Rapid bisulfite conversion and recovery of nucleic acid molecules

Abstract

In various aspects, the present disclosure provides methods for the modification and purification of nucleic acid, e.g., DNA molecules, as well as methods for nucleic acid extraction for further processing or analysis. Also provided are methods for performing a bisulfite conversion reaction on nucleic acid molecules, and performing subsequent desulfonation and purification steps facilitated by a substrate (e.g., magnetic beads) for the purification and recovery of converted nucleic acid molecules. Methods for the separation of nucleic acids, e.g., DNA, from other components of a biological sample are also provided.

Description

Attorney Docket No.202323-624601 RAPID BISULFITE CONVERSION AND RECOVERY OF NUCLEIC ACID MOLECULES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 617,049, filed January 2, 2024, and U.S. Provisional Application No. 63 / 664,683, filed June 26, 2024, which applications are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0001] In various aspects, the presently disclosed subject matter is directed to the extraction, chemical modification, purification, and analysis of nucleic acids, such as DNA. In some embodiments, methods include bisulfite conversion of nucleic acids, e.g., DNA, and subsequent purification using a substrate (e.g., silica-coated magnetic beads) in the presence of a non- chaotropic-based or chaotropic-free binding buffer. BACKGROUND

[0002] DNA methylation plays a role in regulating gene expression. Aberrant DNA methylation has been implicated in many disease processes, including cancer. DNA methylation profiling using methylation sequencing (e.g., whole genome bisulfite sequencing (WGBS)) is increasingly recognized as a valuable diagnostic tool for detection, diagnosis, and / or monitoring of cancer. For example, specific patterns of differentially methylated regions may be useful as molecular markers for various disease states. Detecting and mapping sites of DNA methylation are important to understanding epigenetic gene regulation and providing diagnostic tools for identifying cancers and other disease states associated with changes in gene regulation.

[0003] Mapping methylation sites can be accomplished using, e.g., a bisulfite treatment process described by Frommer, et al. for the detection of 5-methylcytosines in DNA (Proc. Natl. Acad. Sci. USA 89: 1827-31 (1992). The bisulfite method of mapping 5-methylcytosines is based on the observation that cytosine, but not 5-methylcytosine, reacts with hydrogen sulfite ion (also known as bisulfite). Detection is possible because unmethylated cytosines are converted to uracil, and uracil forms base pairs with adenine (thus behaving like thymine), whereas 5-methylcytosine resists conversion by bisulfite treatment and continues to base pair with guanine. This makes the discrimination of methylated cytosines from non-methylated cytosines possible by, e.g., bisulfite genomic sequencing.Attorney Docket No.202323-624601

[0004] Past methods often do not provide accurate quantitative measures of DNA methylation. Additionally, past methods often require long times (e.g., 1-2 days) to complete (e.g., in part due to long incubation times) and do not provide an efficient conversion and recovery of the converted DNA. Methods employing spin columns are labor-intensive and are not readily amenable to automation.

[0005] Some methods for purifying nucleic acids use magnetic beads and use buffers with high concentrations of chaotropic salts to drive absorption of the nucleic acids to the beads. After washing to remove impurities, DNA is eluted from the surface using a high pH, low ionic strength buffer that is compatible with subsequent polymerase-based DNA amplification methods, such as the Polymerase Chain Reaction (PCR). However, residual chaotropic salts and ethanol carried over into the amplification reaction can inhibit polymerase amplification. SUMMARY

[0006] In view of the foregoing, there is a need for improved methods of analyzing methylation in nucleic acids, and particularly for the quantitative assessment of the methylation state of small amounts of DNA and amounts of DNA molecules 500 bp or less in length. Aspects of the present disclosure address this need, and provide other advantages as well.

[0007] In various aspects, the present disclosure provides methods for chemical modification and purification of nucleic acids, e.g., DNA. In some embodiments, the present disclosure provides methods for performing a bisulfite conversion reaction on DNA, performing the subsequent desulfonation and purification steps using a substrate (e.g., silica-coated magnetic beads), and recovering modified DNA from the surface. In various embodiments, the methods disclosed herein provide for the bisulfite conversion of DNA and for subsequent purification of modified DNA using a substrate (e.g., silica-coated magnetic beads) in the presence of a non- chaotropic-based or chaotropic-free binding buffer. In some embodiments, the disclosed methods also include nucleic acid extraction which provides separation of nucleic acids, e.g., DNA, from other components of a biological sample (e.g., a blood sample) using a non-chaotropic-based or chaotropic-free binding buffer.

[0008] In various aspects, the present disclosure is directed to methods for the modification and purification of DNA molecules. In some embodiments, the methods disclosed herein provide for performing a bisulfite conversion reaction on DNA molecules (e.g., single-stranded DNA,Attorney Docket No.202323-624601 “ssDNA”) and performing subsequent desulfonation and purification steps using a substrate (e.g., magnetic beads) for efficient purification and recovery of the converted DNA molecules. In one embodiment, the methods use silica-coated magnetic beads and a non-chaotropic or chaotropic- free binding buffer. In another embodiment, the methods disclosed herein provide for bisulfite conversion and purification of small DNA molecules (e.g., 500 bp or less in length, 400 bp or less in length, 300 bp or less in length, or 200 bp or less in length). Such DNA molecules can be cell free DNA (“cfDNA”) molecules. In various aspects, the subject methods can also be applied using nucleic acids other than DNA, e.g., RNA.

[0009] In accordance with some embodiments, the DNA molecules are subject to bisulfite conversion, e.g., by reaction with a sulfonation reagent such as ammonium hydrogen sulfite (i.e., ammonium bisulfite), sodium hydrogen sulfite (i.e., sodium bisulfite), or by using a commercial kit. In some embodiments, a high concentration (e.g., greater than 45% solution) of ammonium bisulfite is used as a sulfonation reagent, allowing for faster conversion of DNA molecules. In various embodiments, the bisulfite-converted DNA is then added to a binding mixture including a non-chaotropic based or chaotropic-free binding buffer and a substrate (e.g., one or more magnetic beads), and then incubated to bind the DNA molecules to the substrate. In some embodiments, the substrate washing and DNA binding steps are combined in a single step in which an excess amount of binding buffer is added to the substrate followed by addition of the bisulfite-converted DNA. After binding, the binding solution is removed, the substrate is washed, and a desulfonation buffer (e.g., a sodium carbonate / bicarbonate based desulfonation buffer) is added. After desulfonation, the desulfonation buffer is removed, the substrate washed, and the converted DNA molecules eluted using an appropriate elution buffer (e.g., Tris-HCl).

[0010] The presently disclosed subject matter provides methods for treating DNA including contacting a DNA with a bisulfite reagent and binding the DNA to a substrate (e.g., a magnetic bead) by using, e.g., performing in the presence of, a non-chaotropic (or chaotropic-free) binding buffer. Some embodiments provide additional steps, e.g., washing the DNA, e.g., DNA bound to one or more magnetic beads, with a wash buffer. Additional embodiments further provide methods including contacting the DNA with a desulfonation reagent, washing the DNA, e.g., DNA bound to one or more magnetic beads, with a wash buffer, and eluting the DNA with an elution buffer to remove the DNA from the substrate and produce a sample including bisulfite converted and purified DNA molecules for subsequent analysis or implementation.

[0011] One aspect of the present disclosure relates to the bisulfite conversion of DNA fragments, e.g., small DNAs of approximately 500 base pairs or less in length. Accordingly, inAttorney Docket No.202323-624601 some embodiments the DNA subject to bisulfite treatment comprises or consists of a population of DNA strands of 500 or fewer, 400 or fewer, 300 or fewer, or 200 or fewer nucleotides in length and / or cfDNA. In some embodiments the DNA is single stranded. In some aspects, the present disclosure provides for the efficient processing and recovery of DNA, e.g., to provide an improved quantitative measure of cytosine methylation in a sample following a bisulfite reaction. In some embodiments, an amount of treated and recovered DNA in an analytical sample is at least 25%, 30%, 40%, or more (e.g.50% or more) of the amount of DNA in the contacting step that would be expected if recovery were 100% efficient (e.g., after accounting for any requisite concentration or dilution factors). As a method to treat DNA with bisulfite to convert unmethylated cytosines, but not methylcytosines, to uracil, some embodiments provide that an unmethylated cytosine, if present in the DNA, is converted to a uracil. In addition, some embodiments provide that a methylcytosine, if present in the DNA, is not converted to a uracil. The presently disclosed methods are not limited in the types of substrates (e.g., magnetic beads) that are used herein for illustrative purposes, as other suitable substrates may be used. In some embodiments the magnetic beads are silica-coated magnetic bead, and in some embodiments the bead has a diameter of approximately 1 µm, 1 µm or less, or 1.5 µm or less.

[0012] In one aspect, the disclosed subject matter is directed to a method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method including: (a) contacting DNA molecules with a sulfonation reagent to produce sulfonated DNA, wherein the sulfonated DNA includes one or more sulfonated deaminated cytosines; (b) binding the sulfonated DNA to a substrate in a non-chaotropic based binding buffer; optionally wherein the substrate comprises (i) a surface comprising silica, and / or (ii) a magnetic bead; (c) contacting the substrate- bound DNA molecules with a desulfonation reagent; and (d) eluting the DNA molecules from the substrate with an elution buffer.

[0013] In one aspect, the presently disclosed subject matter is directed to a method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method including: (a) producing a solution including single-stranded DNA molecules (ssDNA), such as by adding NaOH to a sample solution including DNA; (b) to the solution including ssDNA, adding a sulfonation reagent to produce a mixture including sulfonated ssDNA molecules, wherein the sulfonated ssDNA molecules include one or more sulfonated deaminated cytosines; (c) combining the sulfonated ssDNA molecules with a substrate and a chaotropic-free binding buffer to produce substrate-bound sulfonated ssDNA molecules; optionally wherein the substrate comprises silica- coated magnetic beads; (d) collecting substrate-bound ssDNA molecules from the chaotropic-free binding buffer, and contacting the collected bead-bound sulfonated ssDNA molecules with aAttorney Docket No.202323-624601 desulfonation reagent to produce substrate-bound converted ssDNA molecules; and (e) eluting converted ssDNA molecules to provide a sample including converted ssDNA molecules, or any combination thereof.

[0014] In one aspect, the present subject matter is directed to a method for bisulfite-converting small DNA molecules, the method including: (a) in a solution combining small DNA molecules with a sulfonation reagent to produce sulfonated small DNA molecules, wherein said small DNA molecules include 300 or fewer bases in length; (b) combining the sulfonated small DNA molecules in the solution of step (a) with a substrate and a non-chaotropic based binding buffer to produce substrate-bound sulfonated small DNA molecules; optionally wherein the substrate comprises silica-coated magnetic beads; (c) collecting substrate-bound sulfonated small DNA molecules from the non-chaotropic binding buffer and contacting the collected substrate-bound sulfonated small DNA molecules with a desulfonation reagent to produce substrate-bound converted small DNA; and (d) eluting converted small DNA molecules to provide a sample including converted small DNA molecules, or any combination thereof.

[0015] In some embodiments, the ssDNA molecules are small ssDNA molecules including 300 bp or less in length.

[0016] In some embodiments, the DNA molecules are denatured to produce single-strand DNA molecules prior to step (a), and wherein the denaturing comprises heat denaturing and / or denaturing using sodium hydroxide (NaOH).

[0017] In one embodiment, the sulfonation reagent comprises from about 45% to about 85% ammonium bisulfite, and optionally from about 0.005 mM to about 1 mM EDTA. In another embodiment, the concentrated sulfonation reagent comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M. In some embodiments, the sulfonation reagent comprises metabisulfite. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0018] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer includes: a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate- KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer, or any combination thereof.Attorney Docket No.202323-624601

[0019] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer comprises KCl at a final concentration ranging from about 1 M to about 4 M (e.g., 2 M to 4 M). In one embodiment, the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M (e.g., 2.7 M to 3.3 M KCl). In some embodiments, the binding buffer comprises KCl at a final concentration of about 3 M.

[0020] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer is a glycine-KCl buffer, and the glycine in the glycine-KCl buffer is at a concentration ranging from about 0.05 M to about 2 M.

[0021] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer is potassium acetate-KCl buffer, and the potassium acetate in the potassium acetate- KCl buffer is at a concentration ranging from about 0.05 M to about 2 M.

[0022] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer is a sodium citrate-KCl buffer, and the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from about 0.05 M to about 2 M.

[0023] In various embodiments, a non-chaotropic buffer component such as the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer, or potassium acetate-KCl buffer has a concentration ranging, for example, from: 0.05 M to 4 M, 0.05 M to 3 M, 0.1 M to 4 M, 0.15 M to 4 M, 0.2 M to 4 M, 0.4 M to 4 M, 0.5 M to 4 M, 0.15 M to 4 M, 0.15 M to 3 M, 0.15 M to 2 M, 0.15 M to 1 M, 0.15 M to 0.5 M, 0.15 M to 0.45 M, 0.35 M to 4 M, or 0.2 M to 0.4 M. In some embodiments, the KCl is at a concentration ranging from 0.05 M to 4 M. In some embodiments, any non-chaotropic based binding buffer or chaotropic-free binding buffer or component thereof, e.g., KCl, described herein can have such a concentration.

[0024] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and the Tris acetate in the Tris acetate- ammonium sulfate buffer can have a concentration ranging from about 0.05 M to about 2 M. In various embodiments, a non-chaotropic based binding buffer or chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, wherein a component, e.g., the Tris acetate, in the Tris acetate-ammonium sulfate buffer can have a concentration ranging, for example, from: .01 M to 5 M, .05 M to 4 M, .05 M to 3 M, .1 M to 4 M, .2 M to 4 M, .4 M to 4 M, .5 M to 4 M, .15 M to 4 M, .15 M to 5 M, .15 M to 3 M, .2 M to 3 M, .35 M to 4 M, .15 M to 4 M, .15 M to 5 M, .15 M to 3 M, .15 M to 2 M, .15 M to 1 M, .15 M to .5 M, .15 M to .45 M, .35 M to 4 M, .2 M to .4 M, or .5 M to 5 M. In some embodiments, the Tris acetate in the Tris acetate-ammonium sulfateAttorney Docket No.202323-624601 buffer comprises a concentration ranging from 0.05 M to 2 M. In some embodiments of ammonium sulfate buffers, ammonium sulfate can have any of the same concentrations in the buffer as those above for Tris acetate. In another embodiment, the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and the ammonium sulfate in the Tris acetate- ammonium sulfate buffer is present at a concentration ranging from about 0.05 M to about 4 M. In some embodiments, the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and the ammonium sulfate in the buffer can have a concentration ranging, for example, from: 0.01 M to 5 M, 0.05 M to 4 M, 0.05 M to 3 M, 0.1 M to 4 M, 0.2 M to 4 M, 0.4 M to 4 M, 0.5 M to 4 M, 0.15 M to 4 M, 0.15 M to 5 M, 0.15 M to 3 M, 0.2 M to 3 M, 0.35 M to 4 M, or 0.5 M to 5 M.

[0025] In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer includes any one or combination of: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; and / or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate. In various embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer has a pH ranging from 2 to 7, such as 2.5 to 7, such as 3 to 7, such as 3.5 to 7, or such as 4 to 7, such as 2 to 6, such as 2.5 to 6, such as 3 to 6, such as 3.5 to 6, such as 4 to 6, or such as 2 to 5, such as 2.5 to 5, such as 3 to 5, such as 3.5 to 5, such as 4 to 5. In some embodiments, the pH ranges from 3.5 to 7.

[0026] In some embodiments, the desulfonation reagent includes ethanol, isopropanol, NaOH, sodium carbonate / sodium bicarbonate and / or boric acid, or any combination thereof. In various embodiments, the desulfonation reagent comprises (i) sodium carbonate and bicarbonate, or (ii) boric acid, but not both.

