High throughput screening of populations carrying naturally occurring mutations

• US 10,233,494 B2
• Filed: 04/24/2018
• Issued: 03/19/2019
• Est. Priority Date: 09/29/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Van Tunen, Adrianus Johannes
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US15/961,542
• Publication Number:
  US 20180245151A1
• Time in Patent Office: 329 Days
• Field of Search: 435 912
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• 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 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Efficient methods are disclosed for the high throughput identification of mutations in genes in members of mutagenized populations. The methods comprise DNA isolation, pooling, amplification, creation of libraries, high throughput sequencing of libraries, preferably by sequencing-by-synthesis technologies, identification of mutations and identification of the member of the population carrying the mutation and identification of the mutation.

Claims

1. A method for detecting a mutation in one or more members of a population, comprising:

(a) providing a plurality of libraries of nucleic acid fragments having reduced complexity, wherein each library of nucleic acid fragments comprise sequences derived from a member of the population,

(b) ligating sequencing adaptors to the plurality of libraries of nucleic acid fragments, and pooling the plurality of libraries of nucleic acid fragments, wherein each library of nucleic acid fragments in the pool is tagged with and identifiable by one or more tag sequences;

(c) performing high-throughput sequencing to sequence at least a portion of the pool of nucleic acid fragments, wherein the high-throughput sequencing is performed on a solid support and comprises annealing the nucleic acid fragments to the solid support and amplifying the annealed nucleic acid fragments; and

(d) clustering/aligning the sequences of the nucleic acid fragments to identify a mutation and a member of the population carrying the mutation using the tag sequences.

2. The method according to claim 1, wherein the population comprising sub-populations of one or more human individuals having naturally-occurring mutations.

3. The method according to claim 2, wherein the naturally-occurring mutations are selected from the group consisting of single nucleotide polymorphisms, nucleotide insertions and deletions, and variations in microsatellite repeat number.

4. The method according to claim 1, wherein the high-throughput sequencing is sequencing-by-synthesis.

5. The method according to claim 1, wherein the high-throughput sequencing is bi-directional sequencing.

6. The method according to claim 1, wherein the solid support comprises one or more beads.

7. The method according to claim 6, wherein step (c) comprises:

(i) annealing the adaptor-ligated nucleic acid fragments to beads, each bead annealing with a single adaptor-ligated nucleic acid fragments;

(ii) emulsifying the beads in water-in-oil microreactors such that each microreactor contains a single bead;

(iii) performing emulsion PCR to amplify adaptor-ligated nucleic acid fragments on the surface of the beads;

(iv) selecting/enriching beads to which are attached the amplified adaptor-ligated nucleic acid fragments;

(v) placing the beads in wells such that each well comprises a single bead; and

generating a pyrophosphate signal.

8. The method according to claim 1, wherein the nucleic acid fragments are sequenced with an average redundancy of at least 50.

9. The method according to claim 1, wherein step (d) comprises aligning the sequences of the nucleic acid fragments to a reference sequence or a consensus sequence.

10. The method according to claim 1, wherein the nucleic acid fragments are each tagged at both ends.

11. The method according to claim 1, wherein the nucleic acid fragments are each tagged with two different tag sequences.

12. The method according to claim 1, wherein the nucleic acid fragments ligated to sequencing adaptors each comprises a sequence derived from a member of the population flanked at both ends by tag sequences.

13. A method of detecting a mutation in one or more members of a population, comprising:

(a) providing a plurality of libraries of amplification products, wherein each library of amplification products is obtained by amplifying nucleic acids isolated from a member of the population,

(b) ligating sequencing adaptors to the plurality of libraries of amplification products, and pooling the plurality of libraries of amplification products, wherein each library of amplification products in the pool is tagged with and identifiable by one or more tag sequences;

(c) performing high-throughput sequencing to sequence at least a portion of the pool of amplification products, wherein the high-throughput sequencing is performed on a solid support and comprises annealing the amplification products to the solid support and amplifying the annealed amplification products; and

(d) clustering/aligning the sequences of the amplification products to identify a mutation and a member of the population carrying the mutation using the tag sequences.

14. The method according to claim 13, wherein the population comprising sub-populations of one or more human individuals having naturally-occurring mutations.

15. The method according to claim 14, wherein the naturally-occurring mutations are selected from the group consisting of single nucleotide polymorphisms, nucleotide insertions and deletions, and variations in microsatellite repeat number.

16. The method according to claim 13, wherein the high-throughput sequencing is sequencing-by-synthesis.

17. The method according to claim 13, wherein the high-throughput sequencing is bi-directional sequencing.

18. The method according to claim 13, wherein the solid support comprises one or more beads.

19. The method according to claim 18, wherein step (c) comprises:

(i) annealing the adaptor-ligated amplification products to beads, each bead annealing with a single adaptor-ligated amplification product;

(ii) emulsifying the beads in water-in-oil microreactors such that each microreactor contains a single bead;

(iii) performing emulsion PCR to amplify adaptor-ligated amplification products on the surface of the beads;

(iv) selecting/enriching beads to which are attached the amplified adaptor-ligated amplification products;

(v) placing the beads in wells such that each well comprises a single bead; and

generating a pyrophosphate signal.

20. The method according to claim 13, wherein the amplification products are sequenced with an average redundancy of at least 50.

21. The method according to claim 13, wherein step (d) comprises aligning the sequences of the amplification products to a reference sequence or a consensus sequence.

22. The method according to claim 13, wherein the amplification products are each tagged at both ends.

23. The method according to claim 13, wherein the amplification products are each tagged with two different tag sequences.

24. The method according to claim 13, wherein the amplification products ligated to sequencing adaptors each comprises a sequence derived from a member of the population flanked at both ends by tag sequences.