Compositions and methods for detection of genomic variance and DNA methylation status
AssigneeSinglera Genomics, Inc.
InventorRui LIU
Life of this patent
Estimating the term…
Abstract
In one aspect, provided herein is an integrated method for simultaneous detection of both a genomic variance and quantification of a DNA methylation state/status on one or more (e.g., hundreds of thousands of) targets, without splitting the limited materials for two different workflows. The present disclosure relates to compositions, kus, devices, and methods for conducting genetic arid genomic analysis, for example, by polynucleotide sequencing in particular aspects, provided herein are compositions, kits, and methods for constructing libraries for simultaneous detection of genomic variants and DNA methylation status on limited DNA inputs, such as circulating polynucleotide fragments in the body of a subject, including circulating tumor DNA.
Claims37
A method for analyzing a first target polynucleotide sequence and a methylation status of a second target polynucleotide sequence in a sample, comprising:
- 1) contacting a sample comprising a polynucleotide with a methylation-sensitive restriction enzyme (MSRE), wherein the MSRE selectively cleaves the polynucleotide at a residue when it is unmethylated or selectively cleaves the polynucleotide at the residue when it is methylated;
- 2) subjecting the sample from step 1) to polynucleotide amplification, using a mixture of:
- i) a first primer set for amplifying a first target polynucleotide sequence in the sample, and
- ii) a second primer set for analyzing a methylation status of a second target polynucleotide sequence in the sample, wherein the methylation status is of a residue in the second target polynucleotide sequence, and one primer of the second primer set hybridizes to the uncleaved second target polynucleotide sequence and together with another primer in the set, amplifies the uncleaved sequence but not the second target polynucleotide sequence cleaved at the residue by the MSRE;
- and
- 3) sequencing polynucleotides amplified in step 2),
- wherein the first target polynucleotide sequence is analyzed using sequencing reads from the amplified first target polynucleotide sequence, and the methylation status of the residue of the second target polynucleotide sequence is analyzed by comparing the observed number of sequencing reads (No) from the amplified second target polynucleotide sequence to a reference number.
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A kit, comprising:
- a methylation-sensitive restriction enzyme (MSRE), wherein the MSRE selectively cleaves at a residue when it is unmethylated or selectively cleaves at the residue when it is methylated;
- a first primer set for amplifying a first target polynucleotide sequence in a sample;
- and/or
- a second primer set for analyzing a methylation status of a second target polynucleotide sequence in the sample, wherein the methylation status is of a residue in the second target polynucleotide sequence, and one primer of the second primer set hybridizes to the uncleaved second target polynucleotide sequence and together with another primer in the set, amplifies the uncleaved sequence but not the second target polynucleotide sequence cleaved at the residue by the MSRE.
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Dependencies are inferred from the claim text.