[0027] In some embodiments, the desulfonation reagent comprises a percentage, e.g., (i) 30% or less, 40% or less, 50%, or less , 60% or less, or 70% or less, or (ii) 30% or more, 40% or more, 50%, or more, 60% or more, or 70% or more, or (iii) from 40% to 60%, from 35% to 65%, or from 30% to 70% ethanol and / or NaOH / HCl, wherein the NaOH includes from 25 mM to 100 mM NaOH and / or 25 mM to 100 mM HCl. In some versions the NaOH has a concentration ranging from 20 mM to 100 mM and the HCl has a concentration ranging from 20 mM to 100 mM. In another embodiment, the desulfonation reagent includes a percentage (e.g., (i) 30% or less, 40% or less, 50%, or less , 60% or less or 70% or less, or (ii) 30% or more, 40% or more, 50%, or more, 60% or more or 70% or more, or (iii) from 40% to 60%, or from 35% to 65%, orAttorney Docket No.202323-624601 from 30% to 70%) ethanol and / or from 40% to 60% of a mixture including sodium carbonate, sodium bicarbonate / carbonate, and / or NaOH / HCl. In some versions of the subject embodiments, the desulfonation reagent is a sodium carbonate / bicarbonate reagent and includes, for example, components which may be ethanol, and sodium carbonate and / or sodium bicarbonate, or any combination thereof. In some versions of the embodiments, the desulfonation reagent does not include NaOH, HCl, or both NaOH and HCl. In various aspects, the desulfonation reagent includes sodium carbonate having a concentration ranging from 25 mM to 50 mM and / or sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

[0028] In some embodiments, a sodium carbonate / bicarbonate reagent includes a percentage, e.g., 30%, 40%, or 50 of ethanol. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.107 M, 0.0201 M, 0.0383 M, 0.480 M, or 0.501 M sodium carbonate, 0.0101 M, 0.0115 M, 0.012 M, 0.0134 M sodium bicarbonate, 0.0301 M, 0.0337 M, 0.0384 M, 0.0422 M, or 0.0489 M NaOH and / or a concentration, 0.0311 M, 0.037 M, or 0.0421 M HCl. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.107 M or less, 0.0201 M or less, 0.0383 M or less, 0.0480 M or less, 0.480 M or less, or 0.501 M or less, 0.0101 M or less, 0.0115 M or less, 0.012 M or less, 0.0134 M or less, 0.0301 M or less, 0.0337 M or less, 0.0384 M or less, 0.0422 M or less, or 0.0489 M or less sodium carbonate and / or sodium bicoarbonate and / or NaOH and / or a concentration, 0.0311 M or less, 0.037 M or less, or 0.0421 M or less HCl. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.107 M or more, 0.0201 M or more, 0.0383 M or more, 0.0480 M or more, 0.480 M or more, or 0.501 M or more, 0.0101 M or more, 0.0115 M or more, 0.012 M or more, 0.0134 M or more, 0.0301 M or more, 0.0337 M or more, 0.0384 M or more, 0.0422 M or more, or 0.0489 M or more sodium carbonate or NaOH and / or a concentration, 0.0311 M or more, 0.037 M or more, or 0.0421 M or more HCl. In various embodiments, the desulfonation reagent has three components: sodium carbonate, sodium bicarbonate and ethanol, e.g., 40% ethanol. In various embodiments, the desulfonation reagent includes sodium carbonate at a concentration ranging, for example, from .03 M to .05 M, .03 M to .04 M, .035 M to .04M, .04 M to .05 M, or .045 M to .05M. In various embodiments, the desulfonation reagent includes sodium bicarbonate at a concentration ranging, for example, from .01 M to .02 M, .005 M to .015 M, .011 to .013 M, or .011 to .012 M. In some versions the sodium carbonate / bicarbonate reagent has a pH around 11, 11.2, 11.7, 12 or 12.3, or a pH of 8 or more, 8.5 or more or 9 or more, or a pH of 12 or less, 11 or less, 10 or less, or 9.5 or less. In various instances the desulfonation reagent has a pH ranging from 11 to 12, such as 11.1 to 12, such as 11.2 to 12, such as 11 to 11.5 or 11 to 11.8, or 11.5 to 11.8, or 11.5 to 12, or 11 to 11.3,Attorney Docket No.202323-624601 or 9 to 10 or 9 to 9.5, or 8.5 to 9.5. In some versions, the methods include adjusting the pH of the solution by adjusting the ratio of sodium carbonate and / or sodium bicarbonate, such as from 5:5, 9:1, 10:0, 10:1, 8:2, 7:3 or 6:3, respectively. In various embodiments, the sodium carbonate / bicarbonate reagent does not include NaOH and / or HCl. In some variations, the sodium carbonate / bicarbonate reagent does not need a step of pH adjustment. In some variations, the sodium carbonate / bicarbonate reagent has only three components. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.010 M to 0.050 M, 0.030 to 0.045 M, or 0.035 to 0.040 M sodium carbonate; and / or 0.01 M to 0.02 M, 0.01 to 0.03 M, or 0.01 to 0.015 sodium bicarbonate; and / or 0.02 M to 0.05, 0.03 M to 0.04 M, or 0.035 M to 0.04 M HCl.

[0029] In some embodiments, the desulfonation reagent includes a percentage (e.g., (i) 30% or less, 40% or less, 50%, or less , 60% or less, or 70% or less, or (ii) 30% or more, 40% or more, 50%, or more, 60% or more, or 70% or more, or (iii) from 40% to 60%, 35% to 45% or from 35% to 65%) of an alcohol such as isopropanol or ethanol and / or a percentage (e.g., (i) 30% or less, 40% or less, 50%, or less , 60% or less, or 70% or less, or (ii) 30% or more, 40% or more, 50%, or more, 60% or more, or 70% or more, or (iii) from 35% to 65% or from 40% to 60%) of boric acid or a mixture including boric acid, NaOH and / or KCl. In some versions of the subject embodiments, the desulfonation reagent is a boric acid reagent and includes, for example, components which may be an alcohol, e.g., isopropanol or ethanol, boric acid, NaOH, KCl, or any combination thereof. In some versions of the embodiments, the desulfonation reagent does not include NaOH, KCl, or both NaOH and KCl.

[0030] In some embodiments, the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the bead bound DNA with the desulfonation reagent in step (c) or (d), or before eluting the DNA molecules from the substrate in step (d) or (e).

[0031] In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer can be used as the washing buffer. In some embodiments, the washing buffer comprises from about 60% to about 90% ethanol.

[0032] In one embodiment, the elution buffer comprises Tris HCl. In one embodiment, the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5.

[0033] In some embodiments, sulfonation reagent and the DNA molecules in step (a) or (b) are incubated for an incubation time, e.g., 120 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, and / or 10Attorney Docket No.202323-624601 minutes or less. An incubation time can range, for example, from 10 minutes to 120 minutes, such as from 10 minutes to 60 minutes, or 10 minutes to 30 minutes. In some embodiments, the sulfonation reagent and the DNA molecules in step (a) or (b) are incubated for an incubation time such as 65 minutes or less, 55 minutes or less, 45 minutes or less, 35 minutes or less, 25 minutes or less, or 15 minutes or less. In some embodiments, the incubation time is 60 minutes or less. In some embodiments, the incubation time is 45 minutes or less. In some embodiments, the incubation time is 30 minutes or less.

[0034] In various embodiments, the methods include steps for isolating nucleic acids from plasma by performing DNA extraction. In some embodiments, such methods include (a) providing a sample of plasma comprising DNA; (b) adding a proteinase to the plasma; (c) binding the DNA from step (b) to a substrate (e.g., a substrate comprising silica, and / or a magnetic bead) in a non-chaotropic based binding buffer; and (d) isolating the nucleic acids by eluting the DNA molecules from the substrate with an elution buffer. In some versions, such methods include performing contaminant removal and / or protein lysis by, e.g., adding one or more chaotropic reagents and / or proteinase to the sample.

[0035] Further provided are kits for performing the bisulfite conversion and purification of DNA molecules for subsequent analysis. In some embodiments, the kit includes a sulfonation reagent, magnetic beads, a non-chaotropic (or chaotropic-free) binding buffer, a wash buffer, and / or an elution buffer. In some embodiments of the kits provided, the non-chaotropic binding buffer comprises a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof. In some embodiments, the sulfonation reagent is an ammonium hydrogen sulfite reagent. In some embodiments, the ammonium bisulfite sulfonation reagent includes 45% or greater ammonium bisulfite.

[0036] In some embodiments, one or more solutions of the kit can be provided by the user of the kit. For example, in some embodiments a wash buffer and / or elution buffer are not included in the kit and can be supplied by the user of the kit. Kits according to embodiments of the present disclosure may further comprise a sample tube, instructions for use, and / or packaging.

[0037] In some aspects, the present disclosure is directed to a kit for treating DNA including: (a) a sulfonation reagent; (b) silica-coated magnetic beads; (c) a non-chaotropic binding buffer; and (d) a desulfonation reagent.Attorney Docket No.202323-624601

[0038] In some aspects, the present disclosure is directed to a kit for treating DNA including: (a) an ammonium bisulfite sulfonation reagent; (b) silica-coated magnetic beads; (c) a chaotropic- free binding buffer; (d) a washing buffer; (e) a desulfonation reagent; and / or (f) an elution buffer, or any combination thereof.

[0039] In some embodiments, the kit includes a denaturing agent to denature double-strand DNA (dsDNA) molecules, wherein the denaturing agent is sodium hydroxide (NaOH).

[0040] In one embodiment, the sulfonation reagent in the kit comprises from about 45% to about 85% ammonium bisulfite, and optionally from about 0.01 mM to about 1 mM EDTA. In some embodiments, the sulfonation reagent in the kit comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M. In some embodiments, the sulfonation reagent comprises metabisulfite. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0041] In one embodiment, the non-chaotropic binding buffer or chaotropic-free binding buffer in the kit is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate- ammonium sulfate buffer or any combination thereof.

[0042] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit comprises KCl at a final concentration ranging from about 1 M to about 4 M (e.g., 2 M to 4 M). In another embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit comprises KCl at a final concentration greater than 2.5 M (e.g., 2.7 M to 3.3 M KCl). In some embodiments, the binding buffer comprises KCl at a final concentration of about 3 M.

[0043] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit is a glycine-KCl buffer, and the glycine in the glycine-KCl buffer is at a concentration ranging from about 0.05 M to about 2 M.

[0044] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit is a potassium acetate-KCl and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration from about 0.05 M to about 2 M.Attorney Docket No.202323-624601

[0045] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit is a sodium citrate-KCl buffer, and the sodium citrate in the sodium citrate-KCl buffer is at a concentration of, for example, from 0.01 M to 4 M, 0.05 M to 2 M, 0.1 M to 2 M, 0.05 M to 1 M, 0.1 M to .5 M, and / or 0.01 M to .3 M. In one embodiment, the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration of from about 0.05 M to about 4 M.

[0046] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit is a Tris acetate-ammonium sulfate buffer, and the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration from about 0.05 M to about 2 M. In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer in the kit is a Tris acetate-ammonium sulfate buffer, and the ammonium sulfate in the Tris acetate- ammonium sulfate buffer is present at a concentration ranging from about 0.05 M to about 4 M.

[0047] In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer in the kit comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate. In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer in the kit has a pH ranging from about 3.5 to about 7.

[0048] In some embodiments, a desulfonation reagent, such as a reagent in a kit, includes ethanol, isopropanol, KCl, NaOH, sodium carbonate, sodium bicarbonate and / or boric acid, or any combination thereof.

[0049] In one embodiment, the desulfonation reagent in the kit comprises from 30% to 70% ethanol, and NaOH / HCl, wherein the NaOH / HCl comprises from about 25 mM to about 100 mM NaOH and about 25 mM to about 100 mM HCl. In one embodiment, the desulfonation reagent in the kit comprises from about 40% to about 60% ethanol and from about 40% to about 60% of a mixture including sodium carbonate, sodium bicarbonate / carbonate, and NaOH / HCl.

[0050] In one embodiment, the desulfonation reagent in the kit comprises from about 40% to about 60% isopropanol and from about 40% to about 60% of a mixture including boric acid, NaOH and KCl.Attorney Docket No.202323-624601

[0051] In one embodiment, the desulfonation reagent in the kit comprises 0.026 M Sodium Carbonate, 0.011 M Sodium Carbonate / bicarbonate, 0.0384 M NaOH and 0.03696 M HCl.

[0052] In one embodiment, the elution buffer comprises Tris HCl. In one embodiment, the elution buffer in the kit comprises 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5.

[0053] In one aspect, embodiments of methods provided herein relate to methods for isolation of nucleic acids, such as small nucleic acids (e.g., double- or single-stranded DNA consisting of 500 or fewer base pairs) or high molecular-weight (HMW) nucleic acids treated with bisulfite for conversion of unmethylated cytosines to uracil. In some embodiments, bisulfite conversion and / or isolation of small molecules of DNA include the use of a non-chaotropic (or chaotropic-free) DNA binding buffer, for example a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer, or any combination thereof. Such buffers can also be applied in an assay step of nucleic acid extraction as well, e.g., extracting DNA, such as cfDNA, from plasma. Isolation of small nucleic acids finds use, for example, in the treatment of DNA with bisulfite reagents for analyzing DNA methylation patterns in cancer screening (see, e.g., United States Patent Application Publication Nos. US 2019 / 0287652 and US 2020 / 0365229, which are herein incorporated by reference for all purposes).

[0054] In some aspects, the present disclosure is related to compositions comprising a sulfonation reagent. In some embodiments, the composition comprises isolated DNA molecules treated with the sulfonation reagent. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M. In some embodiments, the sulfonation reagent comprises metabisulfite. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0055] In one aspect, the present disclosure relates to methods of deaminating methylated cytosines of isolated DNA molecules using a composition or kit disclosed herein. In some embodiments, the method comprises incubating a composition comprising isolated DNA molecules and a sulfonation reagent for a period of time sufficient to deaminate methylated cytosines in the isolated DNA molecules with the sulfonation agent. In some embodiments, the period of time is 60 minutes or less (e.g., 30 minutes or less).Attorney Docket No.202323-624601

[0056] Additional embodiments of the presently disclosed subject matter provided herein will be apparent to persons skilled in the relevant art based on the teachings contained herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The features and advantages of the presently disclosed subject matter will be more clearly understood from the following description taken in conjunction with the accompanying drawings, which are not necessarily drawn to scale, and wherein:

[0058] FIG. 1 is a flowchart illustrating a method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, in accordance with embodiments of the presently disclosed subject matter.

[0059] FIG. 2 is a flowchart illustrating a method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases and for subsequent recovery of converted DNA molecules, in accordance with embodiments of the presently disclosed subject matter.

[0060] FIG. 3 is a flowchart illustrating a method for treating small single-stranded DNA (ssDNA) molecules to convert unmethylated cytosine bases to uracil bases and for subsequent recovery of converted small ssDNA molecules, in accordance with embodiments of the presently disclosed subject matter.

[0061] FIG.4 is a data plot showing bisulfite conversion ratio with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0062] FIG.5 is a data plot showing normalized recovery of DNA post-bisulfite conversion with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0063] FIG.6 is a data plot showing total recovery of DNA following library preparation with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0064] FIG.7 is a data plot showing post-sequencing total DNA fragment count with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.Attorney Docket No.202323-624601

[0065] FIG.8 is a data plot showing post-sequencing abnormal coverage across an enrichment panel with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0066] FIG.9 is a data plot showing post-sequencing coverage of hypermethylated abnormal DNA fragments across an enrichment panel with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0067] FIG.10 is a data plot showing post-sequencing coverage of hypomethylated abnormal DNA fragments across an enrichment panel with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0068] FIG.11 is a data plot showing post-sequencing average DNA fragment length across an enrichment panel with the use of various binding buffers in accordance with embodiments of the presently disclosed subject matter.

[0069] FIG. 12 is a data plot showing post-sequencing average binary target coverage (top panel) and DNA fragment length (bottom panel) for various bisulfite buffers and conversion reaction conditions in accordance with embodiments of the presently disclosed subject matter.

[0070] FIG.13 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel (top panel), across hypermethylated regions (middle panel), and across hypomethylated regions (bottom panel) for various bisulfite buffers and conversion reaction conditions in accordance with embodiments of the presently disclosed subject matter.

[0071] FIG. 14 is a data plot showing post-bisulfite conversion DNA fragment yield across various bead lots with the use of a binding buffer having 0.4 M KCl and 0.25 M glycine in accordance with embodiments of the presently disclosed subject matter, as compared to a ZYMO binding buffer as control. Bead lots are designated by number along the x-axis.

[0072] FIG.15 is a data plot showing post-bisulfite conversion DNA fragment yield of two lots of DYNABEADS with the use of binding buffers having various concentrations of KCl and at various pH values in accordance with embodiments of the presently disclosed subject matter. The letter “d” in a concentration of pH value indicates a decimal point; for example, “0d2MKCL” indicates KCl at a concentration of 0.2 M, and “3d5pH” indicates a pH of 3.5.Attorney Docket No.202323-624601

[0073] FIG.16 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel using two bead lots, and binding buffers having various concentrations of KCl in accordance with embodiments of the presently disclosed subject matter.

[0074] FIGS.17A-17B are data plots showing post-bisulfite conversion fragment yield prior to sequencing (FIG.17A), and post-sequencing coverage of abnormal DNA fragments across an enrichment panel (FIG.17B) using various bead lots, and different binding buffers including 3 M KCl at pH 6 (“pH6+3MKCL”), 0.4 M KCl at pH 5.4 (“pH5d4_0d4MKCL”), and a ZYMO buffer as control.

[0075] FIG.18 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel using various bead lots with a binding buffer having 3 M KCl and no glycine, as compared to a ZYMO buffer as control.

[0076] FIG. 19 shows an illustrative schematic workflow for bisulfite treatment of DNA in accordance with embodiments of the presently disclosed subject matter. DETAILED DESCRIPTION

[0077] In various aspects, the presently disclosed subject matter is directed to the chemical modification and purification of nucleic acids, such as DNA molecules. In some embodiments, the present disclosure provides methods for performing a bisulfite conversion reaction on DNA molecules and performing the subsequent desulfonation and purification steps using a substrate (e.g., magnetic beads). In some embodiments, the methods provide for the use of non-chaotropic (or chaotropic-free) binding buffers that promote a highly stable binding of the converted DNA molecules to the substrate. This facilitates the efficient recovery of bisulfite-treated DNA despite the highly basic reaction conditions of desulfonation that one might have otherwise expected to disrupt the interaction of the DNA with the substrate. In various embodiments, the presently disclosed subject matter provides methods for preparing bisulfite-converted DNA quickly, e.g., in 10, 20, 30, 40, 50, 60, 70, 80, 90, or 120 minutes or less, with high recovery of the converted DNA molecules.

[0078] To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.Attorney Docket No.202323-624601

[0079] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one embodiment” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the disclosed invention(s) may be readily combined, without departing from the scope or spirit of the invention(s). Also, where ranges are listed herein, the provided ranges include the endpoints of the range listed. For example, a listed range of 1 to 3 includes 1 and 3.

[0080] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. Thus, “a” or “an” or “the” can mean one or more than one. For example, “a” widget can mean one widget or a plurality of widgets. The meaning of “in” includes “in” and “on.”

[0081] As used herein, a “DNA fragment” or “small DNA” molecule means DNA molecules that have a length of 500 bp or less. In other embodiments, small DNA molecules may have a length of 400 bp or less, 300 bp or less, or 200 bp or less. In some cases, the small DNA molecules may be in a mixture with longer DNA molecules.

[0082] The term “cell free nucleic acid,” “cell free DNA,” or “cfDNA” refer to polynucleotides present in a sample from a subject or portion thereof that can be isolated or otherwise manipulated without applying a lysis step to the sample as originally collected (e.g., as in lysis for the extraction from cells or viruses). Cell-free nucleic acids are thus unencapsulated or “free” from the cells or viruses from which they originate, even before a sample of the subject is collected. Cell-free polynucleotides may be produced as a byproduct of cell death (e.g. apoptosis or necrosis) or cell shedding, releasing polynucleotides into surrounding body fluids or into circulation. Accordingly, cell-free nucleic acids may be isolated from a non-cellular fraction of blood (e.g. serum or plasma), from other bodily fluids (e.g. urine), or from non-cellular fractions of other types of samples. In some embodiments, cfDNA refers to deoxyribonucleic acid molecules that circulate in a subject’s body (e.g., bloodstream) and may originate from one or more healthy cells and / or from one or more cancer cells.

