HIGH THROUGHPUT SEQUENCE-BASED DETECTION OF SNPS USING LIGATION ASSAYS

• US 20090215633A1
• Filed: 03/01/2007
• Published: 08/27/2009
• Est. Priority Date: 03/01/2006
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Hogers, Rene Cornelis Josephus
• Granted Patent: US 8,460,866 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

Method for the detection the presence or absence of one or more target sequences in one or more samples based on oligonucleotide ligation assays with a variety of ligatable probes containing identifiers and the subsequent identification of the identifiers in the amplicons or ligated probes using high throughput sequencing technologies.

Claims

1. A method for the high throughput detection of one or more target nucleotide sequences in one or more samples comprising the steps of:

(a) providing for each target nucleotide sequence, a first probe and a second probe, wherein the first probe comprises a target specific section at its 3′

end and a first tag section that is non-complementary to the target nucleotide sequence and that comprises a first primer binding sequence, the second probe comprises a second target specific section at its 5′

end and a second tag section that is non-complementary to the target nucleotide sequence and that comprises a second primer binding sequence, wherein the first or second tag section, or both, contain(s) an identifier sequence that is located between the respective first or second primer binding sequence and the respective first or second target specific section,

(b) allowing the first and second target specific section of the respective first and second probe to hybridise to the target sequence,

(c) ligating the first and second probe when the respective target specific sections of the probes are hybridised to essentially adjacent sections on the target sequence to provide ligated probes,

(d) optionally, amplifying the ligated probes with a first and a second primer to provide amplicons,

(e) subjecting the ligated probes or amplicons to high throughput sequencing technology to determine at least part of the nucleotide sequence, at least including the identifier sequence(s), of the ligated probes or the amplicons,

(f) identifying the presence of the target nucleotide sequence in the sample by determination of the presence or absence of the identifier sequence in the nucleotide sequence of step (e).

2. The method according to claim 1, wherein the first and second probe are replaced by a circularizable probe.

3. The method according to claim 1, wherein the first and second probe are Keylock probes, each comprising a clamp section.

4. The method according to claim 1, wherein the first probe is replaced by a first probe that contains a first target specific section and does not contain a first primer binding sequence, and wherein a compound probe is provided that contains a section that is capable of hybridizing to (part of) the first target specific section of the first probe and a primer binding section, and wherein elongation of the compound probe provides an amplifiable elongated compound probe.

5. The method according to claim 1, wherein the first probe contains a first target specific section, does not contain a primer binding sequence and contains an exonuclease resistant section distal from the point of ligation, wherein the second probe comprises a second target specific section and a second tag section that comprises a first and a second primer binding sequence and a intermittently located identifier.

6. The method according to claim 1, wherein the first and second probes anneal to non-adjacent sections on the target sequence and a third probe is provided that anneals to the target sequence adjacent to, and between, the first and the second target specific section.

7. The method according to claim 1, wherein the first and second probes anneal to non-adjacent sections on the target sequence and a polymerase and nucleotides are provided such that the gap between the first and second probe is filled in.

8. The method according to claim 1, wherein the sequencing is carried out by means of high-throughput sequencing.

9. The method according to claim 8, wherein the high-throughput sequencing is performed on a solid support.

10. The method according to claim 8, wherein the high-throughput sequencing is based on Sequencing-by-Synthesis.

11. The method according to claim 8, wherein the high-throughput sequencing comprises the steps of:

annealing the ligated probes or amplicons to beads, each bead annealing with a single ligated probes or amplicons;

emulsifying the beads in micro reactors, each water-in-oil micro reactor comprising a single bead;

performing emulsion PCR to amplify ligated probes or amplicons on the surface of beads;

optionally, selecting/enriching beads containing amplified sequencing-adaptor-ligated fragments;

loading the beads in wells, each well comprising a single bead; and

generating a pyrophosphate signal.

