PAIRED END BEAD AMPLIFICATION AND HIGH THROUGHPUT SEQUENCING

• US 20140031241A1
• Filed: 01/27/2012
• Published: 01/30/2014
• Est. Priority Date: 01/28/2011
• Status: Active Grant

• Current Assignee: The Broad Institute, Inc.
• Original Assignee: The Broad Institute, Inc.
• Inventors: Lennon, Niall J., Nicol, Robert
• Granted Patent: US 9,738,930 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Abstract

The present invention is related to genomic nucleotide sequencing. In particular, the invention describes a paired end sequencing method that enables the sequencing of unique read pairs by co-localizing both 5′ ends on a single emulsion polymerase chain reaction bead. The method may use a customized forked adaptor primer pair that is compatible with massively parallel sequencing techniques. The compositions and methods disclosed herein contemplate sequencing complex genomes, amplified genomic regions, as well as detecting chromosomal structural rearrangements.

Claims

1. A composition comprising a solid support attached to a first emulsion primer and a second emulsion primer, wherein said first emulsion primer is ligated to a first nucleic acid sequence comprising a first portion of a genomic DNA sequence and said second emulsion primer is ligated to a second nucleic acid comprising a second portion of said genomic DNA sequence.

2. The composition of claim 1, wherein said first and second emulsion primers are attached to said solid support at their 5′

ends.

3. The composition of claim 1, wherein said solid support is a bead.

4. The composition of claim 1, wherein said composition further comprises a first high throughput sequencing primer hybridized to said first nucleic acid sequence.

5. The composition of claim 1, wherein said composition further comprises a second high throughput sequencing primer hybridized to said second nucleic acid sequence.

17. The composition of claim 5, wherein said second primer binding sequence is an emulsion primer binding sequence.

6. A method, comprising:

a) providing;

i) a first forked end adapter comprising a first duplex DNA sequence, wherein said first duplex DNA sequence comprises a first nucleic acid sequence ligated to a first primer sequence and a second nucleic acid sequence ligated to a second primer binding sequence;

ii) a second forked end adapter comprising a second duplex DNA sequence, wherein said second duplex DNA sequence comprises a third nucleic acid sequence ligated to a third primer sequence and a fourth nucleic acid sequence ligated to a fourth primer binding sequence;

iii) a genomic DNA fragment capable ligating said first duplex DNA sequence to said second duplex DNA sequence;

iv) a second primer capable of binding to said second primer binding sequence;

v) a fourth primer capable of binding to a fourth primer binding sequence; and

vi) a bead comprising said first primer and said third primer, wherein said first and third primers are attached at the 5′

end.

b) contacting said genomic DNA fragment with said first forked end adapter and said second forked end adapter wherein said genomic DNA fragment ligates between said first duplex sequence and said second duplex sequence, thereby forming a ligated forked adapter primer pair; and

c) emulsion amplifying said ligated forked adapter primer pair with said bead, said second primer and said fourth primer, thereby co-localizing a first doubled stranded bead product and a second double stranded bead product on said bead.

7. The method of claim 6, wherein said method further comprises denaturing said first double stranded bead product and said second double stranded bead product, thereby forming a first single stranded bead product and a second single stranded bead product.

8. The method of claim 7, wherein said method further comprises hybridizing a first sequencing primer to said first single stranded bead product and a second sequencing primer to said second single stranded bead product.

9. The method of claim 8, wherein said method further comprises sequentially sequencing said first single stranded bead product and said second single stranded bead product.

10. The method of claim 9, wherein said sequencing comprises high throughput sequencing.

11. The method of claim 8, wherein said first sequencing primer and said second sequencing primer are high throughput sequencing primers.

12. A kit, comprising:

a) a first container comprising a first forked adapter nucleic acid sequence;

b) a second container comprising a second forked adapter nucleic acid sequence;

c) a third container comprising a plurality of emulsion polymerase chain reaction primers;

d) a fourth container comprising a plurality of high throughput sequencing primers; and

e) instructional materials containing directions providing for the use of said nucleic acids and reagents to perform paired-end sequencing of genomic nucleic acid sequence from a single bead.

13. The kit of claim 12, wherein said first forked adapter comprises an emulsion primer sequence and at least one emulsion primer binding site sequence.

14. The kit of claim 12, wherein said second forked adapter comprises an emulsion primer sequence and at least one emulsion primer binding site sequence, wherein said sequences are different from said first forked adapter.

15. A composition comprising a forked end adapter comprising a duplex DNA sequence, wherein said duplex DNA sequence comprises a first nucleic acid sequence ligated to a primer sequence and a second nucleic acid sequence ligated to a primer binding sequence.

16. The composition of claim 15, wherein said primer sequence is an emulsion primer sequence.

18. A composition comprising a forked end adapter comprising a duplex DNA sequence, wherein said duplex DNA sequence comprises a first nucleic acid sequence ligated to a primer sequence and a second nucleic acid sequence ligated to a barcode sequence.

19. (canceled)

20. A composition comprising a ligated forked adapter pair, wherein said adapter pair comprises a first duplex DNA sequence ligated to a second duplex DNA sequence, said first duplex DNA sequence comprising a first nucleic acid sequence ligated to a first primer sequence and a second nucleic acid sequence ligated to a first primer binding sequence, and said second duplex DNA sequence ligated to a third nucleic acid sequence ligated to a second primer sequence and a fourth nucleic acid sequence ligated to a second primer binding sequence.

21-24. -24. (canceled)

25. A composition comprising a ligated forked adapter pair, wherein said adapter pair comprises a first duplex DNA sequence ligated to a second duplex DNA sequence, said first duplex DNA sequence comprising a first nucleic acid sequence ligated to a first primer sequence and a second nucleic acid sequence ligated to a first primer binding sequence, and said second duplex DNA sequence ligated to a third nucleic acid sequence ligated to a second primer sequence and a fourth nucleic acid sequence ligated to a barcode.

26-28. -28. (canceled)