[0083] The term “circulating tumor DNA” or “ctDNA” refers to nucleic acid fragments that originate from tumor cells or other types of cancer cells, which may be released into a fluid fromAttorney Docket No.202323-624601 an individual's body (e.g., bloodstream) as result of biological processes such as apoptosis or necrosis of dying cells, cell turnover or actively released by viable tumor cells.

[0084] As used herein, the term “genome” refers to the genetic material (e.g., chromosomes) of an organism or a cell.

[0085] As used herein, “sulfonated DNA” refers to the intermediate bisulfite reaction product that is a DNA molecule including cytosines or uracils that are sulfonated as a result of bisulfite treatment. Unmethylated cytosines that have been sulfonated and deaminated are referred to herein as “sulfonated deaminated cytosines,” and may also be referred to individually as “uracil sulfonate” or “uracil sulphonate.” Desulfonation of uracil sulfonate produces uracil.

[0086] As used herein, the terms “hydrogen sulfite” and “bisulfite” are interchangeable.

[0087] As used herein, the “magnetic beads” refers to particles or beads that respond to a magnetic field. Typically, magnetic particles comprise materials that have no magnetic field but that form a magnetic dipole when exposed to a magnetic field, e.g., materials capable of being magnetized in the presence of a magnetic field but that are not themselves magnetic in the absence of such a field. The term “magnetic” as used in this context includes materials that are paramagnetic or superparamagnetic materials. The term “magnetic”, as used herein, also encompasses temporarily magnetic materials, such as ferromagnetic or ferrimagnetic materials with low Curie temperatures, provided that such temporarily magnetic materials are paramagnetic in the temperature range at which silica magnetic particles containing such materials are used according to the present methods to isolate biological materials. In various embodiments, magnetic beads may be replaced with non-magnetic beads, or other substrates, such as columns or filters. In some embodiments, the beads or other substrate comprise a surface made of or coated in silica.

[0088] As used herein, the term “kit” refers to any delivery system for delivering materials (e.g., reaction buffers and materials). In the context of DNA conversion and purification kits, such delivery systems include systems that allow for the storage, transport, or delivery of reagents, buffers and devices (e.g., reaction, binding, wash and / or elution buffers) and / or other supporting materials (e.g., magnetic beads, silica columns, reaction tubes, etc.) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials.Attorney Docket No.202323-624601

[0089] As used herein, the term “chaotropic” includes its commonly understood meaning in the field and refers to the activity of agents such as guanidinium hydrochloride or urea to disrupt hydrogen bonds and destabilize secondary, tertiary, and quaternary structures of biomolecules and the interactions that form them. Likewise, “chaotropic free” refers to compositions (e.g., buffers) that lack chaotropic agents, and are therefore “non-chaotropic.”

[0090] Overview

[0091] In various aspects, methods described herein allow for nucleic acid, such DNA (e.g., cfDNA), extraction from a sample, as well as bisulfite conversion and recovery of converted DNA molecules. In accordance with aspects of the presently disclosed methods, a first step in the process provides for the denaturation of double-stranded DNA (dsDNA) molecules in a sample to produce single-stranded DNA (ssDNA) molecules. Next the denatured, ssDNA molecules are sulfonated using a sulfonation reagent, or more specifically, unmethylated cytosines in the ssDNA molecule are sulfonated, producing sulfonated ssDNA molecules. In some embodiments, methods described herein then provide for on-substrate (e.g., on-bead) desulfonation by first binding sulfonated DNA molecules to a substrate, such as magnetic beads, in the presence of a non- chaotropic (or chaotropic-free) binding buffer and then subsequently desulfonation of the DNA molecules using a desulfonation reagent. In some variations, the substrate-bound DNA can be washed using a washing reagent before and / or after the desulfonation step. The methods described herein provide for recovery and purification of the converted DNA molecules through use of an elution buffer for the elution of converted, single-strand DNA molecules from the substrate. The disclosed methods also include performing extraction by separation of nucleic acids, e.g., DNA, from other aspects of a blood sample such as red blood cells and / or plasma using a non-chaotropic- based or chaotropic-free binding buffer. Performing such extraction can be followed by performing nucleic acid conversion such as by bisulfite conversion or enzymatic conversion.

[0092] Denaturing Double-Stranded DNA Molecules

[0093] In some embodiments, double-stranded DNA (dsDNA) molecules are denatured before the sulfonation reaction to produce single-stranded DNA (ssDNA) molecules. Such a step, taken proactively and / or at a time fully or partially independent of other disclosed steps, is optional and can be skipped (and in some case may occur concurrently with sulfonation due to the conditions thereof, such as concentration of bisulfite reagent and / or incubation temperature). In some aspects dsDNA molecules can be denatured to produce ssDNA molecules using chemicalAttorney Docket No.202323-624601 and / or mechanical means. In some embodiments, dsDNA molecules can be denatured using heat, such as by incubating the dsDNA molecules in a solution at a denaturation temperature of, for example, from 60ºC to 95ºC, 60ºC to 80ºC, 60ºC to 95ºC, 70ºC to 80ºC, 75ºC to 80ºC, 70ºC to 75ºC, 75ºC to 80ºC, 80ºC to 85ºC, 75ºC to 85ºC and / or 60ºC to 70ºC. In some embodiments, dsDNA molecules can be denatured using heat, such as by incubating the dsDNA molecules in a solution at a denaturation temperature of, for example, 65ºC, 75ºC, 85ºC, 95ºC, 60ºC, 70ºC, 80ºC, 90ºC, or any of such temperatures or less. The heat incubation can be applied for a denaturation time ranging, for example, from 2 minutes to 60 minutes, for example from 2 min to 30 min, from 2 min to 20 min, from 2 min to 10 min, or from 2 min to 5 min, from 5 min to 30 min, from 10 min to 30 min, from 15 min to 25 min, from 15 min to 30 min, and / or from 20 min to 30 min. The heat incubation can be applied for a denaturation time of, for example, 1 min, 2 min, 10 min, 15 min, 20 min, 25 min, 30 min, or more. In another embodiment, dsDNA molecules can be denatured to produce ssDNA molecules using a denaturation reagent such as, for example sodium hydroxide (NaOH), ammonium bisulfite, e.g., 60%, 65%, or 70% ammonium bisulfite. For example, the dsDNA molecules can be incubated in the presence of from about 0.05N NaOH to about 5N NaOH for 5 min or more. In some embodiments, the NaOH incubation step can be in the presence from about 0.1N NaOH to about 2N NaOH, from about 0.2N NaOH to about 1N NaOH, or from about 0.3N NaOH to about 0.5N NaOH. In one embodiment, the dsDNA molecules can be denatured in the presence of 0.3N NaOH for 5 min or more. In some aspects, a denaturation step does not include adding a denaturation reagent, e.g., NaOH, to the mixture. In some aspects, a denaturation step does not include heating the sample.

[0094] Sulfonation of DNA Molecules

[0095] Embodiments of the methods described herein provide for sulfonation of DNA molecules in shorter times (e.g., in less than 2 hours, less than 60 min, less than 30 min, less than 20 min, or less than 10 min) by incubation with a concentrated sulfonation reagent (e.g., a high concentration of ammonium bisulfite). In some embodiments, a sulfonation reaction is 60 minutes or less in duration. In some embodiments, a sulfonation reaction is 30 minutes or less in duration. In some embodiments, methods described herein including sulfonation of DNA with a sulfonation reagent, e.g., a concentrated sulfonation reagent, such as ammonium bisulfite (ammonium hydrogen sulfite), provide for efficient sulfonation of DNA in a shorter time than sulfonation with typical bisulfite reagents.

[0096] For example, past methods for the sulfonation of DNA comprise long, typically overnight, incubations in sodium bisulfite, e.g., for 16 hours or more (see, e.g., Frommer M et al.Attorney Docket No.202323-624601 (1992), "A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands" Proc. Natl. Acad. Sci, USA.89:1827-31). However, in some aspects, the presently disclosed subject matter utilizes a concentrated sulfonation reagent. For example, in some aspects of the presently disclosed subject matter, the concentrated sulfonation reagent including ammonium bisulfite has a concentration of at least 45%. In some embodiments, the concentrated sulfonation reagent comprises ammonium bisulfite having a concentration of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or more. In some embodiments, the concentrated sulfonation reagent can further comprise a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). For example, in one embodiment, the concentrated sulfonation reagent comprises from about 45% to about 85% ammonium bisulfite and from about 0.005 mM to about 1 mM EDTA. In some embodiments, the sulfonation reagent comprises ammonium bisulfite having a concentration of about or at least about 65%.

[0097] In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 5.0 M to about 9 M (e.g., about 5.5 M, 6.0 M, 6.5 M, 7.0 M, 7.5 M, 8.0 M, or 8.5 M). In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 6.5 M to about 9 M. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 6.5 M. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 7.5 M. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 9 M. In some embodiments, all of the sulfur in the sulfonation reagent is from one or more sulfites (e.g., sodium bisulfite, ammonium bisulfite, ammonium sulfite monohydrate, or a combination thereof). In some embodiments, the sulfonation reagent comprises metabisulfite. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of about 6.0 M to about 9 M (e.g., about 6.5 M, 7.0 M, 7.5 M, 8.0 M, or 8.5 M). In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of about 7.5 M. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of about 9 M. In some embodiments, the sulfonation reagent comprises a mixture of an ammonium bisulfite solution with one or more of a bisulfite (e.g., sodium bisulfite) and a sulfite (e.g., ammonium sulfite monohydrate).

[0098] In some embodiments, the sulfonation reaction can be incubated at an elevated temperature allowing the reaction to proceed to completion at a more rapid pace. For example, theAttorney Docket No.202323-624601 sulfonation reaction can be incubated at a temperature of from about 60ºC to about 95ºC, from about 65ºC to about 90ºC, from about 70ºC to about 90ºC, or from about 80ºC to about 90ºC. Consequently, methods in accordance with some embodiments described herein reduce the time of the sulfonation reaction and the total time to produce an analytical sample relative to past technologies.

[0099] Magnetic Beads

[0100] In various aspects, methods provided herein relate to the bisulfite treatment and isolation of DNA molecules for a quantitative measure of DNA methylation. In some embodiments, a substrate such as magnetic beads are used for the treatment and isolation of DNA, e.g., beads including a magnetic core and in some versions having an active surface coating for binding DNA molecules, such as a silica coating or silane coating. The use of silica or silane coating allows for more efficient binding DNA and the magnetic core provides an efficient way to concentrate and isolate the beads (and bound DNA) through use of a magnetic field or a magnet.

[0101] The methods disclosed herein are not limited to any particular type of substrate or magnetic bead. Embodiments of the technology described herein may make use of any magnetic beads (e.g., paramagnetic beads) that have an affinity for nucleic acids. In some embodiments, the magnetic beads have a magnetite (e.g., Fe3O4) core and a coating including silicon dioxide (SiO2). The bead structure (e.g., size, porosity, shape) and composition of the solution in which a nucleic acid is bound to the bead can be altered to bind different types of nucleic acids (e.g., DNA or RNA in single stranded, double stranded, or other forms or conformations; nucleic acids derived from a natural source, synthesized chemically, synthesized enzymatically (e.g., by PCR)) and / or different sizes of nucleic acids (e.g., small oligomers, primers, genomic, plasmids, fragments (e.g., consisting of 500, 400, 300, 200 or fewer bases). These characteristics of the beads affect the binding of the nucleic acids to, and elution of DNA from the beads. Related technologies are described, e.g., in U.S. Pat. Nos.6,194,562; 6,270,970; 6,284,470; 6,368,800; 6,376,194, each of which are herein incorporated by reference.

[0102] In some embodiments, the silica-coated magnetic beads can be MagneSil Paramagnetic Particles (Promega, Madison, WI), silica-coated Dynabeads® (Thermo Fisher Scientific, Waltham, MA), SeraSil-Mag™ silica-coated magnetic beads (Cytiva, Marlborough, MA), MagIso™ silica beads (Creative Diagnostics, Shirley, NY), or similar silica-coated magnetic beads.Attorney Docket No.202323-624601

[0103] Methods disclosed herein are not limited to a particular size of magnetic bead. Accordingly, embodiments of the technology may use magnetic beads of a number of different sizes. Smaller beads provide more surface area (per weight unit basis) for adsorption, but smaller beads may be limited in the amount of magnetic material that can be incorporated in the bead core relative to a larger bead. In some embodiments, the particles are distributed over a range of sizes with a defined average or median size appropriate for the technology for which the beads are used. In some embodiments, the particles are substantially of a single particle size distribution.

[0104] In some embodiments, the beads that find use in the present technology have pores that are accessible from the exterior of the particle. Such pores have a controlled size range that is sufficiently large to admit a nucleic acid, e.g., a DNA fragment, into the interior of the particle and to bind to the interior surface of the pores. The pores are designed to provide a large surface area that is capable of binding a nucleic acid. Moreover, in one aspect the technology is not limited to any particular method of nucleic acid (e.g., DNA) binding and / or isolation. Thus, in some embodiments, aspects of the technology relating to the bisulfite reaction are combined with other suitable methods of DNA isolation (e.g., precipitation, column chromatography (e.g., a spin column), etc.).

[0105] Magnetic beads (and bound material) can be removed from a mixture using a magnetic field (e.g., by using a magnet). In some embodiments, other forms of external force in addition to a magnetic field are used to isolate the biological target substance according to embodiments of the present technology. For example, suitable additional forms of external force include, but are not limited to, gravity filtration, vacuum filtration, and centrifugation. Such forces may be used as an alternative to a magnetic field, such as when using non-magnetic beads or other substrates.

[0106] Non-Chaotropic (or Chaotropic-Free) Binding Buffer

[0107] In some embodiments, the presently disclosed subject matter uses a non-chaotropic or chaotropic-free binding buffer, in binding DNA molecules to a substrate (e.g., magnetic beads) for improved isolation and purification of the DNA. Past protocols for recovering and purifying nucleic acids from a solution have used buffers with high concentrations of chaotropic salts, such as guanidinium thiocyanate (GuSCN) or guanidinium hydrochloride (GuHCl). However, the inventors of the presently disclosed subject matter have discovered that the use of non-chaotropic (or chaotropic-free) binding buffers with a substrate (e.g., magnetic beads) provides for improved recovery and purification of DNA molecules, and thus, allows for improved quantitative measuring DNA methylation, as compared with conventional techniques, while avoiding potential negative impacts of residual chaotropic agents in later steps.Attorney Docket No.202323-624601

[0108] Any of a variety of non-chaotropic (or chaotropic-free) salts may be used in the binding buffer, such as potassium chloride (KCl) buffer, glycine-KCl, sodium citrate-KCl, potassium acetate-KCl, Tris acetate-ammonium sulfate, or any combination thereof. In various embodiments, the non-chaotropic (or chaotropic-free) binding buffer is used at an acidic pH, such as from about 3.5 to about 7. In various embodiments, a chaotropic or non-chaotropic buffer component such as KCl in a glycine-KCl buffer, sodium citrate-KCl buffer, or potassium acetate- KCl buffer has a concentration ranging, for example, from: 0.01 M to 5 M, 0.05 M to 4 M, 0.05 M to 3 M, 0.1 M to 4 M, 0.2 M to 4 M, 0.4 M to 4 M, 0.5 M to 4 M, 0.15 M to 4 M, 0.15 M to 5 M, 0.15 M to 3 M, 0.2 M to 3 M, 0.35 M to 4 M,.5 M to 5 M, 0.005 M to 6 M, 0.008 M to 5 M, 0.01 M to 6 M, or 0.01 M to 4.5 M. In various embodiments, a chaotropic or non-chaotropic buffer component such as KCl or ammonium sulfate can have a concentration of 0.1 M or less, 0.2 M or less, 0.3 M or less, 0.4 M or less, or 0.5 M or less, 0.15 M or less, 0.25 M or less, 0.35 M or less, 0.45 M or less, or 0.1 M or more, 0.2 M or more, 0.3 M or more, 0.4 M or more, or 0.5 M or more, 0.15 M or more, 0.25 M or more, 0.35 M or more, or 0.45 M or more. Any non- chaotropic based binding buffer or chaotropic-free binding buffer or component thereof, e.g., KCl, described herein can have such a concentration.

[0109] In one embodiment, the non-chaotropic based binding buffer or chaotropic-free binding buffer comprises KCl at a final concentration ranging from about 1 M to about 4 M (e.g., 2 M to 4 M). In some embodiments, the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M. In some embodiments, the non-chaotropic binding buffer comprises KCl at a final concentration ranging from 2.7 M to 3.3 M. In another embodiment, the non-chaotropic based binding buffer comprises KCl at a final concentration of about 3 M. In some embodiments, the non-chaotropic based binding buffer or chaotropic-free binding buffer is a glycine-KCl buffer having a glycine concentration ranging from about 0.05 M to about 2 M. In some embodiments, the non-chaotropic based binding buffer or chaotropic-free binding buffer is a potassium acetate-KCl buffer having a potassium acetate concentration ranging from about 0.05 M to about 2 M. In some embodiments, the non-chaotropic based binding buffer or chaotropic- free binding buffer is a sodium citrate-KCl buffer having a sodium citrate concentration ranging from about 0.05 M to about 2 M. KCl can be included in the non-chaotropic (or chaotropic-free) binding buffer with a concentration ranging from about 0.05 M to about 4 M.

[0110] In some embodiments, the non-chaotropic (or chaotropic-free) binding buffer or chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer having a Tris acetate concentration ranging from about 0.05 M to about 2 M. In some embodiments, the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer with the Tris acetate-ammoniumAttorney Docket No.202323-624601 sulfate being present at a concentration of from about 0.05 M to about 4 M. In various embodiments, a non-chaotropic based binding buffer or chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, wherein a component, e.g., the Tris acetate, in the Tris acetate- ammonium sulfate buffer can have has a concentration ranging, for example, from: 0.01 M to 5 M, 0.05 M to 4 M, 0.05 M to 3 M, 0.1 M to 4 M, 0.2 M to 4 M, 0.4 M to 4 M, 0.5 M to 4 M, 0.15 M to 4 M, 0.15 M to 5 M, 0.15 M to 3 M, 0.2 M to 3 M, 0.35 M to 4 M, or 0.5 M to 5 M. In embodiments of ammonium sulfate buffers, ammonium sulfate can have any of the same concentrations in the buffer that are above for Tris acetate.

