Whole Genome Mapping by DNA Sequencing With Linked-Paired-End Library

• US 20150344873A1
• Filed: 06/02/2015
• Published: 12/03/2015
• Est. Priority Date: 06/02/2014
• Status: Active Grant

• Current Assignee: Drexel University
• Original Assignee: Drexel University
• Inventors: Xiao, Ming, Sibert, Justin
• Granted Patent: US 9,758,780 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/10 Ploidy or copy number detection
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection

Abstract

The present invention relates to innovative means of generating sequence-linked DNA fragments and subsequent uses of such linked DNA fragments for de novo haplotype-resolved whole genome mapping and massively parallel sequencing. In various embodiments described herein, the methods of the invention relate to methods of generating paired-end nucleic acid fragment sharing common linker nucleic acid sequences using a nicking endonuclease, a T7 endonuclease, a restriction enzyme, or a transposase, methods of analyzing the nucleotides sequences from the linked-paired-end sequenced fragments, and methods of de novo whole genome mapping. Thus, the methods of this invention allow establishing sequence contiguity across the whole genome, and achieving high-quality, low-cost de novo assembly of complex genomes.

Claims

1. A method of generating a linked-paired-end nucleic acid fragment from a DNA sample, the method comprising:

a. contacting a double-stranded DNA template having a first and a second DNA strand with a first nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand;

b. conducting a base extension reaction on the first DNA strand along a corresponding region of the second DNA strand thereby forming a single-stranded flap on the double-stranded DNA template adjacent to the sequence-specific nicking location;

c. contacting the double-stranded DNA template of b) with a second nicking endonuclease to form a cut at a sequence-specific location thereby generating two cut DNA fragments wherein the single stranded flap of b) can hybridize on the complementary strand of one of the two fragments of the cut DNA; and

,

d. conducting a base extension reaction on the second DNA strand of the other fragment of the cut DNA of c) along a corresponding region of the first DNA strand, wherein the reaction starts at the nick and progressing toward the 3′

end of the first DNA strand thereby generating a double-stranded DNA fragment that shares common linker sequences with the double-stranded DNA fragment of c).

2. The method of claim 1, wherein the generated DNA fragments of d) are further processed further using hexamer extension.

3. The method of claim 1, wherein the first and second nicking endonucleases are type II endonucleases.

4. The method of claim 1, wherein the first and second nicking endonucleases comprise one or more endonucleases selected from the group consisting of Nb.BbvCI, Nb.BsmI, NbBsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, and Nt.CviPII.

5. The method of claim 4, wherein the first nicking enzyme and the second nicking enzyme comprise at least one enzyme combination from the group consisting of Nt.BbvcI/Nb.BbvcI respectively and Nb.BbvcI/Nt.BbvcI respectively.

6. The method of claim 1, wherein the base extension reaction of b) comprises contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase.

7. The method of claim 1, wherein the generated fragments are 50 kb or less and the adjacent linker fragments share at least one selected from the group consisting of 100 by or more and 50 by or more.

8. The method of claim 1, wherein the linked-paired-end fragments are sequenced with at least one high throughput next generation sequencing platform selected from the group consisting of Illumina sequencing, SOLiD sequencing, 454 pyrosequencing and Ion Torrent semiconductor sequencing.

9. The method of claim 8, wherein the library preparation for sequencing comprises:

a. circularizing the linked-paired-end fragments;

b. fragmenting and size selecting the fragments of interest; and

,

c. ligating adaptors at a location selected from the group consisting of both ends of the fragments for paired-end sequencing and one end of the fragments for singled-end sequencing.

10. A method of generating a linked-paired-end nucleic acid fragment from a DNA sample, the method comprising:

a. contacting a double-stranded DNA template having a first and a second DNA strand with a first nicking endonuclease to form a nick at a sequence-specific nicking location on the first DNA strand;

b. conducting a base extension reaction on the first DNA strand along a corresponding region of the second DNA strand thereby forming a single-stranded flap on the double-stranded DNA template adjacent to the sequence-specific nicking location;

c. contacting the double-stranded DNA template of b) with a second nicking endonuclease being a T7 endonuclease to generate a cut of the second DNA strand across the nicking sites thereby generating two cut DNA fragments;

a first fragment with a blunt end and a second fragment with an overhang corresponding to the single stranded flap of b); and

,

d. conducting a base extension reaction on the second fragment with the flap overhang from c) wherein the based extension being along the corresponding region of the single stranded flap, and the reaction starting at the nick site of the T7 endonuclease and progressing toward the end of the stranded flap thereby generating a blunt end double-stranded DNA fragment that shares common linker sequences with the first double-stranded DNA fragment of c).

