Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 21, 40, 47, 49, 50, 55, 58, 59, 62, 63, 65, 67, 70, 71, 76, 106, 107, 110, 112, 114, 117, 163 and/or 164 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.

Abstract: Provided herein, among other things, is a method for deaminating a double-stranded nucleic acid. In some embodiments, the method may comprise contacting a double-stranded DNA substrate that comprises cytosines and a double-stranded DNA deaminase having an amino acid sequence that is at least 80% identical to any of SEQ ID NOS: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 19, 24, 26, 27, 28, 33, 40, 49, 50, 63, 95, 96, 97, and/or 99 to produce a deamination product that comprises deaminated cytosines. Enzymes and kits for performing the method are also provided.

Abstract: A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.

Abstract: Among other things, a method for performing multiple enzyme reactions in a single tube is provided. In some embodiments, the method may comprise producing a reaction mix comprising a thermolabile UDG, an AP lyase and DNA fragments that comprise one or more uracil residues, incubating the reaction mix at a relatively low temperature to cleave fragments at the one or more uracil residues, raising the temperature of the reaction mix to a relatively high temperature to inactivate the thermolabile UDG; and deaminating the fragments, thereby converting any cytosine in the fragments of DNA to uracil.

Abstract: A method for identifying the location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and optionally reacting a second portion of the sample with a dioxygenase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase that is more efficient at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: Methods and compositions are provided for identifying any of the presence, location and phasing of methylated and/or hydroxymethylated cytosines in nucleic acids including long stretches of DNA. In some embodiments, the method may comprise reacting a first portion (aliquot) of a nucleic acid sample with a dioxygenase and optionally a glucosyltransferase in a reaction mixture containing the nucleic acid followed by a reaction with a cytidine deaminase to detect and optionally map 5mC in a DNA. Optionally, a second portion can be reacted with glucosyltransferase followed by reaction with a cytidine deaminase to detect and optionally map 5hmC in a DNA.

Abstract: Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.

Abstract: A method for identifying any of the presence, location and phasing of modified cytosines (C) in long stretches of nucleic acids is provided. In some embodiments, the method may comprise (a) reacting a first portion of a nucleic acid sample containing at least one C and/or at least one modified C with a DNA glucosyltransferase and a cytidine deaminase to produce a first product and/or reacting a second portion of the sample with a dioxygenase, optionally a DNA glucosyltransferase and a cytidine deaminase to produce a second product and; (b) comparing the sequences from the first and optionally the second product obtained in (a), or amplification products thereof, with each other and/or an untreated reference sequence to determine which Cs in the initial nucleic acid fragment are modified. A modified TET methylcytosine dioxygenase with improved efficiency compared to unmodified TET2 at converting methylcytosine to carboxymethylcytosine is also provided.

Abstract: 5-methylpyrimidine oxygenases and their use in the modification of nucleic acids are described.

Abstract: Compositions and methods are provided for discrimination between cytosine and modifications thereof using cytidine deaminases and/or oxygenases. Variants of wild type cytidine deaminases are described which show reduced bias with respect to adjacent nucleotides upstream of the cytosine. The methods provide a rapid and convenient use of enzymes to obtain methylomes.