High throughput screening of populations carrying naturally occurring mutations

• US 10,538,806 B2
• Filed: 02/04/2019
• Issued: 01/21/2020
• 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:
  US16/267,123
• Publication Number:
  US 20190153534A1
• Time in Patent Office: 351 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 nucleic acid samples, comprising:

(a) providing a plurality of complexity reduced libraries comprising amplified adapter-ligated nucleic acid fragments, wherein each nucleic acid fragment within a library comprises a sequence derived from a nucleic acid sample and one or more tag sequences for nucleic acid and/or sample identification;

(b) pooling the plurality of libraries of nucleic acid fragments;

(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) aligning the sequences of the nucleic acid fragments and grouping the sequences using the one or more tag sequences to identify a mutation and a nucleic acid and/or a sample comprising the mutation.

2. The method according to claim 1, wherein the mutation is a single nucleotide polymorphism.

3. The method according to claim 1, wherein the mutation is a nucleotide insertion or deletion.

4. The method according to claim 1, wherein the mutation is a microsatellite repeat number variation.

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

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

7. The method according to claim 1, wherein adapters are ligated to create libraries of single-stranded DNA.

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

9. The method according to claim 8, wherein step (c) comprises:

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

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

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

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

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

generating a pyrophosphate signal.

10. The method according to claim 1, wherein the nucleic acid fragments are sequenced with an average redundancy of at least 10.

11. The method according to claim 1, wherein the nucleic acid fragments are sequenced with an average redundancy of at least 25.

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

13. 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.

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

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

16. The method according to claim 1, wherein the nucleic acid fragments each comprises a sequence derived from a nucleic acid sample flanked at both ends by tag sequences.

17. The method according to claim 1, wherein the one or more tag sequences are for nucleic acid identification.

18. The method according to claim 1, wherein the one or more tag sequences are for sample identification.

19. The method according to claim 1, wherein the one or more tag sequences are for nucleic acid and sample identification.