High throughput screening of populations carrying naturally occurring mutations

• US 9,657,335 B2
• Filed: 10/07/2016
• Issued: 05/23/2017
• 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:
  US15/288,253
• Publication Number:
  US 20170022560A1
• Time in Patent Office: 228 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 the detection of a mutation in a target sequence, comprising:

(a) providing a plurality of pools of amplification products each comprising the target sequence amplified from a nucleic acid of a subset of the one or more members of the population, wherein each pool of the amplification products shares a unique pool identifier;

(b) performing high throughput sequencing to sequence the amplification products; and

(c) clustering/aligning the sequences of the amplified products without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis, and detecting the one or more members and/or pools carrying the mutation using the pool identifier.

2. The method according to claim 1, wherein the population comprises sub-populations that comprise 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 amplification products are pooled using a 3D-pooling strategy.

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

6. The method according to claim 1, wherein the high throughput sequencing is performed by pyrosequencing.

7. The method according to claim 1, wherein the high throughput sequencing is performed on a solid support.

8. The method according to claim 7, wherein the solid support is a bead.

9. The method according to claim 7, wherein the high throughput sequencing comprises:

(i) ligating sequencing adaptors to the amplification products;

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

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

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

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

(vi) 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 target sequence that is amplified is from about 80 to about 400 bp.

11. The method according to claim 1, wherein the target sequence that is amplified is from about 90 to about 300 bp.

12. The method according to claim 1, wherein the target sequence that is amplified is from 100 to about 200 bp.

13. The method according to claim 1, wherein the method further comprises fragmenting the amplification products.

14. The method according to claim 1, wherein step (a) comprises amplifying the target sequence from cDNA with a pair of optionally labeled or tagged primers.

15. The method according to claim 14, wherein the method further comprises confirming the mutation by amplifying the target sequence from the identified products of step (c) using the primers of step (a) and determining the sequence of the amplified product.

16. The method according to claim 14, wherein at least one of the primers comprises a gene-specific section, a tag and a sequence primer binding site.

17. The method according to claim 1, wherein the amplified products are sequenced with an average redundancy of at least 4.

18. The method according to claim 1, wherein the amplified products are sequenced with an average redundancy of at least 10.

19. The method according to claim 1, wherein the amplified products are sequenced with an average redundancy of at least 25.

20. The method according to claim 1, wherein the amplified products are sequenced with an average redundancy of at least 50.