim 15 wherein said ligating comprises ligating a plurality of single-stranded oligonucleotides, each comprising a distinctive barcode, to a plurality of nucleic acid fragments to produce a plurality of barcoded templates and pooling said plurality of barcoded templates prior to said amplifying.

24. The method of claim 15 further comprising producing said nucleic acid fragments with a single restriction enzyme.

25. The method of claim 15 further comprising producing said nucleic acid fragments with a restriction enzyme having a degenerate recognition site.

26. A method for genotyping-by-sequencing a sample, the method comprising:

a) amplifying nucleic acid templates comprising single-stranded ends, said templates produced by ligating nucleic acid fragments to a first single-stranded oligonucleotide comprising a first primer binding site and a second single-stranded oligonucleotide comprising a second primer binding site; and

b) obtaining genotype information by sequencing amplicons produced by said amplifying.

27. The method of claim 26 wherein said amplifying comprises using a first amplification primer that hybridizes to the first primer binding site or complement thereof and a second amplification primer that hybridizes to the second primer binding site or complement thereof.

28. The method of claim 26 wherein the first and/or second single-stranded oligonucleotide comprises a barcode.

29. The method of claim 27 wherein the first amplification primer comprises a first sequence that is complementary to the first primer binding site and a second sequence adjacent to the first sequence and comprising a base that is complementary to a subset of nucleic acid fragments.

30. The method of claim 26 wherein obtaining genotype information comprises providing amplicons produced by said amplifying and receiving a nucleotide sequence.

31. The method of claim 26 wherein said genotype information comprises a nucleotide sequence comprising a polymorphism.

32. The method of claim 26 further comprising assigning a genotype to said sample.

33. A composition comprising:

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

i) a restriction fragment;

ii) a first single-stranded oligonucleotide ligated to the restriction fragment; and

ii) a second single-stranded oligonucleotide ligated to the restriction fragment;

b) a first primer capable of hybridizing to said first single-stranded oligonucleotide; and

c) a second primer capable of hybridizing to said second single-stranded oligonucleotide.

34. The composition of claim 33 wherein said first primer is hybridized to said first single-stranded oligonucleotide and said second primer is hybridized to said second single-stranded oligonucleotide.

35. The composition of claim 33 further comprising a polymerase.

36. The composition of claim 33 further comprising an amplicon, wherein said amplicon comprises a sequence of said restriction fragment.

37. The composition of claim 33 wherein said restriction fragment comprises degenerate sticky ends.

38. A method for genotyping-by-sequencing a sample, the method comprising:

a) ligating nucleic acid fragments to a first nucleic acid consisting essentially of a single-stranded oligonucleotide comprising a first primer binding site and a second nucleic acid consisting essentially of a single-stranded oligonucleotide comprising a second primer binding site, wherein said ligating produces templates comprising single-stranded ends;

b) amplifying said templates using a first amplification primer that hybridizes to the first primer binding site or complement thereof and a second amplification primer that hybridizes to the second primer binding site or complement thereof to produce amplicons; and

c) genotyping the sample by acquiring sequence reads from the amplicons.