Strategies for high throughput identification and detection of polymorphisms

• US 10,235,494 B2
• Filed: 04/24/2018
• Issued: 03/19/2019
• 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): Wilder, Cynthia B
• Application Number:
  US15/961,512
• Publication Number:
  US 20180247017A1
• Time in Patent Office: 329 Days
• Field of Search: 435 912
• 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 one or more genetic variations, comprising:

(a) performing reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of complexity reduced nucleic acids, wherein the reproducible complexity reduction comprises enrichment using biotinylated oligonucleotide probes to capture a subset of nucleic acids of each nucleic acid sample;

(b) combining the plurality of libraries to obtain a pool, wherein the complexity reduced nucleic acids of each library in the pool are tagged with a unique sample identifier sequence;

(c) performing high-throughput sequencing to sequence at least a portion of the pool to obtain sequences, wherein the high-throughput sequencing is performed on a solid support and comprises annealing nucleic acids to the solid support and amplifying the annealed nucleic acids;

(d) aligning the sequences obtained by high-throughput sequencing to obtain an alignment, and identifying one or more genetic variations in the plurality of nucleic acid samples using the alignment.

2. The method according to claim 1, wherein the genetic variations are human genetic variations.

3. The method according to claim 2, wherein the genetic variations are selected from the group consisting of single nucleotide polymorphisms and simple sequence repeats.

4. The method according to claim 1, wherein the solid support comprises one or more beads.

5. The method according to claim 1, wherein step (d) comprises comparing the sequences of the complexity reduced nucleic acids to a consensus or reference sequence to identify one or more genetic variations.

6. The method according to claim 1, wherein the reproducible complexity reduction comprises ligating adaptors to both ends of nucleic acids of each nucleic acid sample prior to enrichment using biotinylated oligonucleotide probes.

7. A method for identifying one or more genetic variations, comprising:

(a) performing reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of complexity reduced nucleic acids, wherein the reproducible complexity reduction of each nucleic acid sample comprises (i) ligation of adaptors to nucleic acids to obtain adaptor-ligated nucleic acids, (ii) amplification of the adaptor-ligated nucleic acids using one or more primers to obtain amplified adaptor-ligated nucleic acids, and (iii) enrichment using biotinylated oligonucleotide probes to capture a subset of the amplified adaptor-ligated nucleic acids;

(b) combining the plurality of libraries to obtain a pool, wherein the complexity reduced nucleic acids of each library in the pool are tagged with a unique sample identifier sequence;

(c) performing high-throughput sequencing to sequence at least a portion of the pool to obtain sequences, wherein the high-throughput sequencing is performed on a solid support and comprises annealing nucleic acids to the solid support and amplifying the annealed nucleic acids;

(d) aligning the sequences obtained by high-throughput sequencing to obtain an alignment, and identifying one or more genetic variations in the plurality of nucleic acid samples using the alignment.

8. The method according to claim 7, wherein the genetic variations are human genetic variations.

9. The method according to claim 8, wherein the genetic variations are selected from the group consisting of single nucleotide polymorphisms and simple sequence repeats.

10. The method according to claim 7, wherein the solid support comprises one or more beads.

11. The method according to claim 7, wherein step (d) comprises comparing the sequences of the complexity reduced nucleic acids to a consensus or reference sequence to identify one or more genetic variations.

12. The method according to claim 7, wherein the reproducible complexity reduction comprises ligating adaptors to both ends of the nucleic acids to obtain adaptor-ligated nucleic acids.

13. The method according to claim 7, wherein at least one of the adapters of step (a)(i) comprises a template sequence for hybridizing a primer of step (a)(ii).

14. The method according to claim 7, wherein at least one of the adapters of step (a)(i) comprises a sequence identifier.

