Paired end bead amplification and high throughput sequencing

Patent metadata

Field	Value
Publication number	US-9738930-B2
Application number	US-201213981412-A
Country	US
Kind code	B2
Filing date	Jan 27, 2012
Priority date	Jan 28, 2011
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

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.

First claim

We claim:

1. 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 of 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 said fourth primer binding sequence; and
vi) a bead comprising a first primer having the first primer sequence as recited in i) and a third primer having the third primer sequence as recited in ii), wherein said first and third primers are attached to said bead at their 5′ ends;

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 double stranded bead product and a second double stranded bead product on said bead;
wherein said first primer, second primer, third primer, and fourth primer have distinct, non-complementary sequences and bind to distinct primer binding sequences.

2. The method of claim 1 , 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.

3. The method of claim 2 , 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.

4. The method of claim 3 , wherein said method further comprises sequentially sequencing said first single stranded bead product and said second single stranded bead product.

5. The method of claim 4 , wherein said sequencing comprises high throughput sequencing.

6. The method of claim 3 , wherein said first sequencing primer and said second sequencing primer are high throughput sequencing primers.

Assignees

• Nicol Robert
• Lennon Niall J
• Broad Inst Inc

Inventors

• Nicol Robert
• Lennon Niall J

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2563/159

  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 title

• C40B50/14

  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 · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title