Methods for accurate sequence data and modified base position determination

• US 10,515,714 B2
• Filed: 07/25/2017
• Issued: 12/24/2019
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: Industrial Technology Research Institute
• Original Assignee: Industrial Technology Research Institute
• Inventors: Pan, Chao-Chi, Fann, Jenn-Yeh, Chiou, Chung-Fan, Chien, Hung-Chi, Chen, Hui-Ling
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/659,585
• Publication Number:
  US 20180018425A1
• Time in Patent Office: 882 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

Abstract

Disclosed herein are methods of determining the sequence and/or positions of modified bases in a nucleic acid sample present in a circular molecule with a nucleic acid insert of known sequence comprising obtaining sequence data of at least two insert-sample units. In some embodiments, the methods comprise obtaining sequence data using circular pair-locked molecules. In some embodiments, the methods comprise calculating scores of sequences of the nucleic acid inserts by comparing the sequences to the known sequence of the nucleic acid insert, and accepting or rejecting repeats of the sequence of the nucleic acid sample according to the scores of one or both of the sequences of the inserts immediately upstream or downstream of the repeats of the sequence of the nucleic acid sample.

Claims

1. A method of determining a sequence of a double-stranded nucleic acid sample and a position of at least one modified base in the sequence, comprising:

a. altering the base-pairing specificity of bases of a specific type in a linear pair-locked molecule, the linear pair-locked molecule comprising forward and reverse strands of the double-stranded nucleic acid sample locked together by at least one nucleic acid insert having a known sequence and immediately upstream or downstream of the sample, to produce an altered linear pair-locked molecule;

b. obtaining the sequence data of the altered linear pair-locked molecule, wherein the sequence data comprise at least two copies of the sequence of the altered linear pair-locked molecule;

c. calculating scores of the sequences of at least four inserts contained in the sequence data by comparing the sequences of the at least four inserts to the known sequences of the inserts;

d. accepting or rejecting at least four of the repeats of the sequence of the nucleic acid sample contained in the sequence data according to the scores of at least one of the sequences of the inserts, subject to the condition that at least one sample sequence in each orientation is accepted;

e. compiling an accepted sequence set comprising the at least one sample sequence in each orientation accepted in step (d); and

f. determining the sequence of the double-stranded nucleic acid sample and the position of the at least one modified base in the sequence of the double-stranded nucleic acid sample by comparing the sequences of the forward and reverse strands of the linear pair-locked molecule, using the accepted sequence set.

2. The method of claim 1, further comprising obtaining sequence data of the linear pair-locked molecule via single molecule sequencing, wherein the sequence data comprise sequences of the forward and reverse strands of the linear pair-locked molecule.

3. The method of claim 1, wherein altering the base-pairing specificity of bases of a specific type in the circular pair-locked molecule comprises bisulfite treatment.

4. The method of claim 1, wherein altering the base-pairing specificity of bases of a specific type in the circular pair-locked molecule comprises photochemical transition.

5. The method of claim 1, wherein the nucleic acid insert forms a hairpin with an overhang, and the nucleic acid sample has an overhang compatible with the overhang of the nucleic acid insert.