Random nucleotide mutation for nucleotide template counting and assembly

Patent metadata

Field	Value
Publication number	US-12516367-B2
Application number	US-202117320634-A
Country	US
Kind code	B2
Filing date	May 14, 2021
Priority date	Oct 10, 2014
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

This invention provides a method for sequencing a nucleic acid molecule (NAM) that comprises two or more segments having substantially the same sequence, and that has a length of more than one sequencing read, comprising i) obtaining two more copies of the NAM; ii) subjecting each copy of the NAM in step i) to a mutagenesis that mutates only select nucleic acid positions in the NAMs at a rate of 10% to 90% to produce mutated copies of the NAM (mcNAM); iii) amplifying each of the mcNAMs; iv) obtaining composite sequences of the mcNAMs that are produced by assembling sequence reads of the amplified mcNAMs, such that when taken together, span as much as possible of the entire length of the NAM, thereby sequencing the NAM.

First claim

What is claimed:

1 . A method for sequencing a nucleic acid molecule (NAM) that comprises two or more segments having substantially the same sequence, and that has a length of more than one sequencing read, comprising
i) obtaining two or more copies of the NAM; ii) subjecting each copy of the NAM in step i) to a mutagenesis that mutates only select nucleic acid positions in the NAMs at a rate of 10% to 90% to produce mutated copies of the NAM (mcNAM); iii) amplifying each of the mcNAMs; iv) obtaining composite sequences of the mcNAMs that are produced by assembling sequence reads of the amplified mcNAMs, such that when taken together, span as much as possible of the entire length of the NAM, by
a) aligning the sequence reads according to matching mutation patterns in overlaps of the sequence reads by a scoring method that assigns a weight to the overlaps in the sequence reads that reflects how unlikely it is that the overlaps agree on their mutation patterns by chance, and aligning the sequence reads by selecting overlaps that have the highest assigned weight, and
b) mapping the composite sequences,

thereby sequencing the NAM.

2 . The method of claim 1 , wherein each of the two or more copies of the NAM has a unique primer at its 5′ end and another unique primer at its 3′ end.

3 . The method of claim 1 , wherein the mutagenesis is by cytosine deamination.

4 . The method of claim 1 , wherein each mutable position of the NAMs comprises a cytosine (C),
wherein the cytosine (C) is mutated to a uracil (U) or a thymine (T).

5 . The method of claim 1 , further comprising the step of tagging each NAM or copy thereof.

6 . The method of claim 1 , wherein the NAM is within a mixture of DNA or RNA extracted from a cell.

7 . The method of claim 1 , wherein the NAM is a DNA molecule.

8 . The method of claim 2 , wherein the unique primers of each mcNAM lack a nucleotide that is mutable by the mutagenesis.

9 . The method of claim 1 , wherein the mutagenesis is performed after binding the NAM to a bead or surface.

10 . The method of claim 3 , wherein the cytosine deamination is induced by a bisulfite or a salt thereof.

11 . The method of claim 5 , wherein the tag lacks a nucleotide that is mutable by the mutagenesis.

12 . The method of claim 6 , wherein the DNA or RNA extracted from the cell has been fragmented.

13 . The method of claim 1 , wherein the rate of mutagenizing each mutable position of the NAMs is 25% to 75%.

14 . The method of claim 1 , wherein the rate of mutagenizing each mutable position of the NAMs is 40% to 60%.

15 . The method of claim 1 , wherein the rate of mutagenizing each mutable position of the NAMs is 50%.

Assignees

• Wigler Michael
• Levy Dan
• Cold Spring Harbor Laboratory

Inventors

• Wigler Michael
• Levy Dan

Classifications

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q2531/113

  PCR · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title

• C12Q2521/539

  Deaminase · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title