Methods and compositions for rapid detection and analysis of RNA and DNA cytosine methylation - Patents.com

Abstract

Aspects of the present disclosure relate to methods, compositions and kits for detecting and analyzing the cytosine methylation of DNA and RNA.Certain aspects include methods, compositions and kits that are useful in bisulfite sequencing of methylated nucleic acids from low-input samples, such as methylated nucleic acids including cell-free DNA and cell-free RNA.Methods and compositions for detecting and quantifying 5-hydroxymethylcytosine in DNA are also disclosed. TIFF2025502057000016.tif86129

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 297,165, filed January 6, 2022. U.S. Provisional Patent Application No. 63 / 297,165 is incorporated herein by reference in its entirety.

[0002] Government support statement This invention was made with government support under RM1 HG008935 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] Sequence Listing This application has been submitted in ST26 format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. This ST26 copy was created on January 6, 2023, is named ARCD_P0756WO_Sequence_Listing.xml, and is 59,305 bytes in size.

[0004] I. Field of the Invention Aspects of the invention relate to at least the fields of cell biology and epigenetics. [Background technology]

[0005] II. Background Bisulfite sequencing (BS-seq) is the current gold standard for determining 5mC sequences in DNA and mC sequences in RNA. 5 However, conventional BS-seq has some serious drawbacks, which makes it difficult to determine the m 5 In C sequencing and 5mC sequencing in DNA, m in RNA in low input samples 5However, methods and compositions for detecting and analyzing cytosine methylation in DNA and RNA, including DNA and RNA derived from low-input samples, are of limited use. Summary of the Invention

[0006] overview The present disclosure provides various methods, compositions, systems, and kits for nucleic acid processing and cytosine methylation analysis. Certain aspects of the present disclosure include the use of 5mC and m 5 The present invention relates to certain bisulfite compositions useful in rapid bisulfite treatment of DNA and / or RNA for the detection and analysis of 5hmC. Also disclosed are DNA and RNA processing methods, including the use of the disclosed compositions for cytosine deamination, and preparation of DNA and / or RNA for sequencing and cytosine methylation analysis. Methods for the detection, quantification, and analysis of 5hmC are further disclosed. DNA and RNA processing kits, including bisulfite conversion kits, useful in the preparation of DNA and / or RNA for cytosine methylation analysis are disclosed.

[0007] Aspects of the present disclosure include bisulfite solutions, ammonium sulfite solutions, ammonium bisulfite solutions, sodium bisulfite-free bisulfite solutions, methods for processing nucleic acids, methods for processing DNA, methods for processing RNA, methods for 5mC analysis, m 5The disclosed methods include a method for 5hmC analysis, a method for 5hmC analysis, a bisulfite sequencing method, a methylation analysis method, a bisulfite treatment method, a nucleic acid treatment kit, a DNA treatment kit, and an RNA treatment kit. The disclosed methods may include at least one, two, three, or more of the following steps: making a bisulfite solution, mixing a first ammonium bisulfite solution with a second ammonium bisulfite solution, incubating DNA molecules in the bisulfite solution, incubating RNA molecules in the bisulfite solution, removing DNA molecules from the bisulfite solution, removing RNA molecules from the bisulfite solution, subjecting DNA molecules to alkaline conditions, subjecting RNA molecules to alkaline conditions, treating DNA molecules with an APOBEC deaminase enzyme, detecting nucleotide methylation, quantifying nucleotide methylation, obtaining a sample from a subject, isolating nucleic acid molecules from the sample, sequencing the DNA molecules, and sequencing the RNA molecules. Any one or more of the above steps may be excluded from certain aspects. The composition (e.g., solution) of the present disclosure may include at least one, two, three, or more of the following components: ammonium bisulfite, ammonium sulfite, sodium bisulfite, sodium hydroxide, and APOBEC deaminase enzyme. Any one or more of the above components may be excluded from certain aspects. The kit of the present disclosure may include at least one, two, three, four, or more of the following components: bisulfite solution, sodium bisulfite solution, ammonium bisulfite solution, bisulfite solution without sodium bisulfite, alkaline solution, buffer, instructions for DNA treatment, instructions for DNA treatment, instructions for bisulfite treatment of DNA, and instructions for bisulfite treatment of RNA. Any one or more of the above components may be excluded from certain aspects.