[0111] In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and at least 0.15 M KCl; (iv) 0.25 M glycine and at least 0.4 M KCl; (v) 0.2 M sodium citrate and at least 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0112] Additionally, regarding non-chaotropic (or chaotropic-free) binding buffer implementation, a common problem can arise when residual chaotropic salts and ethanol are carried over into an amplification reaction and inhibit polymerase amplification. Some methods do not require chaotropic salts and organic solvents but utilize a pH dependent anion exchange approach. In some embodiments, nucleic acids bind to silica, independent of chaotropic salts, under acidic conditions in the presence of kosmotropic salts. At low pH and high ionic strength, DNA can also bind to sand, which is similar to silica. To decrease the potential negative impact of residual chaotropic salts on downstream amplification steps post bisulfite conversion, the non- chaotropic, e.g., salt-based, bisulfite conversion binding buffer formulations provided herein were evaluated according to the subject methods to be applied, for example, in a purification step following bisulfite conversion.

[0113] In various embodiments, buffers, such as binding buffers or desulfonation buffers can include concentrated bisulfite solution composed, for example of ammonium bisulfite at a percent such as 45%, sodium bisulfite and / or ammonium sulfite monohydrate.

[0114] Desulfonation of Sulfonated DNA Molecules

[0115] In some embodiments, the presently disclosed subject matter provides for desulfonation of sulfonated DNA molecules bound to a substrate (e.g., magnetic beads), or on- substrate desulfonation. In some embodiments, desulfonation provided herein improves the recovery and purification of DNA molecules while also providing a simple and efficient meansAttorney Docket No.202323-624601 for carrying out the various steps of the presently disclosed subject matter. In some embodiments, the present disclosure provides methods including substrate desulfonation that allows for improved recovery of DNA molecules including small DNA molecules (e.g., DNA molecules 600 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, 100 bp or less, 50 bp or less, 650 bp or less, 550 bp or less, 450 bp or less, 350 bp or less, 250 bp or less, or 150 bp or less in length). Such small DNA molecules can be cell-free DNA (cfDNA) molecules and / or can range from 100 bp to 700 bp, 200 bp to 700 bp, 300 bp to 600 bp, 400 bp to 600 bp, 500 bp to 600 bp, 450 bp to 700 bp, 450 bp to 650 bp, 350 bp to 600 bp, 200 bp to 650 bp, and / or 100 bp to 800 bp, in length. The disclosed subject matter also provides, in some embodiments, for improved recovery of large and / or high molecular weight (HMW) DNA molecules, such as cellular DNA molecules (e.g., DNA molecules isolated from white blood cells (WBCs)). Such HMW DNA molecules can, for example, have a length ranging from 5 Kbp to 40 Kbp, 10 Kbp to 30 Kbp, 15 Kbp to 30 Kbp, 20 Kbp to 30 Kbp, 10 Kbp to 40 Kbp, 20 Kbp to 40 Kbp, 5 Kbp to 35 Kbp, and / or 5 Kbp to 30 Kbp. Such DNA molecules can also, for example, have a length of 5 Kbp or more, 10 Kbp or more, 15 Kbp or more, 20 Kbp or more, 25 Kbp or more, or 30 Kbp or more. In some aspects, bisulfite conversion shears and / or fragments large DNA molecules to, e.g., small DNA fragments, e.g., fragments having a length of 700 bp or less, 600 bp or less, 500 bp or less, or 400 bp or less. Such fragments and / or large DNA molecules may be those described in library preparation method steps described herein. In some aspects, subject nucleic acid molecules, e.g., HMW DNA molecules, are fragments having a length of 2000 bp or more, 1500 bp or more, 1000 bp or more, 700 bp or more, 600 bp or more, or 500 bp or more.

[0116] Any of a variety of desulfonation reagents can be used in the desulfonation of sulfonated, substrate-bound nucleic acid, such as DNA (e.g., ssDNA), or RNA molecules. For example, in some embodiments, the desulfonation reagent includes ethanol, isopropanol, NaOH, sodium carbonate, sodium bicarbonate, HCl, KCl, boric acid, or any combination thereof. In some aspects, HCl is combined in a formulation including sodium carbonate and sodium bicarbonate. In some aspects, KCl is combined in a formulation including boric acid. In some versions, the desulfonation reagent does not include sodium bicarbonate or sodium carbonate. In other embodiments, the desulfonation reagent comprises sodium carbonate and / or bicarbonate and does not include boric acid.

[0117] In some embodiments, the desulfonation reagent comprises an alcohol, e.g., ethanol or isopropanol, based desulfonation buffer having, e.g., an alcohol, e.g., ethanol, concentration of from about 30% to about 70%, from about 40% to about 60%, or from about 40% to about 50%. The desulfonation buffer can further include NaOH and / or HCl. In some embodiments, theAttorney Docket No.202323-624601 desulfonation buffer has a final NaOH concentration ranging from about 25 mM to about 100 mM and / or a final HCl concentration of from about 25 mM to about 100 mM. In some embodiments, the desulfonation reagent comprises from about 40% to about 60% ethanol and from about 40% to about 60% of a mixture including sodium carbonate, sodium bicarbonate / carbonate, and NaOH / HCl.

[0118] In some embodiments, a desulfonation reagent includes an isopropanol based desulfonation buffer having an isopropanol concentration from 30% to 70%, from 40% to 60%, or from 40% to 50%. The desulfonation buffer can further comprise a mixture including boric acid, NaOH and KCl and having a final boric acid concentration of from 30% to 70%, from 40% to 60%, or from 40% to 50%. In some versions of the subject embodiments, the desulfonation reagent is a boric acid reagent and includes, for example, components which may be an alcohol, e.g., isopropanol or ethanol, boric acid, NaOH, KCl, or any combination thereof. In some versions of the embodiments, the desulfonation reagent does not include NaOH, KCl, or both NaOH and KCl. In some versions the desulfonation reagent, e.g., boric acid reagent, includes boric acid having a concentration of 0.0402 M (40.2 mM) or less, KCl having a concentration of 0.0199 M (19.9 mM) or less, NaOH having a concentration of 0.04 M (40 mM) or less, and / or 30%, 40%, 50%, or 60% isopropanol, or any combination thereof. In some versions the desulfonation reagent, e.g., boric acid reagent, includes boric acid having a concentration of 0.0402 M (40.2 mM) or more, KCl having a concentration of 0.0199 M (19.9 mM) or more, NaOH having a concentration of 0.04 M (40 mM) or more, and / or 30%, 40%, 50%, or 60% isopropanol, or any combination thereof. In some versions the desulfonation reagent, e.g., boric acid reagent, includes boric acid having a concentration ranging from 40 mM to 41 mM, 38 to 42 mM, or 35 to 45 mM, KCl having a concentration ranging from 19 mM to 21 mM, 17 mM to 23 mM, or 15 mM to 25 mM, NaOH having a concentration ranging from 38 mM to 42 mM, 35 mM to 45 mM, or 30 mM to 50 mM, and / or 30%, 40%, 50%, or 60% isopropanol, or any combination thereof. In some versions the desulfonation reagent, e.g., boric acid reagent or sodium carbonate / bicarbonate reagent, includes an alcohol, e.g., ethanol or isopropanol, making up 30% or less, 40% or less, 50% or less, or 60% or less of the solution. In some versions the desulfonation reagent, e.g., boric acid reagent or sodium carbonate / bicarbonate reagent, includes an alcohol, e.g., ethanol or isopropanol, making up 30% or more, 40% or more, 50% or more, or 60% or more of the solution. In some versions the desulfonation reagent, e.g., boric acid reagent or sodium carbonate / bicarbonate reagent, includes an alcohol, e.g., ethanol or isopropanol, making up 20%- 70%, 20%-40%, 30%-50%, 40%-60%, 50%-70%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 58%-62%, 50%-65%, 55%-70%, and / or 65%-75% of the solution.Attorney Docket No.202323-624601

[0119] In some versions of the subject embodiments, the desulfonation reagent is a sodium carbonate / bicarbonate reagent and includes, for example, components which may be ethanol, and sodium carbonate and / or sodium bicarbonate, or any combination thereof. Where the desulfonation reagent is a sodium carbonate / bicarbonate reagent, it can include, for example, sodium carbonate having a concentration of 0.0383 M or less, or 0.048 M or less, or 0.026 M or less, sodium bicarbonate having a concentration of 0.0115 M or less, or .011 M or less, or 0.012 M or less, NaOH having a concentration of 0.0384 M or less, HCl having a concentration of 0.037 M or less, or 0.03696 M or less, and / or 30%, 40%, 50%, or 60% ethanol, or any combination thereof. A desulfonation reagent can include, for example, sodium carbonate having a concentration of 0.0383 M or more, 0.026 M or more, or 0.048 M or more, sodium bicarbonate having a concentration of 0.0115 M or more, or .011 M or more, or 0.012 M or more, NaOH having a concentration of 0.0384 M or more, HCl having a concentration of 0.037 M or more, or 0.03696 M or more, and / or 30% or more / less, 40% or more / less, 50% or more / less, or 60% or more / less alcohol, e.g., ethanol or isopropanol, or any combination thereof. In various aspects, the desulfonation reagent includes sodium carbonate having a concentration ranging from 25 mM to 50 mM and / or sodium bicarbonate having a concentration ranging from 1 mM to 25 mM. A desulfonation reagent can include, for example, sodium carbonate having a concentration ranging from, 0.02 M to 0.05 M, 0.01 M to 0.08 M, 0.01 M to 0.04 M, from 0.02 M to 0.04 M., from 0.04 M to 0.05 M, from 0.045 M to 0.055 M, from 0.04 M to 0.06 M, from 0.03 M to 0.06 M, or from 0.01 M to 0.1 M. In addition, a desulfonation reagent can include sodium bicarbonate having a concentration ranging from, for example, 0.005 M to 0.05 M, 0.01 M to 0.012 M, 0.008 M to 0.02 M, 0.008 M to 0.015 M, 0.01 M to 0.02 M, 0.01 M to 0.015 M, 0.005 M to 0.02 M, or 0.005 M to 0.015 M. Furthermore, a desulfonation reagent can include NaOH having a concentration ranging from 0.035 M to 0.045 M, 0.03 M to 0.05 M, 0.025 M to 0.065 M, or 0.035 M to 0.04 M and / or HCl having a concentration ranging from 0.035 M to 0.04 M, 0.03 M to 0.04 M, 0.02 M to 0.05 M, or 0.01 M to 0.06 M. In some versions of the embodiments, the desulfonation reagent does not include NaOH, HCl, or both NaOH and HCl. According to some embodiments, the desulfonation reagent does not require a pH adjustment step. In some versions, the desulfonation reagent has a pH of 10 or more, 11 or more, or 12 or more, or 10 or less, 11 or less, or 11.7 or less, or 12 or less, or 11.5 or more, or 12.5 or more, or 11.5 or less, or 11.7 or less, or 11.9 or less, or 12.2 or less. In various aspects, the desulfonation reagent has a pH ranging from 10 to 14, such as 10 to 12, such as 11 to 12, or such as 11.5 to 12.5, or 11 to 13, or 10 to 13.

[0120] According to some embodiments, a sodium carbonate / bicarbonate reagent includes a percentage, e.g., 30%, 40%, or 50% of ethanol. In various instances the desulfonation reagent hasAttorney Docket No.202323-624601 concentrations of various substances including: e.g., 0.107 M, 0.0201 M, 0.0383 M, 0.480 M, or 0.501 M sodium carbonate, 0.0101 M, 0.0115 M, 0.012 M, 0.0134 M sodium bicarbonate, 0.0301 M, 0.0337 M, 0.0384 M, 0.0422 M, or 0.0489 M NaOH and / or a concentration, 0.0311 M, 0.037 M, or 0.0421 M HCl. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.107 M or less, 0.0201 M or less, 0.0383 M or less, 0.480 M or less, or 0.501 M or less sodium carbonate, 0.0101 M or less, 0.0115 M or less, 0.012 M or less, 0.0134 M or less sodium bicarbonate, 0.0301 M or less, 0.0337 M or less, 0.0384 M or less, 0.0422 M or less, or 0.0489 M or less NaOH and / or a concentration, 0.0311 M or less, 0.037 M or less, or 0.0421 M or less HCl. In various instances the desulfonation reagent has concentrations of various substances including: e.g., 0.107 M or more, 0.0201 M or more, 0.0383 M or more, 0.480 M or more, or 0.501 M or more sodium carbonate, 0.0101 M or more, 0.0115 M or more, 0.012 M or more, 0.0134 M or more sodium bicarbonate, 0.0301 M or more, 0.0337 M or more, 0.0384 M or more, 0.0422 M or more, or 0.0489 M or more NaOH and / or a concentration, 0.0311 M or more, 0.037 M or more, or 0.0421 M or more HCl. In some versions the sodium carbonate / bicarbonate reagent has a pH around 10, 10.7, 11, 11.2, 11.7, 12, or 12.5. In various embodiments, the sodium carbonate / bicarbonate reagent does not include NaOH and / or HCl. In some variations, the sodium carbonate / bicarbonate reagent does not need a step of pH adjustment. In some variations, the sodium carbonate / bicarbonate reagent has only three components.

[0121] Washing Buffer

[0122] In some variations, the methods described herein further comprise contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent and / or before eluting the bound DNA molecules from the magnetic beads. The use of a washing buffer removes impurities and provides a cleaner, more purified converted DNA sample for subsequent analysis.

[0123] Any of a variety of suitable washing buffers can be used in the practice of the presently disclosed subject matter. In some embodiments, the washing buffer comprises from about 60% to about 90% ethanol. In other embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer can be used as the washing buffer.

[0124] Elution Buffer

[0125] In some embodiments, recovery of the converted DNA, e.g., ssDNA, molecules comprises an elution step. In general, any of a variety of suitable buffers that allow for the converted DNA, e.g., ssDNA, molecules to be eluted from the substrate, and subsequentlyAttorney Docket No.202323-624601 recovered, can be used in the practice of the presently disclosed subject matter. In some embodiments, the elution buffer comprises Tris HCl. In one embodiment, the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5.

[0126] In some embodiments, the methods include performing bisulfite conversion as set forth herein and obtaining a recovery of a percentage or less, e.g., 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, of the nucleic acids present in the sample before the process. In some versions of the subject embodiments, the methods include performing bisulfite conversion as set forth herein and obtaining a recovery of a percentage or more, e.g., 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, of the nucleic acids present in the sample before the process. In some embodiments, at least 50% of the bisulfite-treated nucleic acid are recovered in the eluate.

[0127] Kits

[0128] Further provided are kits for performing bisulfite conversion and purification of DNA molecules for subsequent analysis, such as set form in any of the various aspects or embodiments herein. In some embodiments, a kit includes a sulfonation reagent, magnetic beads, a non- chaotropic (or chaotropic-free) binding buffer, a wash buffer, and / or an elution buffer. In some embodiments of the kits provided, the non-chaotropic binding buffer comprises a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof. In some embodiments, the sulfonation reagent is an ammonium hydrogen sulfite reagent. In some embodiments, the ammonium bisulfite sulfonation reagent comprises greater than 45% ammonium bisulfite. In one embodiment, the sulfonation reagent in the kit comprises from about 45% to about 85% ammonium bisulfite, and optionally from about 0.01 mM to about 1 mM EDTA. In another embodiment, the sulfonation reagent in the kit comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA.

[0129] In some embodiments, one or more solutions of the kit can be provided by the user of the kit. For example, in some embodiments a wash buffer and / or elution buffer are not included in the kit and can be supplied by the user of the kit. Kits according to embodiments of the presently disclosed subject matter may further comprise a sample tube, instructions for use, and / or packaging.

[0130] In some embodiments, the presently disclosed subject matter is directed to a kit for treating DNA including: (a) a sulfonation reagent; (b) silica-coated magnetic beads; (c) a non- chaotropic binding buffer; and (d) a desulfonation reagent. In other embodiments, the presentlyAttorney Docket No.202323-624601 disclosed subject matter is directed to a kit for treating DNA including: (a) an ammonium bisulfite sulfonation reagent; (b) silica-coated magnetic beads; (c) a chaotropic-free binding buffer; (d) a washing buffer; (e) desulfonation reagent; and (f) an elution buffer.

[0131] In some embodiments, the kit includes a denaturing agent to denature double-strand DNA (dsDNA) molecules, wherein the denaturing agent is sodium hydroxide (NaOH).

[0132] Processes

[0133] FIG.1 is a block diagram of an example method 100, which is an example of a method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, and subsequent recovery of converted single-stranded DNA (ssDNA) molecules, in accordance with one embodiment of the presently disclosed subject matter. It is notable that any of the disclosed subject matter, where it is described herein as relating or use in association with DNA, e.g., cfDNA, can be applied to other nucleic acids, such as RNA, e.g., cfRNA.

[0134] As shown in FIG.1, at Step 102, a sample is obtained or provided including a plurality of double-strand DNA (dsDNA) molecules. In general, any sample containing double-stranded DNA (dsDNA) molecules can be used. The sample may be a sample selected from the group consisting of blood, plasma, serum, urine, fecal, buffy coat, and saliva samples. Alternatively, the test sample may comprise a sample selected from the group consisting of whole blood, a blood fraction, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, and peritoneal fluid. In one embodiment, the dsDNA sample comprises cell-free DNA (cfDNA). In some embodiments, the dsDNA is circulating tumor DNA (ctDNA).

[0135] In some embodiments, a denaturation step 104 is carried out. In step 104, double- strand DNA (dsDNA) molecules of the sample are denatured to produce single-strand DNA (ssDNA) molecules. In some versions, dsDNA molecules can be denatured using chemical, e.g., a denaturing reagent, and / or heating methods. In one embodiment, the dsDNA is denatured to form ssDNA using sodium hydroxide (NaOH). In another embodiment, the dsDNA is heated (e.g., to temperatures of 60ºC or above) to produce ssDNA molecules. In some aspects, step 104 and / or step 106 includes heating the sample to a denaturation temperature, e.g., 50ºC or above, 55ºC or above, 60ºC or above, 65ºC or above, 70ºC or above 75ºC or above, 80ºC or above, for a denaturation time, e.g., 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. Also, in some embodiments, an independent proactive denaturation step is not carried out. In some versions, step 104 is performed before commencingAttorney Docket No.202323-624601 step 106. Where an independent denaturation step, e.g., denaturing by heating and / or adding a denaturing reagent, is not proactively carried out, denaturation can occur during step 106.

