Strategies for high throughput identification and detection of polymorphisms

• US 9,493,820 B2
• Filed: 02/19/2015
• Issued: 11/15/2016
• 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
• Application Number:
  US14/626,822
• Publication Number:
  US 20150232924A1
• Time in Patent Office: 635 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 in nucleic acid samples, comprising:

(a) providing a plurality of nucleic acid samples;

(b) performing a reproducible complexity reduction on each of the nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein said reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using at least one primer comprising at least one selective nucleotide at its 3′

-end to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence different from the selective nucleotide to indicate origin of each library obtained by the reproducible complexity reduction;

(c) sequencing at least a portion of the 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 plurality of nucleic acid samples in the alignment of step (d).

2. The method according to claim 1, wherein at least one of the primers is phosphorylated.

3. The method according to claim 1, wherein the sequencing step (c) is performed on a solid support such as a bead.

4. The method according to claim 1, wherein sequencing is based on dideoxy chain-terminating sequencing.

5. The method according to claim 3, wherein the sequencing step (c) comprises the steps of:

(i) annealing adapter-ligated fragments to beads, each bead annealing with a single adapter-ligated fragment;

(ii) emulsifying the beads in water-in-oil microreactors, each water-in-oil microreactor comprising a single bead;

(iii) loading the beads in wells, each well comprising a single bead; and

(iv) generating a pyrophosphate signal.

6. The method according to claim 5, wherein, preceding the annealing step, sequencing adaptors are ligated to fragments within the first library and the second library or combination library.

7. The method according to claim 6, wherein the sequencing adaptors carry a 3′

-T overhang.

8. The method according to claim 1, comprising after step (e), a further step of using the one or more polymorphisms determined in step (e) to design detection probes, and wherein high-throughput screening is performed by immobilizing said probes onto an array, followed by contacting of the array comprising the probes with a test library under hybridizing conditions.

9. The method of claim 1, further comprising the steps of:

(f) using the one or more polymorphisms determined in step (e) to design detection probes;

(g) providing a test sample nucleic acid of interest;

(h) performing the complexity reduction of step (b) on the test sample nucleic acid to provide a test library of the test sample; and

(i) subjecting the test library to high-throughput screening to identify the presence, absence or amount of the polymorphisms determined in step (e) using the detection probes designed in step (f).

10. The method of claim 1, wherein step (b) comprises amplifying fragments of the nucleic acid samples using at least one pair of primers each comprising at least one selective nucleotide at its 3′

-end.

11. The method of claim 1, wherein the plurality of libraries obtained by the reproducible complexity reduction are combined to obtain a combined library and sequenced together.

12. The method of claim 1, wherein the unique identifier sequence indicates, independently from sequences of the nucleic acid samples, origin of each library obtained by the reproducible complexity reduction.

13. A method for identifying one or more polymorphisms in nucleic acid samples, comprising:

(a) performing a reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein said reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using at least one primer comprising at least one selective nucleotide at its 3′

-end to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence different from the selective nucleotide to indicate, independent from sequences of the nucleic acid samples, origin of each library obtained by the reproducible complexity reduction;

(b) combining the plurality of libraries obtained by the reproducible complexity reduction to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences;

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

(d) determining one or more polymorphisms between the plurality of nucleic acid samples in the alignment of step (c).