Methods and compositions for amplifying methylated target dna molecules
AssigneeNatera, Inc.
InventorsTing Wang, Trupti KAWLI, Ryan SWENERTON, Bernhard ZIMMERMANN
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Abstract
The present disclosure provides methods and compositions for preparing DNA molecules useful for determining a methylation status of selected regions of the DNA molecules. The methods utilize methylation sensitive restriction enzymes, and typically include one or more amplifications or a selective enrichment of target regions.
Claims129
A method for preparing deoxyribonucleic acid (DNA) molecules useful for determining a methylation status of a genomic region of interest, said method comprising:
- ligating adapters to sample DNA molecules obtained or derived from a first sample from a first subject, thereby forming a plurality of adapted DNA molecules comprising adapted DNA molecules having methylation sensitive restriction enzyme (MSRE) recognition sites;
- contacting the plurality of adapted DNA molecules with one or more MSREs, thereby generating MSRE-exposed, adapted DNA molecules, wherein at least a first plurality of the MSRE-exposed, adapted DNA molecules having one or more unmethylated MSRE recognition sites are cleaved by at least one of the MSREs, and wherein a second plurality of MSRE-exposed adapted DNA molecules are uncleaved by any of the MSREs, thus forming MSRE-exposed, uncleaved adapted DNA molecules;
- and
- performing one or more amplification reactions to amplify one or more target regions from the MSRE-exposed, uncleaved adapted DNA molecules or copies thereof, wherein target region amplicons are formed if the one or more target regions are present in the MSRE-exposed, uncleaved adapted DNA molecules.
A method for preparing DNA molecules useful for determining a methylation status of selected regions of the DNA molecules, comprising:
- ligating adapters to circulating free DNA (cfDNA) obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNA comprising adapted cfDNA having MSRE recognition sites;
- contacting the plurality of adapted cfDNA with one or more MSREs, thereby generating MSRE-exposed, adapted cfDNA, wherein a plurality of the MSRE-exposed, adapted cfDNA are not cleaved by the one or more MSREs, thereby forming MSRE-exposed, uncleaved, adapted cfDNA;
- performing a first amplification step wherein at least a plurality of the MSRE-exposed, uncleaved, adapted cfDNA are amplified;
- performing a second amplification step to amplify a set of target regions from the amplified MSRE-exposed, uncleaved, adapted cfDNA, each target region having one or more MSRE recognition sites, wherein a target region of the set is amplified and thereby generating target region amplicons if the target region is present in one or more of the amplified MSRE-exposed, uncleaved, adapted cfDNA;
- and
- quantifying an amount for at least some of the target region amplicons of the set by performing a next-generation sequencing reaction on clonally amplified target region amplicons, or amplicons derived therefrom.
A method according to 19, wherein the first amplification step comprises one or more PCRs using universal primers.
A method according to 20, wherein the second amplification step comprises one or more PCRs using a set of primer pairs, wherein each pair of the set is designed to amplify one target region having one or more MSRE recognition sites, and wherein at least one primer of each primer pair is a target-specific primer.
A method according to 20, wherein the second amplification step comprises one or more LTC reactions using a set of probe-dependent primer pairs, wherein each pair of the set is designed to amplify one target region having one or more MSRE recognition sites.
A method for preparing DNA molecules useful for determining a methylation status of selected regions of the DNA molecules, comprising:
- ligating adapters to circulating free DNA (cfDNA) obtained or derived from a first liquid sample, thereby forming a plurality of adapted cfDNA comprising adapted cfDNA having MSRE recognition sites;
- contacting the plurality of adapted cfDNA with one or more MSREs, thereby generating MSRE-exposed, adapted cfDNA, wherein a plurality of the MSRE-exposed, adapted cfDNA are not cleaved by the one or more MSREs, thereby forming MSRE-exposed, uncleaved, adapted cfDNA;
- amplifying at least a plurality of the MSRE-exposed, uncleaved, adapted cDNA;
- selectively enriching subsets of amplified MSRE-exposed, uncleaved, adapted cfDNA having one or more target regions using a set of hybrid capture probes, wherein each target region comprises one or more MSRE recognition sites, and wherein each probe of the set is designed to hybridize to one target region;
- and
- quantifying an amount for at least some of the enriched subsets by performing a next-generation sequencing reaction on clonally amplified enriched subsets, or amplicons derived therefrom.
A method according to 23, wherein the amplifying comprises one or more PCRs using universal primers.
The method of 1 or 2, wherein the first sample is a blood sample or a derivative sample thereof.
The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 70 and 500 base pairs in length.
The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 100 and 200 base pairs in length.
The method of any one of the preceding claims, wherein the method further comprises enriching the sample DNA molecules for DNA molecules that are between 130 and 170 base pairs in length.
The method of any one of the preceding claims, wherein the sample DNA molecules or the fragmented DNA molecules, are exposed to one or more polymerases and/or kinases to generate the nucleic acid derivatives generated therefrom.
The method of any one of preceding claims, wherein the ligating comprises ligating adapters to the nucleic acid derivatives generated therefrom.
The method of any one of the preceding claims, wherein the adapters are Y adapters.
The method of any one of the preceding claims, wherein the adapters each comprise a universal priming site.
The method of any one of the preceding claims, wherein the adapters do not include any MSRE recognition site.
The method of any one of the preceding claims, wherein the one or more MSREs are selected from one or more of HpaII, HhaI, HpyCH41V, or BstU1.
The method of any one of the preceding claims, wherein the contacting comprises contacting with two or more MSREs.
The method of any one of the preceding claims, wherein the contacting comprises contacting with three or more MSREs.
The method of any one of the preceding claims, wherein the contacting comprises contacting with four or more MSREs.
The method of any one of the preceding claims, wherein:
- the method further comprises performing the method on a second sample or a second liquid sample, and
- determining the methylation status of the set of target regions in the first sample further comprises comparing the amount of target region amplicons derived from the first sample to the amount of target region amplicons derived from the second sample.
The method of any one of the preceding claims, wherein the method is capable of detecting fully methylated DNA molecules present at 1.0% (by mass) or less in a mixture of DNA molecules.
The method of any one of the preceding claims, wherein the method is capable of differentiating samples having 1.0% (by mass) or less fully methylated DNA molecules from samples having 0% of the fully methylated DNA molecules.
The method of any one of the preceding claims, wherein the method is capable of differentiating samples having 0.1% (by mass) or less fully methylated DNA molecules from samples having 0% of the fully methylated DNA molecules.
The method of any one of the preceding claims, wherein the method is capable of differentiating samples having 0.05% (by mass) or less fully methylated DNA molecules from samples having 0% of the fully methylated DNA molecules.
The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present in 1% or less of the total circulating free DNA (cfDNA) in the sample.
The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present in 0.1% or less of the total circulating free DNA (cfDNA) in the sample.
The method of any one of the preceding claims, wherein circulating tumor DNA (ctDNA) is present in 0.01% or less of the total circulating free DNA (cfDNA) in the sample.
A method according to any one of the preceding claims, wherein the adapters each further comprise a molecular barcode.
The method of any one of the preceding claims, wherein the sample DNA obtained or derived from the first sample comprises a mixture of hypermethylated DNA and hypomethylated DNA.
Dependencies are inferred from the claim text.