[0008] In some aspects, a method for processing DNA includes the steps of: (a) incubating a solution containing DNA molecules and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, said solution not containing sodium bisulfite or added sodium bisulfite; and (b) subjecting the DNA molecule to alkaline conditions Disclosed herein is a method comprising: In some aspects, a method for processing DNA includes the steps of: (a) creating a solution comprising DNA molecules and ammonium bisulfite, said solution not containing sodium bisulfite or added sodium bisulfite; (b) incubating the solution at a temperature of at least 95° C.; and (c) removing the DNA molecules from the solution after a maximum of 12 minutes of (a). A method is also disclosed, including: In some aspects, there is provided a method for processing a nucleic acid sample, comprising: incubating a solution comprising DNA molecules and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, said solution not containing sodium bisulfite or added sodium bisulfite, each of the DNA molecules containing one or more cytosine residues, and after incubating said solution, more than 99% of the DNA molecules do not contain cytosine residues; Further disclosed are methods comprising: In some aspects, the method further comprises subjecting the plurality of DNA molecules to alkaline conditions. In some aspects, the solution does not contain ammonium sulfite or added ammonium sulfite.

[0009] In some aspects, a method for processing RNA comprises: (a) incubating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, said solution not comprising sodium bisulfite or added sodium bisulfite; (b) subjecting the RNA molecule to alkaline conditions Disclosed herein is a method comprising: In some aspects, a method for processing RNA comprises: (a) creating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite, said solution not containing sodium bisulfite or added sodium bisulfite; (b) incubating the solution at a temperature of at least 95° C.; and (c) removing the RNA molecules from the solution after at most 12 minutes of (a). A method is also disclosed, including: In some aspects, there is provided a method for processing a nucleic acid sample, comprising: incubating a solution comprising RNA molecules, ammonium sulfite and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, said solution not comprising sodium bisulfite or added sodium bisulfite, each of the RNA molecules comprising one or more cytosine residues, and after incubating said solution, greater than 99% of the RNA molecules do not comprise a cytosine residue. A method is further disclosed, comprising: In some aspects, the method further comprises subjecting the plurality of RNA molecules to alkaline conditions. In some aspects, the solution comprises 5% to 15% ammonium sulfite by weight. In some aspects, the solution comprises 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% ammonium sulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises about 10% ammonium sulfite by weight.

