Methods for obtaining a single molecule consensus sequence

Patent metadata

Field	Value
Publication number	US-2016237485-A1
Application number	US-201615089071-A
Country	US
Kind code	A1
Filing date	Apr 1, 2016
Priority date	Mar 28, 2008
Publication date	Aug 18, 2016
Grant date	—

Abstract

Methods obtaining a single molecule consensus sequence for a single template molecule, and for obtaining a plurality of single molecule consensus sequences for a plurality of single template molecules is provided. Template molecules having two complementary regions connected with a linker are sequenced. A single read from each template molecule can be obtained, the read containing sequence information for each of the complementary regions. Single molecule consensus sequences can be determined from these reads by comparing the sequence information of the two complementary regions.

First claim

What is claimed is:

1 . A method of obtaining a single molecule consensus sequence for a single template nucleic acid molecule, comprising:
performing a single-molecule sequencing process on a single template molecule, wherein the single template molecule comprises: i) a first strand having a 3′ end and a 5′ end, ii) a second strand, complementary to the first strand, and having a 3′ end and a 5′ end, and iii) a linker that links the 3′ end of the first strand to the 5′ end of the second strand, thereby generating a sequence read from said single template molecule, the read comprising a first sequence corresponding to the first strand, and a second sequence corresponding to the complementary second strand; and comparing the first sequence and second sequence from said sequence read to obtain a single molecule consensus sequence for the single template molecule.

2 . The method of claim 1 wherein the linker comprises a linking oligonucleotide.

3 . The method of claim 2 , wherein said sequence read comprises a sequence of the linking oligonucleotide.

4 . The method of claim 1 , wherein said single-molecule sequencing process comprises a sequencing by synthesis technology.

5 . The method of claim 4 , wherein said sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

6 . The method of claim 1 , wherein said single-molecule sequencing process comprises using a nanopore sensor.

7 . The method of claim 1 , wherein said read the comprises at least 1000 bases.

8 . The method of claim 2 , wherein said linking oligonucleotide comprises a registration sequence.

9 . The method of claim 2 wherein the linking oligonucleotide comprises a barcode sequence.

10 . The method of claim 1 wherein the first strand, the second strand, or both the first strand and the second strand comprise genomic DNA.

11 . The method of claim 1 wherein the first strand, the second strand, or both the first strand and the second strand comprise an amplification product.

12 . The method of claim 1 wherein the first strand, the second strand, or both the first strand and the second strand comprise a PCR amplification product.

13 . A method of obtaining a single molecule consensus sequence for a plurality of single template nucleic acid molecules, comprising:
performing a plurality of single-molecule sequencing processes on a plurality of single template molecules, wherein each single template molecule comprises i) a first strand having a 3′ end and a 5′ end, ii) a second strand, complementary to the first strand, having a 3′ end and a 5′ end, and iii) a linker that links the 3′ end of the first strand to the 5′ end of the second strand, thereby generating a sequence read from each single template molecule, the read for each single template molecule comprising a first sequence corresponding to the first strand, and a second sequence corresponding to the complementary second strand of each single template molecule; and comparing the first sequence and second sequence for each said sequence read to obtain a single molecule consensus sequence for each single template molecule.

14 . The method of claim 13 wherein each linker comprises a linking oligonucleotide.

15 . The method of claim 14 , wherein said single-molecule sequencing of the sequencing template includes sequencing of the linking oligonucleotide.

16 . The method of claim 13 , wherein said single-molecule sequencing processes comprises a sequencing by synthesis technology.

17 . The method of claim 16 , wherein said sequencing by synthesis technology comprises detecting incorporation of each nucleotide incorporated by a polymerase mediated, template dependent sequencing process.

18 . The method of claim 13 , wherein said single-molecule sequencing processes comprises using a nanopore sensor.

19 . The method of claim 13 , wherein said read comprises at least 1000 base pairs.

20 . The method of claim 14 , wherein said linking oligonucleotide comprises a registration sequence.

21 . The method of claim 14 wherein the linking oligonucleotide comprises a barcode sequence.

22 . The method of claim 13 wherein the first strand, the second strand, or both the first strand and the second strand of each template molecule comprise genomic DNA.

23 . The method of claim 13 wherein the first strand, the second strand, or both the first strand and the second strand of each template molecule comprise an amplification product.

24 . The method of claim 1 wherein the first strand, the second strand, or both the first strand and the second strand of each template molecule comprise a PCR amplification product.

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

• G16B30/00 Primary

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

• G16B30/10

  Sequence alignment; Homology search · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title

• C12Q1/6806

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