[0136] At step 106, the ssDNA molecules are contacted with a sulfonation reagent to produce sulfonated DNA molecules. In some versions, and as shown in FIG. 1, steps 104 and 106 are carried out and / or occur simultaneously. In accordance with the presently disclosed subject matter, the sulfonated reagent can be a concentrated sulfonation reagent that allows for rapid sulfonation of DNA molecules. The sulfonation reagent can be any of the sulfonation reagents disclosed herein, including with respect to any of the various aspects or embodiments herein. For example, as described elsewhere herein, the concentrated sulfonation reagent can be ammonium bisulfite having a concentration of at least 45%. In some embodiments, the concentrated sulfonation reagent comprises ammonium bisulfite having a concentration of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or more. The concentrated sulfonation reagent can further comprise a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). In accordance with the presently disclosed subject matter, the use of a concentrated sulfonation reagent (e.g., a high concentration of ammonium bisulfite) provides for efficient sulfonation of DNA in a shorter time than sulfonation with conventional bisulfite reagents. In some embodiments, the sulfonation reaction is carried out with heat to speed up the reaction time. For example, the sulfonation reaction can be allowed to proceed for about 60 min or less at reaction temperatures from about 50ºC to about 80ºC. In other embodiments, the sulfonation reaction can proceed for about 2 min to about 60 min, from about 5 min to about 45 min, from about 5 min to about 30 min, or from about 10 min to about 20min. In an embodiment, the sulfonation reagent comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA, and the sulfonation reaction is carried out in 30 min or less at a temperature of 60ºC or more. In some embodiments, the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of about 5.0 M to about 9 M. In some embodiments, the sulfonation reagent comprises metabisulfite. In some embodiments, the sulfonation reagent comprises a mixture of an ammonium bisulfite solution with one or more of a bisulfite (e.g., sodium bisulfite) and a sulfite (e.g., ammonium sulfite monohydrate).

[0137] At step 108, the sulfonated DNA can be bound to a substrate such as silica-coated magnetic beads in the presence of a non-chaotropic (or chaotropic-free) binding buffer. The use of magnetic beads provides a convenient and simple way to recover or purify the sulfonated DNA from the sulfonation reagent, e.g., through the use of a magnetic separation step or through the use of a magnet. The application of a non-chaotropic (or chaotropic-free) binding buffer can increase the affinity of the sulfonated DNA for the magnetic beads and improve the recovery ofAttorney Docket No.202323-624601 DNA molecules from the reaction. For example, as described elsewhere herein, the non- chaotropic binding buffer or chaotropic-free binding buffer is selected from: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; and (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate, or any combination thereof. A non-chaotropic binding buffer or chaotropic-free binding buffer can have any one or more of the following components: KCl; potassium acetate; glycine, sodium citrate; Tris acetate and ammonium sulfate; Tris acetate; ammonium sulfate, or any combination thereof. In some embodiments, the non-chaotropic binding buffer or chaotropic-free binding buffer has a pH of from 3.5 to 7. The magnetic beads can be, for example, silica-coated magnetic beads, such as, silica-coated Dynabeads™ MyOne™ SILANE (Thermo Fisher Scientific, Waltham, MA), SeraSil-Mag™ silica-coated magnetic beads (Cytiva, Marlborough, MA), MagIso™ silica beads (Creative Diagnostics, Shirley, NY), or similar silica-coated magnetic beads. In various embodiments, a non-chaotropic buffer or chaotropic-free binding buffer has a concentration ranging, for example, from: 0.01 M to 5 M, .05 M to 4 M, .05 M to 3 M, .1 M to 4 M, .2 M to 4 M, .4 M to 4 M, .5 M to 4 M, .15 M to 4 M, .15 M to 5 M, .15 M to 3 M, .2 M to 3 M, .35 M to 4 M, or .5 M to 5 M.

[0138] At step 110, magnetic-bead bound DNA molecules are contacted with a desulfonation reagent allowing for desulfonation of sulfonated uracil bases in the ssDNA molecule. Any of a variety of suitable desulfonation reagents can be used in the practice of the presently disclosed subject matter for desulfonation of sulfonated, bead bound ssDNA molecules. For example, in some embodiments, the desulfonation reagent comprises ethanol, isopropanol, NaOH, sodium carbonate, sodium bicarbonate and / or boric acid. In some embodiments, the desulfonation reagent comprises from about 40% to about 60% ethanol and from about 40% to about 60% of a mixture including sodium carbonate and sodium bicarbonate, and NaOH / HCl. In an embodiment, the desulfonation reagent comprises 38.3 mM sodium carbonate, 11.5 mM sodium bicarbonate, 34.8 mM NaOH, 37 mM HCl and 40% ethanol. In another embodiment, the desulfonation reagent comprises 52 mM sodium carbonate, 13 mM sodium bicarbonate, and 43% ethanol..

[0139] At step 112, the converted ssDNA molecules are recovered from the magnetic beads by eluting the converted ssDNA with an elution buffer. In one embodiment, the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5. In some variations, the magnetic beads and bound, converted ssDNA can be washed with a wash buffer before eluting the converted ssDNA. For example, the beads-ssDNA complex can be washed using a washing buffer including from about 60% to about 90% ethanol.Attorney Docket No.202323-624601

[0140] FIG. 2 is a flowchart illustrating a method 200 for treating double-strand DNA (dsDNA) molecules to convert unmethylated cytosine bases to uracil bases and for subsequent recovery of converted, ssDNA molecules, in accordance with one embodiment of the presently disclosed subject matter.

[0141] At step 202, a sample is obtained or provided containing a plurality of DNA molecules. In general, any sample containing double-stranded DNA (dsDNA) molecules can be used. In one embodiment, the dsDNA sample comprises cell-free DNA (cfDNA). In some embodiments, the dsDNA is circulating tumor DNA (ctDNA). The test sample may be a sample selected from the group consisting of blood, plasma, serum, urine, fecal, and saliva samples. Alternatively, the test sample may comprise a sample selected from the group consisting of whole blood, a blood fraction, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, and peritoneal fluid.

[0142] At step 204, dsDNA molecules are denatured using NaOH to ssDNA molecules. In accordance with this step, dsDNA can be incubated with sodium hydroxide (NaOH) to denature dsDNA molecules. The incubation, or any incubation period described herein, can be from about 2 min to about 60 min, from about 5 min to about 30 min, or from about 10 min to about 20 min at a temperature from about 60ºC to about 95ºC. In some versions of the subject embodiments, and as shown in FIG. 2, the methods include progressing from step 202 to step 206 and / or performing steps 204 and steps 206 simultaneously. In some versions, a proactive denaturation step, e.g., 204, is not conducted. Any incubation described herein can be for a length of time e.g., 60 min or less, 50 min or less, 40 min or less, 30 min or less, 20 min or less, 10 min or less, 65 min or less, 55 min or less, 45 min or less, 35 min or less, 25 min or less, 15 min or less, or 5 min or less. Any incubation described herein can be for a length of time e.g., 60 min or more, 50 min or more, 40 min or more, 30 min or more, 20 min or more, 10 min or more, 65 min or more, 55 min or more, 45 min or more, 35 min or more, 25 min or more, 15 min or more, or 5 min or more.

[0143] At step 206, a sulfonation reagent is added to the solution including ssDNA to produce a mixture including sulfonated ssDNA molecules, wherein the sulfonated ssDNA molecules comprise one or more sulfonated deaminated cytosines. As described elsewhere herein, the sulfonated reagent can be a concentrated sulfonation reagent that allows for rapid sulfonation of DNA molecules. For example, the concentrated sulfonation reagent can be ammonium bisulfite having a concentration of at least 45%, and in some variations, can further comprise a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). In accordance with the presently disclosed subject matter, the use of a concentrated sulfonation reagent (e.g., a high concentration of ammonium bisulfite) provides for efficient sulfonation of DNA in a shorter time thanAttorney Docket No.202323-624601 sulfonation with conventional bisulfite reagents. For example, the sulfonation reaction can be allowed to proceed for about 60 min or less at reaction temperatures from 50ºC to 80ºC. In one embodiment, the sulfonation reagent comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA, and the sulfonation reaction is carried out in 30 min or less at a temperature of 60ºC or more. In some aspects, step 206 includes heating the sample to a temperature, e.g., 50ºC or above, 55ºC or above, 60ºC or above, 65ºC or above, 70ºC or above 75ºC or above, 80ºC or above, for a time, e.g., 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more. In some embodiments, denaturation takes place during step 206.

[0144] At step 208, the sulfonated ssDNA molecules are combined with silica-coated magnetic beads and a chaotropic-free binding buffer and incubated to produce bead-bound sulfonated ssDNA molecules. The use of magnetic beads provides a way to recover or purify the sulfonated DNA from the sulfonation reagent, e.g., through the use of a magnetic separation step or through the use of a magnet. The use of a chaotropic-free binding buffer can increase the affinity of the sulfonated DNA for the silica-coated magnetic beads and improve the recovery of DNA molecules from the reaction. For example, as described elsewhere herein, the chaotropic-free binding buffer is selected from the group consisting of: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; and (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate. In some embodiments, the chaotropic-free binding buffer has a pH of from about 3.5 to about 7. The silica-coated magnetic beads can be, for example, silica-coated Dynabeads™ MyOne™ SILANE (Thermo Fisher Scientific, Waltham, MA), SeraSil-Mag™ silica-coated magnetic beads (Cytiva, Marlborough, MA), MagIso™ silica beads (Creative Diagnostics, Shirley, NY), or similar silica-coated magnetic beads.

[0145] At step 210, bead-bound ssDNA molecules are collected from the chaotropic-free binding buffer and the collected bead-bound sulfonated ssDNA molecules are contacted with a desulfonation reagent. In some embodiments, the bead-bound ssDNA molecules can be collected using a magnetic force, such as a magnet. In some embodiments, the desulfonation reagent comprises ethanol, isopropanol, NaOH, sodium carbonate, sodium bicarbonate and / or boric acid. In some embodiments, the desulfonation reagent comprises from about 40% to about 60% ethanol and from about 40% to about 60% of a mixture including sodium carbonate and sodium bicarbonate, and NaOH / HCl. In one embodiment, the desulfonation reagent comprises 38.3 mM sodium carbonate, 11.5 mM sodium bicarbonate, 34.8 mM NaOH, 37 mM HCl and 40% ethanol.Attorney Docket No.202323-624601 In another embodiment, the desulfonation reagent comprises 52 mM sodium carbonate, 13 mM sodium bicarbonate, and 43% ethanol.

[0146] At step 212, the converted ssDNA molecules are eluted from the silica-coated magnetic beads using an elution buffer. The elution buffer can comprise 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5. In some variations, the magnetic beads and bound, converted ssDNA can be washed with a wash buffer before eluting the converted ssDNA. For example, the beads-ssDNA complex can be washed using a washing buffer including from about 60% to about 90% ethanol.

[0147] FIG. 3 is a flowchart illustrating a method 300 for treating small DNA molecules to convert unmethylated cytosine bases to uracil bases and for subsequent recovery of converted small DNA molecules, in accordance with one embodiment of the presently disclosed subject matter.

[0148] At step 302, a sample is provided or obtained including small double-strand DNA molecules. In general, any sample containing double-stranded DNA (dsDNA) molecules can be used. In one embodiment, the dsDNA sample comprises cell-free DNA (cfDNA). In some embodiments, the dsDNA is circulating tumor DNA (ctDNA). The test sample may be a sample selected from the group consisting of blood, plasma, serum, urine, fecal, and saliva samples. Alternatively, the test sample may comprise a sample selected from the group consisting of whole blood, a blood fraction, a tissue biopsy, pleural fluid, pericardial fluid, cerebral spinal fluid, and peritoneal fluid. In accordance with this embodiment, the sample comprises small dsDNA molecules of approximately 500 base pairs or less in length. In some embodiments the small DNA molecules comprise DNA molecules of 500, 400, 300, 200 or fewer nucleotides in length.

[0149] At step 304, the small, double-strand DNA (dsDNA) molecules are denatured to produce a plurality of single-strand DNA (ssDNA) molecules. Step 304, can be taken and completed before step 306 or can be executed fully or partially simultaneously with step 306. In some aspects, steps 304 and 306 begin at the same time as a result of exposure to denaturation conditions, for example, addition of the sulfonation reagent and / or heating the mixture to a denaturation temperature for a denaturation time. In some versions, step 304 occurs automatically as a condition of other steps, e.g., step 306, and is not a proactive step requiring denaturation reagent addition and / or heating and subsequent cooling exclusively for denaturation. Any of a variety of suitable means for denaturing dsDNA molecules can be used in the practice of the presently disclosed subject matter. For example, dsDNA molecules can be denatured using chemical, mechanical or other means. In one embodiment, the dsDNA is denatured to form ssDNAAttorney Docket No.202323-624601 using sodium hydroxide (NaOH). In another embodiment, the dsDNA is heated (e.g., to temperatures above 60ºC) to produce ssDNA molecules. In some versions of the subject embodiments, and as shown in FIG.3, the methods include progressing from step 302 to step 306 and / or performing steps 304 and steps 306 simultaneously. In some versions, a proactive denaturation step, e.g., 304, is not conducted.

[0150] At step 306, the small ssDNA molecules are combined with a sulfonation reagent to produce sulfonated ssDNA molecules. As described elsewhere herein, the sulfonated reagent can be a concentrated sulfonation reagent that allows for rapid sulfonation of DNA molecules. For example, the concentrated sulfonation reagent can be ammonium bisulfite having a concentration of at least 45%, and in some variations, can further include a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). In accordance with the presently disclosed subject matter, the use of a concentrated sulfonation reagent (e.g., a high concentration of ammonium bisulfite) provides for efficient sulfonation of DNA in a shorter time than sulfonation with conventional bisulfite reagents. For example, the sulfonation reaction can be allowed to proceed for about 60 min or less at reaction temperatures from 50ºC to 80ºC. In one embodiment, the sulfonation reagent comprises 65% ammonium bisulfite, and optionally 0.1 mM EDTA, and the sulfonation reaction is carried out in 30 min or less at a temperature of 60ºC or more. In some embodiments, step 306 includes heating the sample to a temperature, e.g., 50ºC or above, 55ºC or above, 60ºC or above, 65ºC or above, 70ºC or above 75ºC or above, 80ºC or above, for a time, e.g., 10 minutes or more, 15 minutes or more, 20 minutes or more, 25 minutes or more, or 30 minutes or more.

[0151] At step 308, the sulfonated small ssDNA molecules are combined with silica-coated magnetic beads and a non-chaotropic based binding buffer and incubating to produce bead-bound sulfonated small ssDNA molecules.

[0152] As previously described, the use of magnetic beads provides a convenient and simple way to recover or purify sulfonated DNA and the use of a non-chaotropic based binding buffer can increase the affinity of the sulfonated DNA for the silica-coated magnetic beads and improve the recovery of DNA molecules from the reaction. Subject non-chaotropic binding buffers can include one or more of: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate. Silica-coated magnetic beads can include: silica-coated Dynabeads™ MyOne™ SILANE (Thermo FisherAttorney Docket No.202323-624601 Scientific, Waltham, MA), SeraSil-Mag™ silica-coated magnetic beads (Cytiva, Marlborough, MA), MagIso™ silica beads (Creative Diagnostics, Shirley, NY), or similar silica-coated magnetic beads.

[0153] At step 310, bead-bound sulfonated small ssDNA molecules are collected from the non-chaotropic binding buffer and contacting the collected bead-bound sulfonated small DNA molecules with a desulfonation reagent.

[0154] As previously described, the beads and the bead-bound ssDNA molecules can be collected using a magnetic force, such as a magnet. The desulfonation reagent can be ethanol, isopropanol, NaOH, sodium carbonate, sodium bicarbonate and / or boric acid.

[0155] At step 312, the converted small ssDNA molecules are eluted from the silica-coated magnetic beads using an elution buffer, as previously described. In one embodiment, the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from about 6.5 to about 8.5. In some versions, the magnetic beads and bound, converted ssDNA can be washed with a wash buffer before eluting the converted ssDNA.

[0156] In some embodiments, the non-chaotropic (or chaotropic-free) DNA binding buffers described herein are implemented in performing DNA extraction, which refers to recovering nucleic acids, e.g., RNA and / or DNA, such as cfDNA or cfRNA, from a biological sample, e.g., a plasma sample. In some embodiments, DNA extraction includes the use of a non-chaotropic (or chaotropic-free) DNA binding buffer including, for example, a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate- ammonium sulfate buffer, or any combination thereof, or any other buffer described herein. In some versions, a glycine-KCl buffer is used to bind one or more nucleic acids to a bead. Using such buffers to bind the nucleic acids to a bead in order to perform nucleic acid extraction can be executed prior to performing a bisulfite conversion process, such as by converting unmethylated cytosine bases to uracil bases.

[0157] In various embodiments, isolating nucleic acid from plasma, such as by performing DNA extraction, includes performing protein lysis and / or contaminant removal using chaotropic reagents and / or proteinase on a blood plasma sample. Subsequently, nucleic acid capture and recovery may be performed using beads, e.g., magnetic silica beads, or any beads described herein. Any of the buffers described herein, e.g., non-chaotropic (or chaotropic-free), DNA binding buffers described herein may be implemented in association with a substrate (e.g., beads), so as to bind nucleic acids to the substrate and / or remove nucleic acids from the sample.Attorney Docket No.202323-624601

[0158] In some versions, the methods include removing red blood cells from a blood sample to provide plasma by, e.g., centrifuging the sample one, two, or three or more times. In various embodiments, the methods include providing a sample of plasma, e.g, purified plasma, in the presence of one or more non-chaotropic (or chaotropic-free) DNA binding buffers and beads to bind nucleic acids to the beads. Such methods may also include isolating blood sample plasma; adding a proteinase to the plasma; binding the DNA to a magnetic bead in a non-chaotropic based binding buffer; and / or isolating the nucleic acids by eluting the DNA molecules from the magnetic bead with an elution buffer. In some versions, nucleic acid extraction from plasma is performed using a Magmax® cfDNA kit.