[0010] In some aspects, the solution contains 50% to 70% by weight of ammonium bisulfite. In some aspects, the solution contains 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8% by weight. , 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight, 54.2% by weight, 54.3% by weight, 54. 4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight Amount%, 56 wt%, 56.1 wt%, 56.2 wt%, 56.3 wt%, 56.4 wt%, 56.5 wt%, 56.6 wt%, 56.7 wt%, 56.8 wt%, 56.9 wt%, 57 wt%, 57.1 wt%, 57.2 wt%, 57.3 wt%, 57.4 wt%, 5 7.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight, 58.3% by weight, 58.4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight Amount %, 59.1 wt%, 59.2 wt%, 59.3 wt%, 59.4 wt%, 59.5 wt%, 59.6 wt%, 59.7 wt%, 59.8 wt%, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt% , 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 6 3.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64.5% by weight, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65 .3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 6 6.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 67.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68 .5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite, or any range or value derivable therein, or at least 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 50.10%, 50.11%, 50.12%, 50.13%, 50.14%, 50.15%, 50.16%, 50.17%, 50.18%, 50.19%, 50.20%, 50.21%, 50.22%, 50.23%, 50.24%, 50.25%, 50.26%, 50.27%, 50.28%, 50.29%, 50.30%, 50.31%, 50.32%, 50.33%, 50.34%, 50.35%, 50.36%, 50.37%, 50.38%, 50.39%, 50.40%, 50.41%, 50.42%, 50.43%, 50.44%, 50.45%, 50.46%, 50.47%, 50.48%, 50.49%, 50.50%, 50.51%, 50.52%, 50.53%, 50.54%, 50.55%, 50.56%, 50.57%, 50.5 1% by weight, 51.1% by weight, 51.2% by weight, 51.3% by weight, 51.4% by weight, 51.5% by weight, 51.6% by weight, 51.7% by weight, 51.8% by weight, 51.9% by weight, 52% by weight, 52.1% by weight, 52.2% by weight, 52.3% by weight, 52.4% by weight, 52.5% by weight, 52 .6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight, 54.2 weight%, 54.3 weight%, 54.4 weight%, 54.5 weight%, 54.6 weight%, 54.7 weight%, 54.8 weight%, 54.9 weight%, 55 weight%, 55.1 weight%, 55.2 weight%, 55.3 weight%, 55.4 weight%, 55.5 weight%, 55.6 weight%, 55.7 weight%, 55.8 weight%, 55.9 weight%, 56 weight%, 56.1 weight%, 56.2 weight%, 56.3 weight%, 56.4 weight%, 56.5 weight%, 56.6 weight%, 56.7 weight%, 56.8 weight%, 56.9 weight%, 57 weight%, 57.1 weight%, 57.2 weight%, 57.3 weight%, 5 7.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58.4 wt%, 58.5 wt%, 58.6 wt%, 58.7 wt%, 58.8 wt%, 58.9 wt%, 59 wt%, 59.1 wt%, 59.2 wt%, 59.3 wt%, 59.4 wt%, 59.5 wt%, 59.6 wt%, 59.7 wt%, 59.8 wt%, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt% , 60.6 wt%, 60.7 wt%, 60.8 wt%, 60.9 wt%, 61 wt%, 61.1 wt%, 61.2 wt%, 61.3 wt%, 61.4 wt%, 61.5 wt%, 61.6 wt%, 61.7 wt%, 61.8 wt%, 61.9 wt%, 62 wt%, 62.1 wt%, 62.2 wt%, 62.3 wt%, 62.4 wt%, 62.5 wt%, 62.6 wt%, 62.7 wt%, 62.8 wt%, 62.9 wt%, 63 wt%, 63.1 wt%, 63.2 wt%, 63.3 wt%, 63.4 wt%, 63.5 wt%, 63.6 wt%, 63.7 wt %, 63.8 wt%, 63.9 wt%, 64 wt%, 64.1 wt%, 64.2 wt%, 64.3 wt%, 64.4 wt%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 wt%, 66.3 wt%, 66.4 wt%, 66.5 wt%, 66.6 wt%, 66.7 wt%, 66.8 wt%, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 67.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68.5% by weight, 68.6% by weight, 68.7% by weight, 68.8% by weight, 68.9% by weight, 69% by weight, 69.1% by weight, 69.2% by weight, 69.3% by weight, 69.4% by weight, 69.5% by weight, 69.6% by weight, 69.7% by weight, 69.8% by weight, 69.9% by weight, or 70% by weight ammonium bisulfite, or any range or value derivable therein, or up to 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5% by weight , 52.6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight , 54.2% by weight, 54.3% by weight, 54.4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight Amount%, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56.8% by weight, 56.9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight Amount %, 57.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58.4 wt%, 58.5 wt%, 58.6 wt%, 58.7 wt%, 58.8 wt%, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight, 59.9% by weight, 60% by weight, 60.1% by weight, 60.2% by weight, 60.3% by weight, 60.4% by weight, 60.5% by weight, 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8 Weight%, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63 .3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64.5% by weight, 64.6% by weight, 64.7% by weight %, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 Weight%, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises 65% to 67% ammonium bisulfite by weight. In some aspects, the solution comprises about 66.7% ammonium bisulfite by weight.