11. The method of claim 10, wherein the first nicking endonuclease is a type II endonuclease.

12. The method of claim 10, wherein the first nicking endonuclease comprises one or more endonucleases selected from the group consisting of Nb.BbvCI, Nt.BbvCI, Nt.BsmI, Nt.BsmAI, Nt.BstNBI, Nb.BsrDI, Nb.BstI, Nt.BspQI, Nt.Bpu10I and Nt.Bpu10I.

13. The method of claims 10, wherein the base extension reaction comprises contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase.

14. The method of claim 10, wherein the generated fragments are 50 kb or less and the adjacent linker fragments share at least one selected from the group consisting of 100 by or more and 50 by or more.

15. The method of claim 10, wherein the linked-paired-end fragments are sequenced with at least one high throughput next generation sequencing platform selected from the group consisting of Illumina sequencing, SOLiD sequencing, 454 pyrosequencing and Ion Torrent semiconductor sequencing.

16. The method of claim 15, wherein the library preparation for sequencing comprises:

a. circularizing the linked-paired-end fragments;

b. fragmenting and size selecting the fragments of interest; and

,

c. ligating adaptors at a location selected from the group consisting of both ends of the fragments for paired-end sequencing and one end of the fragments for singled-end sequencing.

17. A method of generating a linked-paired-end nucleic acid fragment from a DNA sample, the method comprising:

a. contacting a double-stranded DNA template having a first and a second DNA strand with a restriction endonuclease to form overhang double stranded cuts of the DNA at a sequence-specific location; and

,

b. conducting a base extension reaction on the fragments of the overhang DNA thereby generating blunt end double-stranded DNA fragments that shares common linker sequences between each other.

18. The method of claim 17, wherein the nicking endonuclease is type II endonuclease.

19. The type II endonuclease of claim 18, wherein the endonuclease is from subtype IIb.

20. The method of claim 19, wherein the endonuclease comprises one or more endonucleases selected from the group consisting of BcgI, Bsp24I, BaeI, CjeI, and CjePI.

21. The method of claim 20, wherein the endonuclease enzyme is BaeI.

22. The method of claims 17, wherein the base extension reaction comprises contacting the first DNA strand with a polymerase, one or more nucleotides, and a ligase.

23. The method of claim 17, wherein the generated fragments are 50 kb or less and the adjacent linker fragments share at least one selected from the group consisting of 100 by or more and 50 by or more.

24. The method of claim 17, wherein the linked-paired-end fragments are sequenced with at least one high throughput next generation sequencing platform selected from the group consisting of Illumina sequencing, SOLiD sequencing, 454 pyrosequencing and Ion Torrent semiconductor sequencing.

25. The method of claim 24, wherein the library preparation for sequencing comprises:

a. circularizing the linked-paired-end fragments;

b. fragmenting and size selecting the fragments of interest; and

,

c. ligating adaptors at a location selected from the group consisting of both ends of the fragments for paired-end sequencing and one end of the fragments for singled-end sequencing.

26. A method of whole genome mapping, the method comprising

a. constructing a linked-paired-end sequencing library wherein the linked DNA fragments are used;

and,

b. mapping the sequence reads of the sequenced libraries to the reference genome map.

27. The method of claim 26, wherein the genome mapping comprises a haplotype sequence assembly.

28. The method of claim 27, wherein the haplotype comprises the human major histocompatibility (MHC) region.

29. A method of analyzing computationally the nucleotides sequences from linked-paired-end sequenced fragments, the method comprising performing a pairwise comparison of all overlap nucleotides sequence read, and correlating the nucleotides sequence read to a sequence assembly, a genetic or cytogenetic map, a structural pattern, a structural variation, physiological characteristic, a methylation pattern, an epigenomic pattern, a location of a CpG island, a single nucleotide polymorphism (SNP), a copy number variation (CNV), or a combination thereof.