[0159] In various instances, the methods include isolating nucleic acids from plasma by performing (1) nucleic acid isolation, e.g., DNA, extraction by, for example: isolating blood sample plasma; adding a proteinase to the plasma; binding the DNA from the proteinase-treated sample to a magnetic bead in a non-chaotropic based binding buffer; and / or isolating the nucleic acids by eluting the DNA molecules from the magnetic bead with an elution buffer; and then subsequently performing (2) a bisulfite conversion process by, for example, treating nucleic acid molecules to convert unmethylated cytosine bases to uracil bases, the method including: contacting nucleic acid molecules with a sulfonation reagent to produce sulfonated nucleic acid, wherein the sulfonated nucleic acid includes one or more sulfonated deaminated cytosines; binding said sulfonated nucleic acid to a silica-coated magnetic bead in a binding buffer, wherein the binding buffer is a non-chaotropic based binding buffer; contacting the magnetic-bead bound nucleic acid molecules with a desulfonation reagent; and / or eluting the nucleic acid molecules from the magnetic beads with an elution buffer, or any combination thereof. In some embodiments, the methods include isolating nucleic acids from plasma by performing (1) nucleic acid isolation, e.g., DNA, extraction, and then (2) treating nucleic acid molecules to convert unmethylated cytosine bases to uracil bases.

[0160] In various embodiments, the methods, such as methods for isolating nucleic acids, e.g,. DNA, from plasma, include steps, e.g., sequential steps, of performing (1) nucleic acid denaturation; (2) sulfonation and / or hydrolytic deamination; (3) nucleic acid binding to beads, e.g., magnetic beads; (4) desulfonation; and / or (5) elution. Denaturation can be performed by example by adding a reagent such as NaOH to a sample. Step (2) can be performed by adding a formulated ammonium bisulfite solution. Step (3) can be performed by running the sample over beads, e.g., magnetic beads, such as Dynabeads® in a container along with any of the buffers, e.g., binding buffers described herein. One or more, e.g., two washes of the beads can be performed before step (4). Step (4) can be performed by adding any of the buffers, e.g,Attorney Docket No.202323-624601 desulphonation buffers, described herein. One or more, e.g., two washes of the beads can be performed before step (5). Step (5) can be performed by adding a reagent such as Tris-HCl to the sample. Any one or combination of such steps can be performed for a length of time, e.g, 60 min or less, 30 min or less, 20 min or less, 15 min or less, 10 min or less, 5 min or less, or 3 min or less. Any one or combination of such steps can be performed for a length of time, e.g, 60 min or more, 30 min or more, 20 min or more, 15 min or more, 10 min or more, 5 min or more, or 3 min or more. Any one or combination of such steps can be performed for a length of time ranging, for example, from 3 min to 30 min, such as 5 min to 20 min, or 5 min to 15 min. ILLUSTRATIVE EMBODIMENTS

[0161] The present disclosure provides the following illustrative embodiments.

[0162] Embodiment 1: A method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method comprising: (a) contacting DNA molecules with a sulfonation reagent to produce sulfonated DNA comprising one or more sulfonated deaminated cytosines; (b) binding the sulfonated DNA to a substrate in a non-chaotropic based binding buffer; optionally wherein the substrate comprises (i) a surface comprising silica, and / or (ii) a magnetic bead; (c) contacting the substrate-bound DNA molecules with a desulfonation reagent; and (d) eluting the DNA molecules from the substrate with an elution buffer.

[0163] Embodiment 2: The method according to embodiment 1, wherein the ssDNA molecules are ssDNA molecules having a length of 400 bp or less.

[0164] Embodiment 3: The method according to any one of the preceding embodiments, further comprising denaturing the DNA molecules to produce single-strand DNA molecules prior to step (a), optionally wherein the denaturing comprises heating, adding sodium hydroxide (NaOH), or both heating and adding NaOH.

[0165] Embodiment 4: The method according to any one of the preceding embodiments, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.005 mM to 1 mM EDTA.

[0166] Embodiment 5: The method according to embodiment 4, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.Attorney Docket No.202323-624601

[0167] Embodiment 6: The method according to any one of the preceding embodiments, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer, or any combination thereof.

[0168] Embodiment 7: The method according to embodiment 6, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

[0169] Embodiment 8: The method according to embodiment 6, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

[0170] Embodiment 9: The method according to embodiment 6, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0171] Embodiment 10: The method according to embodiment 6, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0172] Embodiment 11: The method according to embodiment 6, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0173] Embodiment 12: The method according to any one of embodiments 6 or 9-11, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

[0174] Embodiment 13: The method according to embodiment 6, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

[0175] Embodiment 14: The method according to any one of embodiments 6 or 13, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

[0176] Embodiment 15: The method according to any one of embodiments 1-6, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25Attorney Docket No.202323-624601 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0177] Embodiment 16: The method according to any one of the preceding embodiments, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

[0178] Embodiment 17: The method according to any one of the preceding embodiments, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate and sodium bicarbonate.

[0179] Embodiment 18: The method according to embodiment 17, wherein the desulfonation reagent comprises sodium carbonate and sodium bicarbonate.

[0180] Embodiment 19: The method according to embodiment 17, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol, (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

[0181] Embodiment 20: The method according to embodiment 17, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0182] Embodiment 21: The method according to embodiment 17, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% isopropanol, and (ii) from 30% to 70% of a mixture comprising boric acid, NaOH, and KCl.

[0183] Embodiment 22: The method according to embodiment 17, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

[0184] Embodiment 23: The method according to any one of the preceding embodiments, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (c) or before eluting the DNA molecules from the substrate in step (d).

[0185] Embodiment 24: The method according to embodiment 23, wherein the non- chaotropic binding buffer is used as the washing buffer.Attorney Docket No.202323-624601

[0186] Embodiment 25: The method according to embodiment 23, wherein the washing buffer comprises from 60% to 90% ethanol.

[0187] Embodiment 26: The method according to any one of the preceding embodiments, wherein the elution buffer comprises Tris HCl.

[0188] Embodiment 27: The method according to any one of the preceding embodiments, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH ranging from 6.5 to 8.5.

[0189] Embodiment 28: The method according to any one of the preceding embodiments, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 60 minutes or less.

[0190] Embodiment 29: The method according to any one of the preceding embodiments, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 45 minutes or less.

[0191] Embodiment 30: The method according to any one of the preceding embodiments, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 30 minutes or less.

[0192] Embodiment 31: A method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method comprising: (a) adding NaOH to a sample solution comprising DNA to produce a solution comprising single-stranded DNA molecules (ssDNA); (b) adding a sulfonation reagent to the solution comprising ssDNA to produce a mixture comprising sulfonated ssDNA molecules, wherein the sulfonated ssDNA molecules comprise one or more sulfonated deaminated cytosines; (c) combining the sulfonated ssDNA molecules with a substrate and a chaotropic-free binding buffer to produce substrate-bound sulfonated ssDNA molecules; optionally wherein the substrate comprises silica-coated magnetic beads; (d) collecting substrate- bound ssDNA molecules from the chaotropic-free binding buffer, and contacting the collected bead-bound sulfonated ssDNA molecules with a desulfonation reagent to produce substrate-bound converted ssDNA molecules; and (e) eluting converted ssDNA molecules to provide a sample comprising converted ssDNA molecules.

[0193] Embodiment 32: The method of embodiment 31, wherein the ssDNA molecules comprise small ssDNA molecules comprising 300 bp or less in length.Attorney Docket No.202323-624601

[0194] Embodiment 33: The method to embodiment 31, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.05 mM to 1 mM EDTA.

[0195] Embodiment 34: The method according to embodiment 31, wherein the sulfonation reagent comprises 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

[0196] Embodiment 35: The method according to any one of embodiments 31-34, wherein the chaotropic-free binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

[0197] Embodiment 36: The method according to embodiment 35, wherein the chaotropic- free binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

[0198] Embodiment 37: The method according to embodiment 35, wherein the chaotropic- free binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

[0199] Embodiment 38: The method according to embodiment 35, wherein the chaotropic- free binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0200] Embodiment 39: The method according to embodiment 35, wherein the chaotropic- free binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0201] Embodiment 40: The method according to embodiment 35, wherein the chaotropic- free binding buffer is sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0202] Embodiment 41: The method according to any one of embodiments 35 or 38-40, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

[0203] Embodiment 42: The method according to embodiment 35, wherein the chaotropic- free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601

[0204] Embodiment 43: The method according to embodiment 35 or 42, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

[0205] Embodiment 44: The method according to any one of embodiments 31-35, wherein the chaotropic-free binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0206] Embodiment 45: The method according to any one of embodiments 31-44, wherein the chaotropic-free binding buffer has a pH ranging from 3.5 to 7.

[0207] Embodiment 46: The method according to any one of embodiments 31-45, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0208] Embodiment 47: The method according to embodiment 46, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0209] Embodiment 48: The method according to embodiment 46, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol; (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

[0210] Embodiment 49: The method according to embodiment 46, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0211] Embodiment 50: The method according to embodiment 46, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% isopropanol, and (ii) from 30% to 70% of a mixture comprising boric acid, NaOH, and KCl.

[0212] Embodiment 51: The method according to embodiment 46, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.Attorney Docket No.202323-624601

[0213] Embodiment 52: The method according to any one of embodiments 31-51, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (d) or before eluting the DNA molecules from the substrate in step (e).

[0214] Embodiment 53: The method according to embodiment 52, wherein the chaotropic- free binding buffer is used as the washing buffer.

[0215] Embodiment 54: The method according to embodiment 52, wherein the washing buffer comprises from 60% to 90% ethanol.

[0216] Embodiment 55: The method according to any one of embodiments 31-54, wherein the elution buffer comprises Tris HCl.

[0217] Embodiment 56: The method according to any of embodiments 31-55, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH ranging from 6.5 to 8.5.

[0218] Embodiment 57: The method according to any one of embodiments 31-56, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 60 minutes or less.

[0219] Embodiment 58: The method according to any one of embodiments 31-56, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 45 minutes or less.

[0220] Embodiment 59: The method according to any one of embodiments 31-56, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 30 minutes or less.

[0221] Embodiment 60: A method for bisulfite-converting small DNA molecules, the method comprising: (a) in a solution combining small DNA molecules with a sulfonation reagent to produce sulfonated small DNA molecules, wherein said small DNA molecules comprise 300 or fewer bases in length; (b) combining the sulfonated small DNA molecules in the solution of step (a) with a substrate and a non-chaotropic based binding buffer to produce substrate-bound sulfonated small DNA molecules; optionally wherein the substrate comprises silica-coated magnetic beads; (c) collecting substrate-bound sulfonated small DNA molecules from the non- chaotropic binding buffer, and contacting the collected bead-bound sulfonated small DNA molecules with a desulfonation reagent to produce substrate-bound converted small DNA; and (d) eluting converted small DNA molecules to provide a sample comprising converted small DNA molecules.Attorney Docket No.202323-624601

[0222] Embodiment 61: The method according to embodiment 60, wherein the small DNA molecules are denatured to produced small single-stranded DNA (ssDNA) molecules prior to step (a), and wherein the denaturing comprises heat denaturing and / or denaturing using sodium hydroxide (NaOH).

[0223] Embodiment 62: The method according to any one of embodiments 60-61, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.005 mM to 1 mM EDTA.

[0224] Embodiment 63: The method according to embodiment 62, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

[0225] Embodiment 64: The method according to any one of embodiments 60-63, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

[0226] Embodiment 65: The method according to embodiment 64, wherein the non- chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

[0227] Embodiment 66: The method according to embodiment 64, wherein the non- chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentrating ranging from 2.7 M to 3.3 M.

[0228] Embodiment 67: The method according to embodiment 64, wherein the non- chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine- KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0229] Embodiment 68: The method according to embodiment 64, wherein the non- chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0230] Embodiment 69: The method according to embodiment 64, wherein the non- chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601

[0231] Embodiment 70: The method according to any one of embodiments 64 or 67-69, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

[0232] Embodiment 71: The method according to embodiment 64, wherein the non- chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

[0233] Embodiment 72: The method according to any one of embodiments 64 or 71, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

[0234] Embodiment 73: The method according to any one of embodiments 60-72, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0235] Embodiment 74: The method according to any one of embodiments 60-73, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

[0236] Embodiment 75: The method according to any one of embodiments 60-74, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0237] Embodiment 76: The method according to embodiment 75, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0238] Embodiment 77: The method according to embodiment 75, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol; (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.Attorney Docket No.202323-624601

[0239] Embodiment 78: The method according to embodiment 75, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0240] Embodiment 79: The method according to embodiment 75, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH, and KCl.

[0241] Embodiment 80: The method according to embodiment 75, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

[0242] Embodiment 81: The method according to any one of embodiments 60-80, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (c) or before eluting the DNA molecules from the substrate in step (d).

[0243] Embodiment 82: The method according to embodiment 81, wherein the non- chaotropic binding buffer is used as the washing buffer.

[0244] Embodiment 83: The method according to embodiment 81, wherein the washing buffer comprises from 60% to 90% ethanol.

[0245] Embodiment 84: The method according to any one of embodiments 60-83, wherein the elution buffer comprises Tris HCl.

[0246] Embodiment 85: The method according to any one of embodiments 60-84, wherein the elution buffer comprises 10 mM Tris HCl at a pH ranging from 6.5 to 8.5.

[0247] Embodiment 86: The method according to any one of embodiments 60-85, wherein the sulfonation reagent is brought into contact with the small DNA molecules in step (a) for 60 minutes or less.

[0248] Embodiment 87: The method according to any one of embodiments 60-85, wherein the sulfonation reagent and the small DNA molecules in step (a) are incubated for 45 minutes or less.Attorney Docket No.202323-624601

[0249] Embodiment 88: The method according to any one of embodiments 60-85, wherein the sulfonation reagent and the small DNA molecules in step (a) are incubated for 30 minutes or less.

[0250] Embodiment 89: The method according to any one of embodiments 1-3, 6-32, 35-61, or 64-88, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

[0251] Embodiment 90: The method according to any one of embodiments 1-89, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0252] Embodiment 91: A kit for treating DNA comprising: (a) a sulfonation reagent; (b) silica-coated magnetic beads; (c) a non-chaotropic binding buffer; and (d) a desulfonation reagent.

[0253] Embodiment 92: The kit of embodiment 91, wherein the kit further comprises a denaturing agent to denature double-strand DNA (dsDNA) molecules, and wherein the denaturing agent is sodium hydroxide (NaOH).

[0254] Embodiment 93: The kit according to embodiment 91, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.01 mM to 1 mM EDTA.

[0255] Embodiment 94: The kit according to embodiment 91, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

[0256] Embodiment 95: The kit according to any one of embodiments 91-94, wherein the non- chaotropic binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

[0257] Embodiment 96: The kit according to embodiment 95, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

[0258] Embodiment 97: The kit according to embodiment 95, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.Attorney Docket No.202323-624601

[0259] Embodiment 98: The kit according to embodiment 95, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0260] Embodiment 99: The kit according to embodiment 95, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0261] Embodiment 100: The kit according to embodiment 95, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0262] Embodiment 101: The kit according to any one of embodiments 95 or 98-100, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

[0263] Embodiment 102: The kit according to embodiment 95 wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

[0264] Embodiment 103: The kit according to any one of embodiments 95 or 102, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

[0265] Embodiment 104: The kit according to embodiment 91, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0266] Embodiment 105: The kit according to any one of embodiments 91-104, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

[0267] Embodiment 106: The kit according to embodiment 91, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid, or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.Attorney Docket No.202323-624601

[0268] Embodiment 107: The kit according to embodiment 106, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0269] Embodiment 108: The kit according to embodiment 106, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol; (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

[0270] Embodiment 109: The kit according to embodiment 106, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0271] Embodiment 110: The kit according to embodiment 106, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH and KCl.

[0272] Embodiment 111: The kit according to embodiment 106, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

[0273] Embodiment 112: A kit for treating DNA comprising: (a) an ammonium bisulfite sulfonation reagent; (b) silica-coated magnetic beads; (c) a chaotropic-free binding buffer; (d) a washing buffer; (e) a desulfonation reagent; and (f) an elution buffer.

[0274] Embodiment 113: The kit of embodiment 112, wherein the kit further comprises a denaturing agent to denature double-strand DNA (dsDNA) molecules, and wherein the denaturing agent is sodium hydroxide (NaOH).

[0275] Embodiment 114: The kit of embodiment 112, wherein the ammonium bisulfite sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.01 mM to 1 mM EDTA.

[0276] Embodiment 115: The kit according to embodiment 112, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

[0277] Embodiment 116: The kit according to any one of embodiments 112-115, wherein the chaotropic-free binding buffer is selected from the group consisting of a potassium chloride (KCl)Attorney Docket No.202323-624601 buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

[0278] Embodiment 117: The kit according to embodiment 116, wherein the chaotropic-free binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

[0279] Embodiment 118: The kit according to embodiment 116, wherein the chaotropic-free binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

[0280] Embodiment 119: The kit according to embodiment 116, wherein the chaotropic-free binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl binding buffer is at a concentration ranging from 0.05 M to 2 M.

[0281] Embodiment 120: The kit according to embodiment 116, wherein the chaotropic-free binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0282] Embodiment 121: The kit according to embodiment 116, wherein the chaotropic-free binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate- KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0283] Embodiment 122: The kit according to any one of embodiments 116 or 119-121, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

[0284] Embodiment 123: The kit according to embodiment 116, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

[0285] Embodiment 124: The kit according to any one of embodiments 116 or 123, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

[0286] Embodiment 125: The kit according to embodiment 116, wherein the chaotropic-free binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 MAttorney Docket No.202323-624601 KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

[0287] Embodiment 126: The kit according to any one of embodiments 116-125, wherein the chaotropic-free binding buffer has a pH ranging from 3.5 to 7.

[0288] Embodiment 127: The kit according to embodiment 116, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid, or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0289] Embodiment 128: The kit according to embodiment 127, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0290] Embodiment 129: The kit according to embodiment 127, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol, (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

[0291] Embodiment 130: The kit according to embodiment 127, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

[0292] Embodiment 131: The kit according to embodiment 127, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH and KCl.