12. The method according to claim 8, wherein the high-throughput sequencing comprises the steps of:

annealing the ligated probes or amplicons to beads, each bead annealing with a single ligated probes or amplicons;

emulsifying the beads in micro reactors, each water-in-oil micro reactor comprising a single bead;

performing emulsion PCR to amplify ligated probes or amplicons on the surface of beads;

optionally, selecting/enriching beads containing amplified sequencing-adaptor-ligated fragments;

loading the beads in wells, each well comprising a single bead; and

determine the nucleotide sequence of the amplified ligated probes or amplicons using labelled reversible terminator nucleotides.

13. The method according to claim 8, wherein the high-throughput sequencing comprises the steps of:

annealing the ligated probes or amplicons to a surface containing first and second primers or first and second primer binding sequences respectively,

performing bridge amplification to provide clusters of amplified ligated probes or amplicons,

determine the nucleotide sequence of the amplified ligated probes or amplicons using labelled reversible terminator nucleotides.

14. The method according to claim 8, wherein the high-throughput sequencing comprises the steps of:

annealing the ligated probes or amplicons to a surface containing first and second primers or first and second primer binding sequences respectively,

performing bridge amplification to provide clusters of amplified ligated probes or amplicons,

generating a pyrophosphate signal.

15. The method according to claim 1, wherein the identifier is a sample identifier.

16. The method according to claim 1, wherein the identifier is a combined locus/allele identifier.

17. The method according to claim 1, wherein the identifier comprises a sample identifier and a locus/allele identifier.

18. The method according to claim 1, wherein the identifier is from 1-30 bp, preferably from 2-16, more preferably from 3-8, most preferably from 4-6 bp.

19. The method according to claim 1, wherein the primer comprises an 3′

anchor sequence.

20. The method according to claim 1, wherein the identifier does not contain 2 or more identical consecutive bases.

21. The method according to claim 1, wherein for two or more samples or for two or more locus/allele combinations, the corresponding identifiers contain at least two different nucleotides.

22. The method according to claim 1 wherein for two or more samples or for two or more locus/allele combinations, the identifier sequences are used to genotype the sample(s) for one or more sequences and/or polymorphisms, such as SNPs and/or indels.

23. (canceled)

24. A kit comprising a first probe and a second probe, wherein the first probe comprises a target specific section at its 3′

end and a first tag section that is non-complementary to the target nucleotide sequence and that comprises a first primer binding sequence, the second probe comprises a second target specific section at its 5′

end and a second tag section that is non-complementary to the target nucleotide sequence and that comprises a second primer binding sequence, wherein the first or second tag section, or both, contain(s) an identifier sequence that is located between the respective first or second primer binding sequence and the respective first or second target specific section.

25. A kit according to claim 24, wherein the first and second probe are replaced by a circularizable probe.

26. A kit according to claim 24, wherein the first and second probe are Keylock probes, each comprising a clamp section.

27. A kit according to claim 24, wherein the first probe is replaced by a first probe that contains a first target specific section and does not contain a first primer binding sequence, and wherein a compound probe is provided that contains a section that is capable of hybridizing to (part of) the first target specific section of the first probe and a primer binding section, and wherein elongation of the compound probe provides an amplifiable elongated compound probe.

28. A kit according to claim 24, wherein the first probe contains a first target specific section, does not contain a primer binding sequence and contains an exonuclease resistant section distal from the point of ligation, wherein the second probe comprises a second target specific section and a second tag section that comprises a first and a second primer binding sequence and a intermittently located identifier.

29. A kit according to claim 24, wherein the first and second probes anneal to non-adjacent sections on the target sequence and a third probe is provided that anneals to the target sequence adjacent to, and between, the first and the second target specific section.

30. A kit according to claim 24, wherein the first and second probes anneal to non-adjacent sections on the target sequence and a polymerase and nucleotides are provided such that the gap between the first and second probe is filled in.