Sequencing using concatemers of copies of sense and antisense strands

Patent metadata

Field	Value
Publication number	US-2016376647-A1
Application number	US-201615242201-A
Country	US
Kind code	A1
Filing date	Aug 19, 2016
Priority date	Mar 28, 2008
Publication date	Dec 29, 2016
Grant date	—

Abstract

Methods and systems for single molecule sequencing using concatemers of copies of sense and antisense strands. Concatemers are provided, for example, by carrying out rolling circle amplification on a circular molecule having sense and antisense regions to produce repeated copies of the sense and antisense regions connected by linking regions. The circular molecules can be produced by ligating hairpin adapters to each end of a double-stranded nucleic acid having a sense and antisense strand. The ligations can be carried out, for example using blunt end ligation. In some cases, a single molecule consensus sequence for a single template molecule is obtained. A single read from each template molecule can be obtained by comparing the sequence information of the sense and antisense regions.

First claim

What is claimed is:

1 . A method of obtaining nucleotide sequence information, the method comprising:
a) fragmenting a nucleic acid sample to provide at least one double-stranded nucleic acid fragment comprising a double-stranded region comprising a sense strand and a complementary antisense strand; b) ligating a first hairpin adapter to a first end of said fragment and ligating a second hairpin adapter to a second end of said fragment, thereby providing a circular nucleic acid; c) performing a rolling circle replication process with said circular nucleic acid as a template, thereby producing a concatemer comprising repeating copies of said sense and antisense strands; and d) performing a real time, single-molecule sequencing process that generates a single sequencing read from said concatemer, said single sequencing read comprising at least one first sequence corresponding to said sense strand and at least one second sequence corresponding to said antisense strand.

2 . The method of claim 1 , further comprising comparing said at least one first sequence and said at least one second sequence to obtain a single molecule consensus sequence.

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

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

5 . The method of claim 1 , wherein said single-molecule sequencing process is performed using a sequencing by synthesis technology.

6 . The method of claim 1 , wherein said first and second hairpin adapters are identical.

7 . The method of claim 1 , wherein said first and second hairpin adapters have blunt ends.

8 . The method of claim 1 , wherein said first hairpin adapter or said second hairpin adapter, or both said first hairpin adapter and said second hairpin adapter, comprise a registration sequence.

9 . The method of claim 1 , wherein the nucleic acid sample comprises genomic DNA.

10 . The method of claim 1 , wherein the nucleic acid sample comprises amplification products.

11 . The method of claim 1 , wherein the double-stranded region comprises at least 1000 base pairs.

12 . A method of obtaining nucleotide sequence information, the method comprising:
performing a single-molecule sequencing process on a single template molecule, wherein the template molecule comprises a linear concatemer nucleic acid strand comprising repeating, alternating copies of a sense region and a complementary antisense region, wherein first linking regions are present between 3′ ends of the sense regions and 5′ ends of the antisense regions, and wherein second linking regions are present between 3′ ends of the antisense regions and 5′ ends of the sense regions, thereby generating a sequence read from the single template molecule, the read comprising at least one first sequence corresponding to the sense region, and at least one second sequence corresponding to the complementary antisense region.

13 . The method of claim 12 , further comprising comparing the at least one first sequence and the at least one second sequence from the sequence read to obtain a single molecule consensus sequence for the single template molecule.

14 . The method of claim 12 , wherein the single-molecule sequencing process comprises a sequencing by synthesis technology.

15 . The method of claim 14 , wherein the sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

16 . The method of claim 12 , wherein the single-molecule sequencing process is performed using a nanopore sensor.

17 . The method of claim 12 , wherein the sequence read comprises at least 1000 bases.

18 . The method of claim 12 , wherein the first linking region or the second linking region, or both the first linking region and the second linking region, comprise a registration sequence.

19 . The method of claim 12 , wherein the first and second linking regions are identical.

20 . A system for sequencing template nucleic acid molecules comprising:
a plurality of template nucleic acid molecules, each template nucleic acid molecule comprising a linear concatemer nucleic acid strand comprising repeating, alternating copies of a sense region and a complementary antisense region, wherein first linking regions are present between 3′ ends of the sense regions and 5′ ends of the antisense regions, and wherein second linking regions are present between 3′ ends of the antisense regions and 5′ ends of the sense regions; a sequencing instrument in contact with the plurality of template nucleic acid molecules that performs at least one single-molecule sequencing process on each of the plurality of template molecules, each of the at least one single-molecule sequencing process consisting of sequencing only one single template molecule of the plurality of template molecules, thereby generating a sequence read from each of the plurality of template molecules, the sequence read comprising sequences of both the sense region and the antisense region of each of the plurality of template molecules; and a computer that determines the sequence of each of the plurality of template molecules by analyzing the sequence read from each of the plurality of template molecules.

21 . The system of claim 20 wherein the computer compares the sequences of the sense region and the antisense region from each of the plurality of template molecules, thereby improving sequencing accuracy.

22 . The system of claim 20 , wherein the at least one single-molecule sequencing process is performed using a nanopore sensor.

23 . The system of claim 20 , wherein the at least one single-molecule sequencing process is performed using a sequencing by synthesis technology.

24 . The system of claim 23 , wherein the sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

Assignees

• Pacific Biosciences California Inc

Inventors

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

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• C12Q1/689

  for bacteria · CPC title

• G16B30/10

  Sequence alignment; Homology search · CPC title

• G16B30/00 Primary

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title