[0293] Embodiment 132: The kit according to embodiment 127, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

[0294] Embodiment 133: The kit according to any one of embodiments 112-132, wherein the chaotropic-free binding buffer is used as the washing buffer.

[0295] Embodiment 134: The kit according to any one of embodiments 112-133, wherein the washing buffer comprises from 60% to 90% ethanol.

[0296] Embodiment 135: The kit according to any one of embodiments 112-134, wherein the elution buffer comprises Tris HCl.Attorney Docket No.202323-624601

[0297] Embodiment 136: The kit according to embodiment 135, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from 6.5 to 8.5.

[0298] Embodiment 137: The kit according to any one of embodiments 91, 92, 95-113, or 116-136, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

[0299] Embodiment 138: The kit according to any one of embodiments 91-137, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0300] Embodiment 139: The method according to any one of embodiments 1-59, wherein the DNA molecules are high molecular weight (HMW) DNA molecules; optionally wherein the HMW DNA have a length of at least 5 Kb.

[0301] Embodiment 140: A method for isolating nucleic acids from plasma, the method comprising: (a) providing a sample of plasma comprising DNA molecules; (b) adding a proteinase to the plasma; (c) binding the DNA from step (b) to a substrate in a non-chaotropic based binding buffer; optionally wherein the substrate comprises (i) a surface comprising silica, and / or (ii) a magnetic bead; and (d) isolating the nucleic acids by eluting the DNA molecules from the substrate with an elution buffer.

[0302] Embodiment 141: The method according to embodiment 140, further comprising adding one or more chaotropic reagents to the sample.

[0303] Embodiment 142: The method according to embodiments 140 or 141, further comprising treating the eluted DNA to convert unmethylated cytosine bases to uracil bases.

[0304] Embodiment 143: The method according to any one of embodiments 140-142, wherein the DNA comprises cfDNA molecules having a length of 400 bp or less.

[0305] Embodiment 144: The method according to any one of embodiments 140-143, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer, or any combination thereof.

[0306] Embodiment 145: The method according to embodiment 144, wherein the non- chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.Attorney Docket No.202323-624601

[0307] Embodiment 146: The method according to embodiment 144, wherein the non- chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

[0308] Embodiment 147: The method according to embodiment 144, wherein the non- chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine- KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0309] Embodiment 148: The method according to embodiment 144, wherein the non- chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0310] Embodiment 149: The method according to embodiment 144, wherein the non- chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

[0311] Embodiment 150: The method according to embodiment 144, wherein the non- chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

[0312] Embodiment 151: The method according to any one of embodiments 140-150, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

[0313] Embodiment 152: The method according to any one of embodiments 140-151, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before eluting the DNA molecules from the substrate in step (d).

[0314] Embodiment 153: The method according to embodiment 152, wherein the non- chaotropic binding buffer is used as the washing buffer.

[0315] Embodiment 154: The method according to embodiment 152 or 153, wherein the washing buffer comprises from 60% to 90% ethanol.

[0316] Embodiment 155: The method according to any one of embodiments 140-154, wherein the elution buffer comprises Tris HCl.

[0317] Embodiment 156: The method according to any one of embodiments 140-155, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from 6.5 to 8.5.Attorney Docket No.202323-624601

[0318] Embodiment 157: A composition comprising isolated DNA molecules and a sulfonation reagent, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

[0319] Embodiment 158: The composition of embodiment 157, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

[0320] Embodiment 159: A method of deaminating methylated cytosines comprising incubating the composition of embodiment 157 or 158 for a period of time sufficient to deaminate methylated cytosines in the isolated DNA molecules with the sulfonation agent; optionally wherein the period of time is 60 minutes or less. EXAMPLES

[0321] Example 1: Rapid bisulfite conversion and recovery of DNA molecules using various binding buffers

[0322] To evaluate the recovery of converted DNA molecules using methods of the presently disclosed subject matter, a variety of non-chaotropic or chaotropic-free binding buffers were tested and pre-, and post-sequencing data compared to a commercially available kit for bisulfite conversion, EZ DNA Methylation-Lightning™ kit (available from Zymo Research Corp (Irvine, Calif.).

[0323] In addition to the Zymo kit binding buffer, the following binding buffers were tested: (1) 4.1 M GuHCl + 41.1% v / v ethanol; (2) 7M GuHCl + 50 mM Tris-HCl (pH 5.4) (alcohol free); (3) 7 M GuHCl; (4) 2 M GuSCN + 14.4 M trisodium citrate and 41.1% v / v ethanol; (5) 2 M GuSCN + 14.4 M trisodium citrate and 42.8% v / v methanol; (6) 2 M GuSCN + 14.4 M trisodium citrate and 42.8% v / v 1:2 methanol / ethanol; (7) 2M GuSCN + 14.4 M trisodium citrate and 42.8% v / v 1:3 methanol / ethanol (8) 3M GuSCN + 20 mM trisodium citrate (alcohol free); (9) 6 M sodium perchlorate in 50 mM Tris-HCl (pH 5.4); (10) 0.25 M glycine + 400 mM KCl; (11) 0.25 M potassium acetate + 0.15 M KCl (pH 5.4); (12) 0.2 M sodium citrate + 400 mM KCl (pH 5.4); (13) 0.25 M Tris Acetate buffer (pH 4) + 200 mM ammonium sulfate; (14) 0.25 M Tris Acetate buffer + 400 mM ammonium sulfate (pH 5.4); and (15) 0.2 M Tris acetate + 300 mM ammonium sulfate (pH 5.4).Attorney Docket No.202323-624601

[0324] In carrying out this experiment, test samples were obtained including double-strand DNA (dsDNA) molecules. The dsDNA molecules in the test samples were first denatured using 0.32 N NaOH to produce test samples including single-stranded DNA (ssDNA) molecules. The ssDNA sample was contacted with a sulfonation reagent including 65% ammonium bisulfite and 0.1 mM EDTA for 30 min. at 70ºC. Next, the silica-coated magnetic beads (Dynabeads®MyOne Silane (Thermo Fisher Scientific, Waltham, MA)) in the presence of one of the above-noted non- chaotropic binding buffers. Binding incubation was conducted 5 min at room temperature. After incubation the beads were pulled down with a magnet and the supernatant discarded. Magnetization was conducted for 18 min at room temperature. The ssDNA bound beads were then resuspending in a desulfonation buffer including 40% ethanol in a sodium bicarbonate alkaline buffer with final concentration 0.026 M Sodium Carbonate, 0.011 M Sodium Carbonate / bicarbonate, 0.0384 M NaOH, and 0.03696 M HCl and incubated for 13 minutes at room temperature. After incubation the beads were pulled down with a magnet and the supernatant discarded and the beads washed with a washing buffer including 80% ethanol in water. Elution mixing time was 4 min at 1500 rpm. This was followed by 2 min elution incubation. After washing and heat drying the ssDNA bound beads were resuspended in an elution buffer including 10 mM Tris HCl pH 8.0 and allowed to incubate at room temperature for 4 min. with shaking at 1500 rpms. The beads were again exposed to a magnet and the supernatant containing converted ssDNA molecules collected. The collected ssDNA molecules were analyzed and pre- sequencing, and post-sequencing performance metrics are shown in FIGS.4-11.

[0325] FIG.5 is a data plot showing normalized recovery of DNA post bisulfite conversion with the use of various binding buffers. As shown in FIG.5, with the exception of the guanidine thiocyanate based binding buffers, the normalized concentration across various binding buffers was similar. FIGS.5 and 6 provide pre-sequencing performance metrics data. It is notable that FIG.5 represents the pre-sequencing metric of post bisulfite conversion yield and / or recovery. In turn FIG.6 represents a pre-sequencing metric of library prep yield.

[0326] FIG.6 is a data plot showing total recovery of DNA post GRAIL® MethylSeq library preparation with the use of various binding buffers. Total DNA yield was determined using Fragment Analyzer®for DNA quantification (Agilent, Santa Clara, CA). As shown in FIG.6, total recovered DNA was determined to be between approximately 6000 ng and 9000 ng depending on the binding buffer used.

[0327] To evaluate post-sequencing performance of the bisulfite conversion and recovery of converted ssDNA, the recovered ssDNA molecules were enriched using an enrichment panel (see,Attorney Docket No.202323-624601 e.g., WO 2019 / 195268, WO 2020 / 069350, and WO 2020 / 154682, which are each incorporated by reference for all purposes herein), a sequencing library prepared and sequenced using an Illumina (San Diego, CA) sequencing platform, and sequence reads analyzed using a bioinformatic pipeline. Post-sequencing performance metrics are shown in FIGS.4 and 7-11.

[0328] FIG.7 is a data plot showing post-sequencing total DNA fragment count with the use of various binding buffers. As shown in FIG.7, with the exception of the sodium perchlorate based binding buffer, fragment count across various binding buffers was similar.

[0329] FIG.4 is a data plot showing bisulfite conversion ratio with the use of various binding buffers. As shown in FIG.4, with the exception of the sodium perchlorate binding buffer, bisulfite conversion was consistent across the various binding buffers tested at >99%.

[0330] FIG.8 is a data plot showing post-sequencing coverage of abnormal fragments across an enrichment panel with the use of various binding buffers. As shown in FIG. 8, with the exception of the sodium perchlorate based binding buffer, the coverage of abnormal methylated sequence reads per region across the targeted enrichment panel was similar.

[0331] FIG.9 is a data plot showing post-sequencing coverage of hypermethylated abnormal DNA fragments across an enrichment panel with the use of various binding buffers. As shown in FIG.9, again with the exception of the sodium perchlorate based binding buffer, the coverage of hypermethylated abnormal sequence reads per region across the targeted enrichment panel was similar.

[0332] FIG.10 is a data plot showing post-sequencing coverage of hypomethylated abnormal DNA fragments across an enrichment panel with the use of various binding buffers. As shown in FIG.10, again with the exception of the sodium perchlorate based binding buffer, the coverage of hypomethylated abnormal sequence reads per region across the targeted enrichment panel was similar.

[0333] FIG.11 is a data plot showing post-sequencing average DNA fragment length across an enrichment panel with the use of various binding buffers. As shown in FIG.11, the fragment length of sequence reads recovered using the various binding buffers tested was similar at around 175 bp.

[0334] Surprisingly, the inventors discovered that binding buffers without chaotropic agents (i.e., chaotropic-free) had comparable performance metrics across the various binding buffers tested (Glycine, Potassium acetate, Sodium citrate and Tris acetate with KCl or AmmoniumAttorney Docket No.202323-624601 sulfate) as those seen with conventional chaotropic based binding buffers, such as guanidine hydrochloride and guanidine thiocyanate.

[0335] Example 2: Optimization of bisulfite conditions for rapid bisulfite conversion and recovery of DNA molecules

[0336] To optimize the recovery of converted DNA molecules, a variety of sulfonation reagents and conditions for bisulfite conversion were tested and pre-, and post-sequencing data compared to a commercially available kit for bisulfite conversion, EZ DNA Methylation- Lightning™ kit (available from Zymo Research Corp (Irvine, Calif.)).

[0337] In addition to the ZYMO kit conversion reagent, the sulfonation reagents for bisulfite conversion listed in Table 1 were prepared and tested (with the total concentration of sulfur from bisulfite, sulfite, and / or metabisulfite as indicated). Assays were performed similarly as in Example 1. Table 1: Tested bisulfite conversion reagents.Attorney Docket No.202323-624601

[0338] In addition, different bisulfite conversion times and incubation temperatures were also tested. Collected ssDNA molecules at the end of the procedure were analyzed, and results for pre- sequencing and post-sequencing performance metrics are shown in FIGS.12 and 13.

[0339] FIG. 12 is a data plot showing post-sequencing average binary target coverage (top panel) and DNA fragment length (bottom panel) for various bisulfite buffers and conversion reaction conditions. Higher levels of binary target coverage and DNA fragment length were observed for bisulfite conversion at 70 ℃ across various sulfonation reagents (as compared to 80 ℃).

[0340] FIG.13 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel (top panel), across hypermethylated regions (middle panel), and across hypomethylated regions (bottom panel) for various bisulfite buffers and conversion reaction conditions. Higher levels abnormal methylation coverage, as well as coverage of hypermethylated and hypomethylated regions, were observed for bisulfite conversion at 70 ℃ across various sulfonation reagents (as compared to 80 ℃), as well as for 20-minute bisulfite reactions as compared to 30-minute reactions.

[0341] Example 3: Optimization of binding buffers for rapid bisulfite conversion and recovery of DNA molecules

[0342] To optimize the recovery of converted DNA molecules using methods of the presently disclosed subject matter, a variety of non-chaotropic based and chaotropic based binding buffers were tested, and pre- and post-sequencing data compared to a commercially available kit for bisulfite conversion, EZ DNA Methylation-Lightning™ kit (available from Zymo Research Corp (Irvine, Calif.).

[0343] In addition to the ZYMO kit binding buffer, the non-chaotropic buffers prepared and tested are listed in Table 2. Chaotropic buffers were tested as well for the sake of comparison, with a buffer comprising 7M guanidine hydrochloride performing comparably to control. Table 2. Non-chaotropic based binding buffers.Attorney Docket No.202323-624601

[0344] Experiments were performed similarly as in Example 1, with silica-coated magnetic beads (DynabeadsTMMyOneTMSilane (Thermo Fisher Scientific, Waltham, MA)) added in the presence of one of the binding buffers. DNA was denatured with a 10 uL solution of 0.32 N NaOH. 100 uL of 65% ammonium bisulfite with 0.1 mM EDTA was added for bisulfite conversion. A solution of 450 uL of a binding buffer with 15 uL DYNABEADS (3.2% bead concentration) was added to bind the converted DNA. Bead-bound DNA was then treated with a desulphonation buffer (52 mM sodium carbonate, 13 mM sodium bicarbonate, and 43% ethanol), and the beads were washed with a wash buffer of 80% ethanol. DNA was eluted from the beads in elution buffer (10 mM Tris-HCl (pH 8)). Collected ssDNA molecules after the procedure were analyzed, and pre-sequencing and post-sequencing performance metrics are shown in FIGS.14-18.

[0345] FIG. 14 is a data plot showing post-bisulfite conversion DNA fragment yield across various bead lots with the use of a binding buffer having 0.4 M KCl and 0.25 M glycine. As shown in FIG.14, yield was comparable to control (ZYMO buffer) for multiple bead lots when using a buffer with 0.4 M KCl and 0.25 M glycine, but showed inconsistent performance across bead lots.

[0346] One bead lot showing higher yield performance with the 0.4 M KCl buffer (“L052L”), and one bead lot showing lower yield performance with the 0.4 M KCl buffer (“L008VM”) were selected to evaluate effects of different conditions on yield consistency. FIG. 15 is a data plot showing post-bisulfite conversion fragment yield with these two bead lots using binding buffers with various KCl concentrations and at various pH values. As shown in FIG.15, binding buffers with KCl at concentrations ranging from 2 M to 4 M led to greater consistency in post-bisulfite conversion fragment yield between bead lots by substantially improving recovery with the L008VM bead lot.

[0347] FIG.16 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel using two bead lots, and binding buffers having various concentrations of KCl. As shown in FIG.16, binding buffers with KCl at concentrations ranging from 2 M to 4M led to improvement in the reproducibility of post-sequencing coverage despite variation in bead lots, with 4 M KCl showing the highest reproducibility level.Attorney Docket No.202323-624601

[0348] FIGS.17A-17B are data plots showing post-bisulfite conversion fragment yield prior to sequencing (FIG.17A), and post-sequencing coverage of abnormal DNA fragments across an enrichment panel (FIG.17B) using various bead lots, and different binding buffers including 3 M KCl at pH 6 (“pH6+3MKCL”), 0.4 M KCl at pH 5.4 (“pH5d4_0d4MKCL”), and a ZYMO buffer as control. The KCl buffers also included 0.25 mM glycine. As shown in FIGS.17A-17B, use of a binding buffer including 3 M KCl and 0.25 M glycine led to more reproducible post-bisulfite conversion fragment yield and post-sequencing coverage across multiple bead lots, including lot L008VM (which underperformed at lower KCl concentrations).

[0349] FIG.18 is a data plot showing post-sequencing coverage of abnormal DNA fragments across an enrichment panel using various bead lots with a binding buffer having 3 M KCl and no glycine, as compared to a ZYMO buffer as control. As shown in FIG.18, use of a binding buffer including 3 M KCl led to more reproducible coverage despite variation in bead lots, even though glycine was not included.

[0350] Additional considerations

[0351] It is to be understood that the figures and descriptions of the present disclosure have been simplified to illustrate elements that are relevant for a clear understanding of the present disclosure, while eliminating, for the purpose of clarity, many other elements found in a typical system. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present disclosure. However, because some of such elements and steps do not facilitate a better understanding of the present disclosure, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to variations and modifications of such elements and methods known to those skilled in the art.

[0352] As used herein any reference to “one embodiment” or “an embodiment” means that a particular element, feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment, thereby providing a framework for various possibilities of described embodiments to function together.

[0353] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having” or any other variation thereof, are intended to cover a non-exclusive inclusion. ForAttorney Docket No.202323-624601 example, a process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Further, unless expressly stated to the contrary, “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0354] In addition, use of the “a” or “an” are employed to describe elements and components of the embodiments herein. This is done merely for convenience and to give a general sense of the invention(s). This description should be read to include one or at least one and the singular also includes the plural unless it is obvious that it is meant otherwise.

[0355] The terms “commonly,” and “typically” are not utilized herein to limit the scope of the claimed embodiments or to imply that certain features are critical or essential to the structure or function of the claimed embodiments. These terms are intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present disclosure.

[0356] The term “substantially” is utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation and to represent the degree by which a quantitative representation may vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0357] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact but may be approximate and / or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter.

[0358] As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generallyAttorney Docket No.202323-624601 acceptable in the art. In embodiments, about means a range extending to + / - 10% of the specified value. In embodiments, about includes the specified value.

[0359] Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.1, 2.2, 3.25, 4.1, and the like) and any range within that range.

