Strategies for high throughput identification and detection of polymorphisms

• US 9,447,459 B2
• Filed: 01/28/2014
• Issued: 09/20/2016
• Est. Priority Date: 06/23/2005
• Status: Active Grant

• Current Assignee: Keygene N.V.
• Original Assignee: Keygene N.V.
• Inventors: Van Eijk, Michael Josephus Theresia, Van Der Poel, Henricus Johannes Adam
• Primary Examiner(s): BOESEN, CHRISTIAN C
• Application Number:
  US14/166,218
• Publication Number:
  US 20140213462A1
• Time in Patent Office: 966 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2539/107 Representational Difference Analysis [RDA]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 30/10 by measuring physical properties, e.g. mass
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• 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

The invention relates to a method for the high throughput identification of single nucleotide polymorphisms by performing a complexity reduction on two or more samples to yield two or more libraries, sequencing at least part of the libraries, aligning the identified sequences and determining any putative single nucleotide polymorphisms, confirming any putative single nucleotide polymorphism, generating detection probes for the confirmed single nucleotide polymorphisms, subjection a test sample to the same complexity reduction to provide a test library and screen the test library for the presence or absence of the single nucleotide polymorphisms using the detection probe.

Claims

1. A method for identifying one or more polymorphisms, comprising:

(a) ligating sequencing adapters carrying a 3′

-T overhang to at least a first library of amplified fragments comprising sequences of a first nucleic acid sample and a second library of amplified fragments comprising sequences of a second nucleic acid sample to obtain first and second libraries of sequencing adapter-ligated fragments;

(b) combining the first and second libraries of sequencing adapter-ligated fragments;

(c) sequencing at least a portion of the combined libraries to obtain sequences;

(d) aligning the sequences obtained in step (c) to obtain an alignment; and

(e) determining one or more polymorphisms between the first and second nucleic acid samples in the alignment of step (d).

2. The method according to claim 1, wherein the first and second libraries of sequencing adapter-ligated fragments are each tagged with a unique identifier sequence.

3. The method according to claim 1, wherein step (d) comprises sequencing on a solid support.

4. The method according to claim 3, wherein the solid support comprises beads.

5. The method according to claim 4, wherein the sequencing adapters are capable of annealing to the beads.

6. The method according to claim 5, further comprising annealing the combined libraries of sequencing adapter-ligated fragments to the beads for emulsion polymerisation and subsequent high-throughput sequencing.

7. The method according to claim 1, wherein step (e) comprises comparing sequences of the first and second nucleic acid samples to identify a nucleotide sequence variant.

8. The method according to claim 7, wherein the first and second nucleic acid samples comprise plant DNA.

9. The method according to claim 7, wherein the first and second nucleic acid samples comprise human DNA.

10. The method according to claim 1, wherein step (e) comprises comparing sequences of the first and second nucleic acid samples to identify a single nucleotide polymorphism (SNP).

11. The method according to claim 1, wherein the amplified fragments are obtained by PCR amplification.

12. A method for identifying one or more nucleotide sequence variants, comprising:

(a) performing a complexity reduction on one or more nucleic acid samples of interest to provide at least a first amplified fragment comprising a first identifier sequence in addition to sample sequences and a second amplified fragment comprising a second identifier sequence in addition to sample sequences, wherein the first identifier sequence is different from the second identifier sequence and distinguishes the first amplified fragment from the second amplified fragment,

(b) ligating sequencing adapters carrying a 3′

-T overhang to at least the first amplified fragment and the second amplified fragment to obtain a pool of sequencing adapter-ligated fragments;

(c) sequencing at least a portion of the pool to obtain sequences;

(d) aligning the sequences obtained in step (c) to obtain an alignment; and

(e) determining one or more nucleotide sequence variants from the one or more of nucleic acid samples in the alignment of step (d).