Compositions and methods for nucleic acid sequencing

Patent metadata

Field	Value
Publication number	US-9542527-B2
Application number	US-201414497067-A
Country	US
Kind code	B2
Filing date	Sep 25, 2014
Priority date	Mar 28, 2008
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

Compositions and methods for nucleic acid sequencing include template constructs that comprise double stranded portions in a partially or completely contiguous constructs, to provide for redundant sequence determination through one or both of sequencing sense and antisense strands, and iteratively sequencing the entire construct multiple times. Additional sequence components are also optionally included within such template constructs. Methods are also provided for the use and preparation of these constructs as well as sequencing compositions for their application.

First claim

What is claimed is:

1. A method of obtaining a sequence of an individual double-stranded nucleic acid template molecule, the method comprising:
(a) fragmenting a nucleic acid sample, thereby providing a plurality of double-stranded nucleic acid molecules, said double-stranded nucleic acid molecules comprising (i) a first strand, and (ii) a second strand, which is complementary to the first strand;
(b) ligating a hairpin loop to a first end of said double-stranded nucleic acid molecules to covalently link said first strand and said second strand at said first end of said double-stranded nucleic acid molecules, thereby producing a plurality of double-stranded nucleic acid template molecules, wherein said first strand and said second strand at said first end of each of said plurality of double-stranded nucleic acid template molecules is linked by said hairpin loop;
(c) identifying individual nucleotides of said first strand and said second strand of an individual double-stranded nucleic acid template molecule from said plurality of double-stranded nucleic acid template molecules produced in step (b) using a real time, single-molecule sequencing process, said single-molecule sequencing process consisting of sequencing said individual double-stranded nucleic acid template molecule, thereby generating a single sequencing read from said individual double-stranded nucleic acid template molecule, wherein said single sequencing read comprises a first nucleotide sequence of said first strand and a second nucleotide sequence of said second strand; and
(d) obtaining a sequence of said individual double-stranded nucleic acid template molecule by analyzing said single sequencing read using both said first nucleotide sequence of said first strand and said second nucleotide sequence of said second strand, which includes comparing the first nucleotide sequence and the second nucleotide sequence from said individual double-stranded nucleic acid template molecule.

2. The method of claim 1 , wherein said real time, single-molecule sequencing process comprises performing a polymerase mediated template directed sequencing process.

3. The method of claim 1 , wherein said real time, single-molecule sequencing process is performed using an electrochemical system.

4. The method of claim 1 , wherein said real time, single-molecule sequencing process is performed using a nanopore sensor.

5. The method of claim 1 , wherein said plurality of double-stranded nucleic acid molecules comprise genomic DNA.

6. The method of claim 1 , wherein said individual double-stranded nucleic acid template molecule comprises at least 500 base pairs.

7. The method of claim 1 , wherein said hairpin loop comprises a registration sequence.

8. The method of claim 7 , wherein said registration sequence is used for providing a basis for alignment of the first nucleotide sequence of the first strand to the second nucleotide sequence of the second strand.

9. The method of claim 1 , wherein said hairpin loop comprises a barcode sequence.

10. The method of claim 1 , wherein said individual double-stranded template molecule comprises a single-stranded segment.

11. The method of claim 10 , wherein the single-stranded segment is an initiation point for the real-time, single molecule sequencing process.

Assignees

• Pacific Biosciences California Inc

Inventors

• Travers Kevin
• Otto Geoff
• Turner Stephen
• Heiner Cheryl
• Ma Congcong

Classifications

• C12Q2525/155

  incorporating/generating a new priming site · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2525/301

  Hairpin oligonucleotides · CPC title

• C12Q1/6806

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

• G06F19/22 Primary

  Physics · mapped topic