Random nucleotide mutation for nucleotide template counting and assembly
AssigneeCold Spring Harbor Laboratory
InventorsMichael Wigler, Dan Levy
Life of this patent
Estimating the term…
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.
Claims23
1-7. (canceled)
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, and
- b) mapping the composite sequences,
- thereby sequencing the NAM
10-14. (canceled)
(canceled)
21-22. (canceled)
A kit for determining the number of NAMs in a group of NAMs comprising:
- a) a mutagen;
- and
- b) instructions for performing mutagenesis resulting in suboptimal mutagenesis,
- wherein the mutagen is a bisulfite or a salt thereof, or a deamination agent.
24-27. (canceled)
A composition of matter comprising two or more copies of a nucleic acid molecule (NAM) comprising two or more segments having substantially the same sequence, and that has a length of more than one sequencing read, wherein each copy of the NAM has a unique primer at its 5′ end and another unique primer at its 3′ end, and is subjected to a mutagenesis that mutates each mutable position in the NAMs at a rate of 10% to 90% to produce mutated copies of the NAM (mcNAM), wherein the unique primers of each mcNAM lack a nucleotide that is mutable by the mutagenesis.
29-30. (canceled)
Dependencies are inferred from the claim text.