HIGH THROUGHPUT SCREENING OF POPULATIONS CARRYING NATURALLY OCCURRING MUTATIONS

• US 20170166962A1
• Filed: 02/16/2017
• Published: 06/15/2017
• Est. Priority Date: 09/29/2005
• Status: Abandoned Application

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: van EIJK, Michael Josephus Theresia, van TUNEN, Adrianus Johannes
• Application Number:
  US15/434,788
• Publication Number:
  US 20170166962A1
• Time in Patent Office: Days
• 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 of detecting a genetic variation, comprising:

(a) amplifying tagged nucleic acid molecules in a plurality of nucleic acid molecules to generate amplification products, each of the tagged nucleic acid molecules comprising one or more tag sequences;

(b) sequencing the amplification products to generate a set of sequence reads;

(c) grouping and aligning the set of sequence reads to a reference sequence using the one or more tag sequences; and

(d) detecting the genetic variation using the grouped and aligned sequence reads.

2. The method of claim 1, wherein the genetic variation is naturally occurring.

3. The method of claim 2, wherein the genetic variation is a single nucleotide polymorphism (SNP), a small insertion or a deletion, or a variation in microsatellite repeat number.

4. The method of claim 1, wherein the nucleic acid molecules are human derived.

5. The method of claim 1, wherein the nucleic acid molecules are genomic DNA molecules.

6. The method of claim 1, wherein both ends of each of the plurality of nucleic acid molecules are tagged.

7. The method of claim 1, wherein the sequencing is performed using high-throughput sequencing.

8. The method of claim 1, wherein the sequencing is bi-directional sequencing.

9. The method of claim 1, wherein the sequencing is sequencing-by-synthesis.

10. The method of claim 1, wherein the sequencing is performed on a solid support.

11. The method of claim 1, wherein the sequencing comprises performing a multi-fold redundant sequencing of the amplification products.

12. The method of claim 11, wherein the multi-fold redundant sequencing comprises a redundancy of at least 2.

13. The method of claim 11, wherein the multi-fold redundancy permits distinction between a sequencing error and a genetic variation.

14. The method of claim 11, wherein the detection of the genetic variation comprises detecting a sequence change multiple times.

15. The method of claim 1, wherein aligning comprises comparing the sequence reads to a consensus sequence.

16. The method of claim 1, wherein the genetic variation is detected without the use of an enzyme which recognizes and cuts single nucleotide sequence mismatches and without performing heteroduplex analysis.

17. The method of claim 1, wherein the detection is performed in silico.

18. The method of claim 1, further comprising preparing the nucleic acid molecules for sequencing by ligating adaptors to create a library of single stranded DNA.

19. The method of claim 1, further comprising tagging nucleic acid molecules by amplification or ligation.

20. The method of claim 1, wherein the sequencing comprises detecting a light signal indicative of a sequencing reaction.

21. The method of claim 1, wherein the alignment is performed using a computer program.

22. The method of claim 1, wherein a plurality of genetic variations are detected.

23. A method of detecting a genetic variation, comprising:

(a) amplifying tagged nucleic acid molecules in a plurality of nucleic acid molecules having the genetic variation to generate amplification products, each of the tagged nucleic acid molecules comprising a tag sequence at each end;

(b) performing high-throughput sequencing-by-synthesis of the amplification products of step (a) to generate a set of sequence reads;

(c) grouping and aligning the set of sequence reads to a reference sequence using the tag sequence at each end; and

(d) detecting the genetic variation using grouping and alignment information from step (c).