Directed assembly of amplicons to enhance read pairing signature with massively parallel short read sequencers

• US 20090036325A1
• Filed: 05/21/2008
• Published: 02/05/2009
• Est. Priority Date: 05/25/2007
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Sheridan, John Andrew, Smith, Douglas R., McKernan, Kevin J.
• Application Number:
  US12/154,294
• Publication Number:
  US 20090036325A1
• Time in Patent Office: Days
• US Class Current: 506/26
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

The present teachings relate to improved methods, kits, and compositions for making nucleic acid libraries and sequencing nucleic acids. In some embodiments, directionally defined concatamers are generated, facilitating sequencing efforts.

Claims

1. A method of obtaining an informative desired subset of a complex nucleic acid mixture comprising;

amplifying the desired subset with a plurality of primer pairs to form a plurality of first amplicons, wherein each of the plurality of primer pairs comprises a forward primer and a reverse primer,

(a) wherein the forward primer comprises a first asymmetric adapter, and,

(b) wherein the reverse primer comprises a second asymmetric adapter;

cleaving the first asymmetric adapter and the second asymmetric adapter of the plurality of first amplicons to form a plurality of sticky fragments comprising a first sticky end and a second sticky end;

ligating the plurality of sticky fragments to form a concatamer comprising a plurality of sticky fragments bearing a determined orientation;

cleaving the concatamer to form a plurality of cleavage products; and

,

obtaining an informative desired subset of the complex nucleic acid mixture.

2. The method of claim 1

(a) wherein the concatamer comprises a first sticky fragment with a downstream end ligated to an upstream end of a second sticky fragment, and,

(b) wherein the concatamer does not comprise the upstream end of the first sticky fragment ligated to the downstream end of the second sticky fragment.

3. The method of claim 2 further comprising;

amplifying the informative desired subset in an emulsion PCR, wherein the emulsion PCR comprises primer-immobilized beads, to form a collection of amplicon-bearing beads;

immobilizing the amplicon-bearing beads on a solid support; and

,

sequencing the amplicon of each amplicon-bearing bead.

4. The method according to claim 3 wherein the sequencing comprising ligation-sequencing.

5. The method according to claim 3 wherein the sequencing comprises sequencing by synthesis.

6. A method of forming a concatamer containing sticky fragments comprising;

amplifying a complex nucleic acid sample with a plurality of primer pairs to form a plurality of first amplicons, wherein each of the plurality of primer pairs comprises a forward primer and a reverse primer,

(a) wherein the forward primer comprises a first asymmetric adapter, and,

(b) wherein the reverse primer comprises a second asymmetric adapter;

cleaving the first asymmetric adapter and the second asymmetric adapter of the plurality of first amplicons to form a plurality of sticky fragments comprising a first sticky end and a second sticky end; and

,

ligating the plurality of sticky fragments to form a concatamer comprising a plurality of sticky fragments bearing a determined orientation,

wherein the ligating does not comprise a vector, and

wherein the first asymmetric adapter, the second asymmetric adapter, or both the first and the second asymmetric adapter comprise a phosphorothiolate and wherein the cleaving comprises treating with aqueous Silver.