15. A method for identifying one or more genetic variations, comprising:

(a) performing reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of complexity reduced nucleic acids, wherein the reproducible complexity reduction comprises enrichment using biotinylated oligonucleotide probes to capture a subset of nucleic acids of each nucleic acid sample;

(b) ligating sequencing adapters carrying a 3′

-T overhang to the plurality of libraries of complexity reduced nucleic acids;

(c) combining the plurality of libraries to obtain a pool, wherein the complexity reduced nucleic acids of each library in the pool are tagged with a unique sample identifier sequence;

(d) performing high-throughput sequencing to sequence at least a portion of the pool to obtain sequences, wherein the high-throughput sequencing is performed on a solid support and comprises annealing nucleic acids to the solid support and amplifying the annealed nucleic acids;

(e) aligning the sequences obtained by high-throughput sequencing to obtain an alignment, and identifying one or more genetic variations in the plurality of nucleic acid samples using the alignment.

16. The method according to claim 15, wherein the genetic variations are human genetic variations.

17. The method according to claim 16, wherein the genetic variations are selected from the group consisting of single nucleotide polymorphisms and simple sequence repeats.

18. The method according to claim 15, wherein the solid support comprises one or more beads.

19. The method according to claim 15, wherein step (e) comprises comparing the sequences of the complexity reduced nucleic acids to a consensus or reference sequence to identify one or more genetic variations.

20. The method according to claim 15, wherein the reproducible complexity reduction comprises ligating adaptors to both ends of nucleic acids of each nucleic acids sample prior to enrichment using biotinylated oligonucleotide probes.

21. The method according to claim 15, wherein step (b) comprises ligating the sequencing adapters carrying a 3′

-T overhang to the complexity reduced nucleic acids carrying a 3′

-A overhang.

22. A method for identifying one or more genetic variations, comprising:

(a) performing reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of complexity reduced nucleic acids, wherein the reproducible complexity reduction of each nucleic acid sample comprises (i) ligation of adaptors to nucleic acids to obtain adaptor-ligated nucleic acids, (ii) amplification of the adaptor-ligated nucleic acids using one or more primers to obtain amplified adaptor-ligated nucleic acids, and (iii) enrichment using biotinylated oligonucleotide probes to capture a subset of the amplified adaptor-ligated nucleic acids;

(b) ligating sequencing adapters carrying a 3′

-T overhang to the plurality of libraries of complexity reduced nucleic acids;

(c) combining the plurality of libraries to obtain a pool, wherein the complexity reduced nucleic acids of each library in the pool are tagged with a unique sample identifier sequence;

(d) performing high-throughput sequencing to sequence at least a portion of the pool to obtain sequences, wherein the high-throughput sequencing is performed on a solid support and comprises annealing nucleic acids to the solid support and amplifying the annealed nucleic acids;

(e) aligning the sequences obtained by high-throughput sequencing to obtain an alignment, and identifying one or more genetic variations in the plurality of nucleic acid samples using the alignment.

23. The method according to claim 22, wherein the genetic variations are human genetic variations.

24. The method according to claim 23, wherein the genetic variations are selected from the group consisting of single nucleotide polymorphisms and simple sequence repeats.

25. The method according to claim 22, wherein the solid support comprises one or more beads.

26. The method according to claim 22, wherein step (e) comprises comparing the sequences of the complexity reduced nucleic acids to a consensus or reference sequence to identify one or more genetic variations.

27. The method according to claim 22, wherein the reproducible complexity reduction comprises ligating adaptors to both ends of the nucleic acids to obtain adaptor-ligated nucleic acids.

28. The method according to claim 22, wherein step (b) comprises ligating the sequencing adapters carrying a 3′

-T overhang to the complexity reduced nucleic acids carrying a 3′

-A overhang.

29. The method according to claim 22, wherein at least one of the adapters of step (a)(i) comprises a template sequence for hybridizing a primer of step (a)(ii).

30. The method according to claim 22, wherein at least one of the adapters of step (a)(i) comprises a sequence identifier.