Methods of determining nucleic acid structural information
AssigneeBionano Genomics, Inc.
InventorsMichael G. Saghbini, Henry B. Sadowski, Goran Pljevaljcic, Alex R. Hastie, Han Cao
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Abstract
Methods of double-stranded nucleic acid sequence determination and assembly that are able to identify insertions, deletions, repeat region sizes and genomic rearrangements, for example, are disclosed herein, which can use relatively large labeled nucleic acid fragments to analyze the structure of even larger genetic regions. In some embodiments these methods involve the use of certain parameters which unexpectedly improve overall method performance. In some embodiments these methods involve sample labeling that does not result in the formation of single-stranded nucleic acid fragment labeling intermediaries.
Claims23
A method of analyzing nucleic acid molecules without compromising strand phosphodiester bond integrity, comprising:
- providing a plurality of nucleic acid molecules comprising a plurality of occurrences of a DNA repeated motif, wherein an input coverage is 50-fold or less, wherein the DNA repeated motif comprises a methyltransferase recognition sequence and the DNA repeated motif is selected to have an average repeat frequency of about 5 sites to about 35 sites per 100 Kb in the plurality of nucleic acid molecules;
- covalently labeling the plurality of nucleic acid molecules in a sequence-specific fashion at the DNA repeated motif with a label, wherein the covalent labeling is effected with a methyltransferase in the presence of a modified cofactor, and wherein the modified cofactor comprises modified S-adenosyl methionine (SAM), and wherein the modified cofactor comprises a tag that is detectable, transferable, or both;
- and
- detecting the label to identify patterns of the DNA repeated motif of the plurality of nucleic acid molecules,
- wherein the covalently labeled nucleic acid molecules have a length of at least 150 kb and wherein the strand phosphodiester bonds of the plurality of nucleic acid molecules are intact.
A method of analyzing nucleic acid molecules without compromising strand phosphodiester bond integrity, comprising:
- providing a plurality of nucleic acid molecules comprising a plurality of occurrences of a first DNA repeated motif and a second DNA repeated motif, wherein an input coverage is 50-fold or less, wherein the first DNA repeated motif, the second DNA repeated motif, or both have an average repeat frequency of about 5 sites to about 35 sites per 100 Kb in the plurality of nucleic acid molecules;
- covalently labeling the plurality of nucleic acid molecules in a sequence-specific and CpG-methylation-insensitive fashion at the first DNA repeated motif with a first label, wherein the first DNA repeated motif comprises a first methyltransferase recognition sequence and the covalent labeling is effected with a CpG-methylation-insensitive methyltransferase in the presence of a first modified cofactor,
- covalently labeling the plurality of nucleic acid molecules in a sequence-specific and CpG-methylation-dependent fashion at the second DNA repeated motif with a second label, wherein the second DNA repeated motif comprises a second methyltransferase recognition sequence and the covalent labeling is effected with a CpG-methylation-dependent methyltransferase in the presence of a second modified cofactor;
- and
- detecting the first label and the second label to identify patterns of the first DNA repeated motif and the second DNA repeated motif of the plurality of nucleic acid molecules, respectively,
- wherein the covalently labeled nucleic acid molecules have a length of at least 150 kb and the strand phosphodiester bonds of the plurality of nucleic acid molecules are intact.
Dependencies are inferred from the claim text.