Random nucleotide mutation for nucleotide template counting and assembly

Patent metadata

Field	Value
Publication number	US-11008606-B2
Application number	US-201515515913-A
Country	US
Kind code	B2
Filing date	Oct 9, 2015
Priority date	Oct 10, 2014
Publication date	May 18, 2021
Grant date	May 18, 2021

Abstract

A method for determining the number of nucleic acid molecules (NAMs) in a group of NAMs, comprising
i) obtaining an amplified and mutagenized group of NAMs that was produced by
a. subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90% thus forming a group of mutagenized NAMs (mNAMs), and b. amplifying the group of mNAMs;
ii) obtaining sequences of the mNAMs in the group of amplified mNAMs; and iii) counting the number of different sequences obtained in step (ii) to determine the number of unique mNAMs in the group of mNAMS,

thereby determining the number of NAMs in the group of NAMs.

First claim

What is claimed:

1. A method for determining the number of nucleic acid molecules (NAMs) in a group of NAMs, comprising
i) obtaining an amplified and mutagenized group of NAMs that was produced by
a. subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90% thus forming a group of mutagenized NAMs (mNAMs), and
b. amplifying the group of mNAMs;

ii) obtaining sequences of the mNAMs in the group of amplified mNAMs; and
iii) counting the number of different sequences obtained in step (ii) to determine the number of unique mNAMs in the group of mNAMS,
thereby determining the number of NAMs in the group of NAMs.

2. The method of claim 1 , wherein obtaining sequences in step (ii) comprises obtaining composite sequences produced by assembling sequence reads of the mNAMs by
a) aligning the sequence reads according to matching mutation patterns in overlaps of the sequence reads, thereby obtaining composite sequences, and
b) mapping the composite sequences; and
wherein step (iii) comprises counting the number of jointly overlapping different composite sequences obtained in step (ii).

3. The method of claim 1 , wherein a sub-group of NAMs in the group of NAMs is determined to have substantially the same nucleotide sequence,
wherein the sub-group of NAMs is determined to have nucleotide sequences that are at least 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.9, or 99.9% identical; or
wherein the nucleotide sequences of a sub-group of NAMs comprise a stretch of consecutive nucleotides having a sequence which includes at least two mutable positions and is i) identical to the sequence of a stretch of consecutive nucleotides within another NAM within the sub-group of NAMs, or ii) determined to have at least 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.9, or 99.9% identical to the sequence of a stretch of consecutive nucleotides within another NAM within the sub-group of NAMs.

4. The method of claim 1 , wherein the counting comprises counting the number of different sequences that are determined to have substantially the same sequence except for their mutable positions, thereby determining the number of NAMs in the group of NAMs that had substantially the same sequence; or
wherein the counting comprises counting the number of different sequences which lack substantially the same sequence in any stretch including at least two mutable positions, thereby determining the number of NAMs without substantially the same sequence in the group of NAMs.

5. The method of claim 1 , wherein
a) the mutagenesis is by cytosine deamination,
b) each mutable position of the NAMs comprises a cytosine and the cytosine (C) is mutated to a uracil (U) or a thymine (T),
c) each NAM in the group of NAMs has a unique primer at its 5′ end and another unique primer at its 3′ end,
d) the NAM is within a mixture of DNA or RNA extracted from a cell, wherein the DNA or RNA extracted from the cell has been fragmented,
e) the NAM is a DNA molecule, or an RNA molecule,
f) one or more NAMs in the group of NAMs has a length of one sequencing read length,
g) one or more NAMs in the group of NAMs has a length of two or more sequencing read lengths, and/or
h) the number of NAMs in the group of NAMs is about 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10-10000, wherein if the number of NAMs in the group of NAMs is greater than 10000, then diluting the group of NAMs.

6. The method of claim 1 , further comprising the step of tagging each NAM or copy thereof,
wherein the tag lacks a nucleotide that is mutable by the mutagenesis.

Assignees

• Wigler Michael
• Levy Dan
• Cold Spring Harbor Laboratory

Inventors

• Wigler Michael
• Levy Dan

Classifications

• C12Q1/6851

  Quantitative amplification · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• C12Q2531/113

  PCR · CPC title

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title