COMPOSITIONS AND METHODS FOR CO-AMPLIFYING SUBSEQUENCES OF A NUCLEIC ACID FRAGMENT SEQUENCE

• US 20140243242A1
• Filed: 08/08/2012
• Published: 08/28/2014
• Est. Priority Date: 08/08/2011
• Status: Active Grant

• Current Assignee: The Broad Institute, Inc.
• Original Assignee: The Broad Institute, Inc.
• Inventors: Nicol, Robert, Lennon, Niall J.
• Granted Patent: US 10,364,464 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/50 Detection characterised by immobilisation to a surface
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present invention is related to genomic nucleotide sequencing. In particular, the invention describes a single reaction method to co-amplify multiple subsequences of a nucleic acid fragment sequence (i.e., for example, at least two read pairs from a single library insert sequence). Nucleic acid fragment sequences may include, but are not limited to, localizing library insert sequences and/or unique read pair sequences in specific orientations on a single emulsion polymerase chain reaction bead. Methods may include, but are not limited to, annealing, melting, digesting, and/or reannealing high throughput sequencing primers to high throughput sequencing primer binding sites. The compositions and methods disclosed herein contemplate sequencing complex genomes, amplified genomic regions, as well as detecting chromosomal structural rearrangements that are compatible with massively parallel high throughput sequencing platforms as well as ion semiconductor matching sequencing platforms (i.e., for example, Ion Torrent platforms).

Claims

1. A method, comprising:

a) providing;

i) a solid substrate that can be attached to at least one labeled nucleic acid sequence;

ii) a plurality of nucleic acid sequences each further comprising at least one primer sequence, wherein said plurality of nucleic acid sequences comprise labeled nucleic acid sequences each further comprising an attachment feature; and

ii) a plurality of nucleic acid fragments, wherein said plurality of nucleic acid fragments comprise a plurality of subsequences;

b) annealing said plurality of nucleic acid fragments to said at least one primer sequence of said at least one nucleic acid sequences under conditions that generate a plurality of amplified nucleic acid fragments, wherein said amplified nucleic acid fragments are labeled fragments which comprise labeled nucleic acid sequences; and

c) combining said labeled nucleic acid sequences with the solid substrate under conditions that attach.

2. The method of claim 1, wherein said label of the labeled nucleic acid sequence comprises a label selected from the group consisting of biotin, universal primers, and nucleic acid barcodes.

3. The method of claim 1, wherein the plurality of nucleic acid sequences comprises a mixture of labeled and non-labeled nucleic acid sequences.

4. The method of claim 3, wherein the ratio of the mixture of labeled and non-labeled nucleic acid sequences is between 30:

70 and 70;

30.

5. The method of claim 1, wherein said labeled nucleic acid sequence is attached to the solid substrate by complementary hybridization, ligation or chemical bond.

6. The method of claim 1, wherein said labeled nucleic acid sequence is attached to the solid substrate with a linker.

7. The method of claim 6, wherein said linker comprises a streptavidin molecules, a nucleic acid sequence, a thioester linker sequence or a ribonucleic acid linker sequence.

8. The method of claim 1, wherein said solid substrate is selected from the group consisting of a bead and a microwell or a surface.

9. The method of claim 1, wherein said solid substrate is combined with the labeled fragments before amplification.

10. The method of claim 1, wherein said primer sequence comprises at least one random primer sequence selected from the group consisting of hexamer, heptamer, octomer and nonomer.

11. The method of claim 1, wherein said plurality of nucleic acid fragments are derived from a biological sample selected from the group consisting of a single genome, a single nucleic acid library, and a single nucleic acid library insert sequence.

12. The method of claim 1, wherein each of said plurality of nucleic acid fragments is circularized.

13. The method of claim 1, wherein the plurality of nucleic acid fragments is ligated to at least one barcode.

14. The method of claim 13, wherein barcoded nucleic acid fragment is amplified.

15. The method of claim 1, wherein said plurality of subsequences comprise a first subsequence having a first read pair sequence.

16. The method of claim 1, wherein said plurality of subsequences comprise a second subsequence having a second read pair sequence.

17. The method of claim 16, wherein said first read pair sequence comprises a first high throughput sequencing primer binding site.

18. The method of claim 17, wherein said second read pair sequence comprises a second high throughput sequencing primer binding site.

19. The method of claim 1, wherein said method further provides at least one primer selected from the group consisting of a first high throughput sequencing primer and a second high throughput sequencing primer.

20. The method of claim 16, wherein said method further comprises the step of annealing said first high throughput sequencing primer binding site to said first high throughput sequencing primer, under conditions such that said first read sequence is amplified.

21. The method of claim 16, wherein said method further comprises the step of annealing said second high throughput sequencing primer binding site to said second high throughput sequencing primer, under conditions such that said second read sequence is amplified.

22. The method of claim 16, wherein said first and second high throughput sequencing primers are selected from the group consisting of 454 sequencing primers, Illumina sequencing primers, SOLiD sequencing primers, and ion semiconduction sequencing primers.

23. A kit comprising:

a) a first container comprising a solid substrate capable of attaching at least one nucleic acid fragment sequence by a linker, wherein said nucleic acid fragment sequence comprises a plurality of subsequences;

b) a second container comprising a first high throughput sequencing primer capable of amplifying a first subsequence of said plurality of subsequences;

c) a third container comprising a second high throughput sequencing primer capable of amplifying a second subsequence of said plurality of subsequences;

d) a fourth container comprising at least one linker; and

e) instructions for co-amplifying said first and second subsequences with said first and second high throughput sequencing primers in a single reaction mixture.

24-33. -33. (canceled)