[0360] While particular embodiments and applications have been illustrated and described, it is to be understood that the disclosed embodiments are not limited to the precise construction and components disclosed herein. Various modifications, changes and variations, which will be apparent to those skilled in the art, may be made in the arrangement, operation and details of the method and apparatus disclosed herein without departing from the spirit and scope defined in the appended claims. Although the invention(s) have been disclosed in connection with specific preferred aspects or embodiments, the invention(s) as claimed are not unduly limited to such specific aspects or embodiments.

Claims

Attorney Docket No.202323-624601 CLAIMS We claim:

1. A method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method comprising: (a) contacting DNA molecules with a sulfonation reagent to produce sulfonated DNA comprising one or more sulfonated deaminated cytosines; (b) binding the sulfonated DNA to a substrate in a non-chaotropic based binding buffer; optionally wherein the substrate comprises (i) a surface comprising silica, and / or (ii) a magnetic bead; (c) contacting the substrate-bound DNA molecules with a desulfonation reagent; and (d) eluting the DNA molecules from the substrate with an elution buffer.

2. The method according to claim 1, wherein the ssDNA molecules are ssDNA molecules having a length of 400 bp or less.

3. The method according to claim 1, further comprising denaturing the DNA molecules to produce single-strand DNA molecules prior to step (a), optionally wherein the denaturing comprises heating, adding sodium hydroxide (NaOH), or both heating and adding NaOH.

4. The method according to claim 1, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.005 mM to 1 mM EDTA.

5. The method according to claim 4, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

6. The method according to claim 1, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate- ammonium sulfate buffer, or any combination thereof.Attorney Docket No.202323-624601 7. The method according to claim 6, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

8. The method according to claim 6, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

9. The method according to claim 6, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

10. The method according to claim 6, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

11. The method according to claim 6, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

12. The method according to claim 6, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

13. The method according to claim 6, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate- ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

14. The method according to claim 6, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

15. The method according to claim 1, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl, (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi)Attorney Docket No.202323-624601 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

16. The method according to claim 1, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

17. The method according to claim 1, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate and sodium bicarbonate.

18. The method according to claim 17, wherein the desulfonation reagent comprises sodium carbonate and sodium bicarbonate.

19. The method according to claim 17, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol, (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

20. The method according to claim 17, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

21. The method according to claim 17, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% isopropanol, and (ii) from 30% to 70% of a mixture comprising boric acid, NaOH, and KCl.

22. The method according to claim 17, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

23. The method according to claim 1, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (c) or before eluting the DNA molecules from the substrate in step (d).Attorney Docket No.202323-624601 24. The method according to claim 23, wherein the non-chaotropic binding buffer is used as the washing buffer.

25. The method according to claim 23, wherein the washing buffer comprises from 60% to 90% ethanol.

26. The method according to claim 1, wherein the elution buffer comprises Tris HCl.

27. The method according to claim 1, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH ranging from 6.5 to 8.

5.

28. The method according to claim 1, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 60 minutes or less.

29. The method according to claim 1, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 45 minutes or less.

30. The method according to claim 1, wherein the sulfonation reagent and the DNA molecules in step (a) are incubated for 30 minutes or less.

31. A method for treating DNA molecules to convert unmethylated cytosine bases to uracil bases, the method comprising: (a) adding NaOH to a sample solution comprising DNA to produce a solution comprising single-stranded DNA molecules (ssDNA); (b) adding a sulfonation reagent to the solution comprising ssDNA to produce a mixture comprising sulfonated ssDNA molecules, wherein the sulfonated ssDNA molecules comprise one or more sulfonated deaminated cytosines; (c) combining the sulfonated ssDNA molecules with a substrate and a chaotropic-free binding buffer to produce substrate-bound sulfonated ssDNA molecules; optionally wherein the substrate comprises silica-coated magnetic beads; (d) collecting substrate-bound ssDNA molecules from the chaotropic-free binding buffer, and contacting the collected bead-bound sulfonated ssDNA molecules with a desulfonation reagent to produce substrate-bound converted ssDNA molecules; andAttorney Docket No.202323-624601 (e) eluting converted ssDNA molecules to provide a sample comprising converted ssDNA molecules.

32. The method according to claim 31, wherein the ssDNA molecules comprise small ssDNA molecules comprising 300 bp or less in length.

33. The method according to claim 31, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.05 mM to 1 mM EDTA.

34. The method according to claim 31, wherein the sulfonation reagent comprises 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

35. The method according to claim 31, wherein the chaotropic-free binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

36. The method according to claim 35, wherein the chaotropic-free binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

37. The method according to claim 35, wherein the chaotropic-free binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

38. The method according to claim 35, wherein the chaotropic-free binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

39. The method according to claim 35, wherein the chaotropic-free binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601 40. The method according to claim 35, wherein the chaotropic-free binding buffer is sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

41. The method according to claim 35, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

42. The method according to claim 35, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate- ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

43. The method according to claim 35, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate- ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

44. The method according to claim 31, wherein the chaotropic-free binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

45. The method according to claim 31, wherein the chaotropic-free binding buffer has a pH ranging from 3.5 to 7.

46. The method according to claim 31, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

47. The method according to claim 46, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

48. The method according to claim 46, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol;Attorney Docket No.202323-624601 (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

49. The method according to claim 46, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

50. The method according to claim 46, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% isopropanol, and (ii) from 30% to 70% of a mixture comprising boric acid, NaOH, and KCl.

51. The method according to claim 46, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

52. The method according to claim 31, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (d) or before eluting the DNA molecules from the substrate in step (e).

53. The method according to claim 52, wherein the chaotropic-free binding buffer is used as the washing buffer.

54. The method according to claim 52, wherein the washing buffer comprises from 60% to 90% ethanol.

55. The method according to claim 31, wherein the elution buffer comprises Tris HCl.

56. The method according to claim 31, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH ranging from 6.5 to 8.

5.

57. The method according to claim 31, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 60 minutes or less.Attorney Docket No.202323-624601 58. The method according to claim 31, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 45 minutes or less.

59. The method according to claim 31, wherein the sulfonation reagent and the ssDNA molecules in step (b) are incubated for 30 minutes or less.

60. A method for bisulfite-converting small DNA molecules, the method comprising: (a) in a solution combining small DNA molecules with a sulfonation reagent to produce sulfonated small DNA molecules, wherein said small DNA molecules comprise 300 or fewer bases in length; (b) combining the sulfonated small DNA molecules in the solution of step (a) with a substrate and a non-chaotropic based binding buffer to produce substrate-bound sulfonated small DNA molecules; optionally wherein the substrate comprises silica-coated magnetic beads; (c) collecting substrate-bound sulfonated small DNA molecules from the non- chaotropic binding buffer, and contacting the collected bead-bound sulfonated small DNA molecules with a desulfonation reagent to produce substrate-bound converted small DNA; and (d) eluting converted small DNA molecules to provide a sample comprising converted small DNA molecules.

61. The method according to claim 60, wherein the small DNA molecules are denatured to produced small single-stranded DNA (ssDNA) molecules prior to step (a), and wherein the denaturing comprises heat denaturing and / or denaturing using sodium hydroxide (NaOH).

62. The method according to claim 60, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.005 mM to 1 mM EDTA.

63. The method according to claim 62, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

64. The method according to claim 60, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KClAttorney Docket No.202323-624601 buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate- ammonium sulfate buffer or any combination thereof.

65. The method according to claim 64, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

66. The method according to claim 64, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentrating ranging from 2.7 M to 3.3 M.

67. The method according to claim 64, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

68. The method according to claim 64, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

69. The method according to claim 64, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

70. The method according to claim 64, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

71. The method according to claim 64, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate- ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

72. The method according to claim 64, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.Attorney Docket No.202323-624601 73. The method according to claim 60, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

74. The method according to claim 60, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

75. The method according to claim 60, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

76. The method according to claim 75, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

77. The method according to claim 75, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol; (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

78. The method according to claim 75, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

79. The method according to claim 75, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises: (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH, and KCl.

80. The method according to claim 75, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.Attorney Docket No.202323-624601 81. The method according to claim 60, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before contacting the substrate-bound DNA with the desulfonation reagent in step (c) or before eluting the DNA molecules from the substrate in step (d).

82. The method according to claim 81, wherein the non-chaotropic binding buffer is used as the washing buffer.

83. The method according to claim 81, wherein the washing buffer comprises from 60% to 90% ethanol.

84. The method according to claim 60, wherein the elution buffer comprises Tris HCl.

85. The method according to claim 60, wherein the elution buffer comprises 10 mM Tris HCl at a pH ranging from 6.5 to 8.

5.

86. The method according to claim 60, wherein the sulfonation reagent is brought into contact with the small DNA molecules in step (a) for 60 minutes or less.

87. The method according to claim 60, wherein the sulfonation reagent and the small DNA molecules in step (a) are incubated for 45 minutes or less.

88. The method according to claim 60, wherein the sulfonation reagent and the small DNA molecules in step (a) are incubated for 30 minutes or less.

89. The method according to any one of claims 1-3, 6-32, 35-61, or 64-88, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

90. The method according to any one of claims 1-88, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.Attorney Docket No.202323-624601 91. A kit for treating DNA comprising: (a) a sulfonation reagent; (b) silica-coated magnetic beads; (c) a non-chaotropic binding buffer; and (d) a desulfonation reagent.

92. The kit according to claim 91, wherein the kit further comprises a denaturing agent to denature double-strand DNA (dsDNA) molecules, and wherein the denaturing agent is sodium hydroxide (NaOH).

93. The kit according to claim 91, wherein the sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.01 mM to 1 mM EDTA.

94. The kit according to claim 91, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

95. The kit according to claim 91, wherein the non-chaotropic binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer or any combination thereof.

96. The kit according to claim 95, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

97. The kit according to claim 95, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

98. The kit according to claim 95, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

99. The kit according to claim 95, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601 100. The kit according to claim 95, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

101. The kit according to claim 95, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

102. The kit according to claim 95 wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate- ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

103. The kit according to claim 95, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

104. The kit according to claim 91, wherein the non-chaotropic binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

105. The kit according to claim 91, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

106. The kit according to claim 91, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid, or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

107. The kit according to claim 106, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.Attorney Docket No.202323-624601 108. The kit according to claim 106, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol; (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

109. The kit according to claim 106, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

110. The kit according to claim 106, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH and KCl.

111. The kit according to claim 106, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

112. A kit for treating DNA comprising: (a) an ammonium bisulfite sulfonation reagent; (b) silica-coated magnetic beads; (c) a chaotropic-free binding buffer; (d) a washing buffer; (e) a desulfonation reagent; and (f) an elution buffer.

113. The kit according to claim 112, wherein the kit further comprises a denaturing agent to denature double-strand DNA (dsDNA) molecules, and wherein the denaturing agent is sodium hydroxide (NaOH).

114. The kit according to claim 112, wherein the ammonium bisulfite sulfonation reagent comprises from 45% to 85% ammonium bisulfite, and optionally from 0.01 mM to 1 mM EDTA.Attorney Docket No.202323-624601 115. The kit according to claim 112, wherein the sulfonation reagent comprises from 55% to 75% ammonium bisulfite, and optionally EDTA having a concentration ranging from 0.05 mM to 0.5 mM.

116. The kit according to claim 112, wherein the chaotropic-free binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate- ammonium sulfate buffer or any combination thereof.

117. The kit according to claim 116, wherein the chaotropic-free binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

118. The kit according to claim 116, wherein the chaotropic-free binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

119. The kit according to claim 116, wherein the chaotropic-free binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl binding buffer is at a concentration ranging from 0.05 M to 2 M.

120. The kit according to claim 116, wherein the chaotropic-free binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

121. The kit according to claim 116, wherein the chaotropic-free binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

122. The kit according to claim 116, wherein the KCl in the glycine-KCl buffer, sodium citrate-KCl buffer or potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 4 M.

123. The kit according to claim 116, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate- ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601 124. The kit according to claim 116, wherein the chaotropic-free binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the ammonium sulfate in the Tris acetate-ammonium sulfate buffer is present at a concentration ranging from 0.05 M to 4 M.

125. The kit according to claim 116, wherein the chaotropic-free binding buffer comprises: (i) 3 M KCl; (ii) 0.25 M glycine and 3 M KCl; (iii) 0.25 M potassium acetate and 0.15 M KCl; (iv) 0.25 M glycine and 0.4 M KCl; (v) 0.2 M sodium citrate and 0.4 M KCl; (vi) 0.25 M Tris acetate and 0.2 M ammonium sulfate; (vii) 0.25 M Tris acetate and 0.4 M ammonium sulfate; or (viii) 0.2 M Tris acetate and 0.3 M ammonium sulfate.

126. The kit according to claim 116, wherein the chaotropic-free binding buffer has a pH ranging from 3.5 to 7.

127. The kit according to claim 116, wherein the desulfonation reagent comprises an alcohol, and (i) boric acid, or (ii) sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

128. The kit according to claim 127, wherein the desulfonation reagent comprises sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

129. The kit according to claim 127, wherein the alcohol is ethanol, and further wherein the desulfonation reagent comprises: (i) from 30% to 70% ethanol, (ii) NaOH having a concentration ranging from 20 mM to 100 mM; and (iii) HCl having a concentration ranging from 20 mM to 100 mM.

130. The kit according to claim 127, wherein the desulfonation reagent comprises (i) from 40% to 60% ethanol, and (ii) from 40% to 60% of a mixture comprising sodium carbonate, sodium bicarbonate, or both sodium carbonate and sodium bicarbonate.

131. The kit according to claim 127, wherein the alcohol is isopropanol, and further wherein the desulfonation reagent comprises (i) from 40% to 60% isopropanol, and (ii) from 40% to 60% of a mixture comprising boric acid, NaOH and KCl.Attorney Docket No.202323-624601 132. The kit according to claim 127, wherein the desulfonation reagent comprises (i) sodium carbonate having a concentration ranging from 25 mM to 50 mM, and (ii) sodium bicarbonate having a concentration ranging from 1 mM to 25 mM.

133. The kit according to claim 112, wherein the chaotropic-free binding buffer is used as the washing buffer.

134. The kit according to claim 112, wherein the washing buffer comprises from 60% to 90% ethanol.

135. The kit according to claim 112, wherein the elution buffer comprises Tris HCl.

136. The kit according to claim 135, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from 6.5 to 8.

5.

137. The kit according to any one of claims 91, 92, 95-113, or 116-136, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

138. The kit according to any one of claims 91-136, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

139. The method according to any one of claims 1-59, wherein the DNA molecules are high molecular weight (HMW) DNA molecules; optionally wherein the HMW DNA have a length of at least 5 Kb.

140. A method for isolating nucleic acids from plasma, the method comprising: (a) providing a sample of plasma comprising DNA molecules; (b) adding a proteinase to the plasma; (c) binding the DNA from step (b) to a substrate in a non-chaotropic based binding buffer; optionally wherein the substrate comprises (i) a surface comprising silica, and / or (ii) a magnetic bead; andAttorney Docket No.202323-624601 (d) isolating the nucleic acids by eluting the DNA molecules from the substrate with an elution buffer.

141. The method according to claim 140, further comprising adding one or more chaotropic reagents to the sample.

142. The method according to claim 140, further comprising treating the eluted DNA to convert unmethylated cytosine bases to uracil bases.

143. The method according to claim 140, wherein the DNA comprises cfDNA molecules having a length of 400 bp or less.

144. The method according to claim 140, wherein the non-chaotropic based binding buffer is selected from the group consisting of a potassium chloride (KCl) buffer, a glycine-KCl buffer, a sodium citrate-KCl buffer, a potassium acetate-KCl buffer, a Tris acetate-ammonium sulfate buffer, or any combination thereof.

145. The method according to claim 144, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration ranging from 2 M to 4 M.

146. The method according to claim 144, wherein the non-chaotropic based binding buffer comprises KCl at a final concentration greater than 2.5 M; optionally wherein the KCl is at a final concentration ranging from 2.7 M to 3.3 M.

147. The method according to claim 144, wherein the non-chaotropic based binding buffer is a glycine-KCl buffer, and wherein the glycine in the glycine-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

148. The method according to claim 144, wherein the non-chaotropic based binding buffer is a potassium acetate-KCl buffer, and wherein the potassium acetate in the potassium acetate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.

149. The method according to claim 144, wherein the non-chaotropic based binding buffer is a sodium citrate-KCl buffer, and wherein the sodium citrate in the sodium citrate-KCl buffer is at a concentration ranging from 0.05 M to 2 M.Attorney Docket No.202323-624601 150. The method according to claim 144, wherein the non-chaotropic based binding buffer is a Tris acetate-ammonium sulfate buffer, and wherein the Tris acetate in the Tris acetate-ammonium sulfate buffer comprises a concentration ranging from 0.05 M to 2 M.

151. The method according to claim 140, wherein the non-chaotropic binding buffer has a pH ranging from 3.5 to 7.

152. The method according to claim 140, wherein the method further comprises contacting the substrate-bound DNA with a washing buffer before eluting the DNA molecules from the substrate in step (d).

153. The method according to claim 152, wherein the non-chaotropic binding buffer is used as the washing buffer.

154. The method according to claim 152, wherein the washing buffer comprises from 60% to 90% ethanol.

155. The method according to claim 140, wherein the elution buffer comprises Tris HCl.

156. The method according to claim 140, wherein the elution buffer comprises 5 mM to 10 mM Tris HCl at a pH of from 6.5 to 8.

5.

157. A composition comprising isolated DNA molecules and a sulfonation reagent, wherein the sulfonation reagent comprises a total concentration of sulfur from one or more sulfites of: (i) 6.5 M to 9 M; (ii) about 6.5 M; (iii) about 7.5 M; or (iv) about 9 M.

158. The composition of claim 157, wherein the sulfonation reagent comprises metabisulfite, and optionally wherein the sulfonation reagent comprises a total concentration of sulfur from metabisulfite and one or more sulfites of: (i) 7.5 M to 9 M; (ii) about 7.5 M; or (iii) about 9 M.

159. A method of deaminating methylated cytosines comprising incubating the composition of claim 157 or 158 for a period of time sufficient to deaminate methylated cytosines in the isolated DNA molecules with the sulfonation agent; optionally wherein the period of time is 60 minutes or less.

Images