Methods of sequencing and identifying the position of a modified base in a nucleic acid

Patent metadata

Field	Value
Publication number	US-9951383-B2
Application number	US-201615271915-A
Country	US
Kind code	B2
Filing date	Sep 21, 2016
Priority date	Dec 11, 2008
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Methods are provided that include linking forward and reverse strands of a double stranded segment to form circular template molecules, and obtaining sequence data from the circular template molecule to identify modified bases, for example including the use of bisulfite treatment. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

First claim

What is claimed is:

1. A method of determining a sequence of a double-stranded nucleic acid and the position of at least one methylated cytosine in the sequence, comprising:
a) ligating forward and reverse strands of the double-stranded nucleic acid together to form a circular template molecule comprising a double-stranded segment having each end capped with a hairpin structure;
b) obtaining sequence data of the circular template molecule via single molecule sequencing, wherein the sequence data comprises sequences of the forward and reverse strands of the double-stranded nucleic acid contained in the circular template molecule; and
c) determining the position of the at least one methylated cytosine in the sequence of the double-stranded nucleic acid by comparing the sequences of the forward and reverse strands of the double-stranded nucleic acid contained in the circular template molecule, wherein at least one base in the circular template molecule is a uracil and wherein the at least one uracil is produced with bisulfite treatment prior to the single molecule sequencing.

2. The method of claim 1 , wherein the double-stranded nucleic acid comprises DNA.

3. The method of claim 1 , wherein the circular molecule comprises a DNA/RNA hybrid.

4. The method of claim 1 wherein single molecule sequencing comprises detecting the incorporation of nucleotides into a growing strand.

5. The method of claim 4 wherein the incorporation of nucleotides into a growing strand is detected in real time.

6. The method of claim 1 wherein the double-stranded nucleic acid has not been amplified.

7. The method of claim 1 wherein the double-stranded nucleic acid has been amplified using a maintenance methyltransferase.

8. The method of claim 1 , wherein the single molecule sequencing is carried out with a polymerase enzyme.

9. The method of claim 8 , wherein the polymerase enzyme is a DNA polymerase.

10. The method of claim 1 , wherein the double-stranded nucleic acid comprises RNA.

Assignees

• Pacific Biosciences California Inc

Inventors

• Turner Stephen
• Sorenson Jon
• Travers Kevin
• Heiner Cheryl

Classifications

• C12Q2527/113

  Time · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q2537/164

  Methylation detection other then bisulfite or methylation sensitive restriction endonucleases · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title