Strategies for high throughput identification and detection of polymorphisms

• US 9,898,577 B2
• Filed: 10/10/2017
• Issued: 02/20/2018
• 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:
  US15/729,328
• Publication Number:
  US 20180025111A1
• Time in Patent Office: 133 Days
• Field of Search: None
• 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 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 the reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using one or more primers to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence to indicate origin of each library obtained by the reproducible complexity reduction; (b) combining the plurality of libraries to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences; (c) aligning the sequences to obtain an alignment; and (d) identifying one or more polymorphisms in the plurality of nucleic acid samples.

Claims

1. A method for identifying genetic variation in nucleic acid samples, comprising:

(a) performing a reproducible complexity reduction on a plurality of nucleic acid samples, wherein the reproducible complexity reduction comprises enrichment using biotinylated capture oligonucleotide hybridization probes to capture a subset of fragments of the nucleic acid samples, and amplifying these fragments using one or more primers to obtain a pool of amplified fragments, wherein the amplified fragments are tagged with a unique identifier sequence to indicate sample origin of the amplified fragments;

(b) sequencing at least a portion of the pool of the amplified fragments to obtain sequences, wherein the sequencing step (b) is performed on a solid support;

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

(d) identifying genetic variation in the plurality of nucleic acid samples using the alignment of step (c).

2. The method of claim 1, wherein the genetic variation is a single nucleotide polymorphism (SNP), a small insertion or a deletion, or a variation in microsatellite repeat number.

3. The method of claim 1, wherein the nucleic acid samples are human nucleic acid samples.

4. The method according to claim 2, wherein the solid support is a bead.

5. The method according to claim 1, wherein the sequencing step (b) comprises dideoxy chain-terminating sequencing.

6. The method according to claim 1, wherein the sequencing step (b) comprises high-throughput sequencing.

7. The method according to claim 4, wherein the sequencing step (b) comprises:

(i) annealing amplified fragments to beads, each bead annealing with a single amplified 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.

8. The method according to claim 7, wherein, preceding the annealing step, sequencing adaptors are ligated to the amplified fragments.

9. The method according to claim 8, wherein the sequencing adaptors carry a 3′

-T overhang.

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

11. The method according to claim 1, wherein the nucleic acid molecules are tagged using amplification or ligation.

12. The method according to claim 1, wherein the unique identifier sequence indicates, independently from sequences of the nucleic acid samples, sample origin of each amplified fragment.