[0011] In some aspects, the solution does not contain added sodium bisulfite. In some aspects, the solution has a level of ammonium sulfite and / or ammonium bisulfite of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 100%, 100%. 5%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or a derivative thereof at levels above any range of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 0.10%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.75%, 0.90%, 0.95 ... Does not contain sodium bisulfite at a level equal to 0%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any range derivable therein.In some aspects, the solutions may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0. at levels exceeding 70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% concentration, or any range derivable therein; Approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.2 0%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0 Does not contain sodium bisulfite at a level equal to .80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% concentration, or any range derivable therein.

[0012] In some aspects, the solution does not contain added ammonium sulfite. In some aspects, the solution is at a level of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 100%, 100%. %, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any range derivable therein. at or near the level of approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, Does not contain ammonium sulfite at a level equal to 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any range derivable therein.In some aspects, the solutions may be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0. At levels exceeding 70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% concentration, or any range derivable therein; is approximately 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, Does not contain ammonium sulfite at a level equal to 0.80%, 0.85%, 0.90%, 0.95%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% concentration, or any range derivable therein.

[0013] In some aspects, the solution does not contain ammonium sulfite or added ammonium sulfite. In some aspects, the solution is 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10-18 M, 1x10 -19 M, 1x10 -20 M or lower, or at 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less ammonium sulfite by weight.

[0014] In some aspects, the solution does not contain sodium bisulfite or added sodium bisulfite. In some aspects, the solution is 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10-17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or lower, or at 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less sodium bisulfite by weight.

[0015] In some aspects, the solution has a bisulfite concentration of 6.5M to 10M, or any range or value derivable therein. In some aspects, the solution has a bisulfite concentration of 8M to 10M. In some aspects, the solution has a bisulfite concentration of 9M to 10M. In some aspects, the solution has a bisulfite concentration of 6.5M to 7.5M. In some aspects, the solution is 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M, 10. a solution of bisulfite salt at 1M, 10.2M, 10.3M, 10.4M, or 10.5M, or any range or value derivable therein, or at least 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, a solution of bisulfite salt at 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M, 10.1M, 10.2M, 10.3M, 10.4M, or 10.5M, or any range or value derivable therein, or at most 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M, 10.1M, 10.2M, 10.3M, 10.4M, or 10.5M, or any range or value derivable therein; In some aspects, the solution is about 7.0M bisulfite solution. In some aspects, the solution is about 7.0M bisulfite solution.In some aspects, the solution is about a 9.5M bisulfite solution.

[0016] In some aspects, the solution has a pH of 4.8 to 5.4. In some aspects, the pH of the solution is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or more, or any range or value derivable therein, or is at least 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or more, or any range or value derivable therein, and is up to 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or more, or any range or value derivable therein. In some aspects, the pH of the solution is about 5.1.

[0017] In some aspects, the method comprises incubating the solution at a temperature of 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, 99.9°C, or any range or value derivable therein, at a temperature of at least 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, 99.9°C, or any range or value derivable therein, or at a temperature of up to 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, 99.9°C, or any range or value derivable therein. In some aspects, the method comprises incubating the solution at a temperature of at least 98°C. In some aspects, the method includes incubating the solution for 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, or 4 minutes, or any range or value derivable therein, for at least 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, or 4 minutes, or any range or value derivable therein, or for up to 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, or 4 minutes, or any range or value derivable therein. In some aspects, the method includes incubating the solution for up to 10 minutes. In some aspects, the method includes incubating the solution for up to 8 minutes.

[0018] In some aspects, also disclosed herein is a DNA processing kit comprising: (a) a solution comprising ammonium bisulfite having a bisulfite concentration of 6.5M to 10M, the solution not comprising sodium bisulfite or added sodium bisulfite; and (b) instructions for processing a DNA sample. In some aspects, the solution does not comprise ammonium sulfite or added ammonium sulfite. In some aspects, the kit further comprises an alkaline solution. In some aspects, the kit further comprises one or more buffer solutions. Any one or more of the aforementioned components may be excluded from certain aspects.

[0019] Further disclosed herein in some aspects is an RNA processing kit comprising: (a) a solution containing ammonium sulfite and ammonium bisulfite at a bisulfite concentration of 6.5 M to 8 M, the solution containing no sodium bisulfite or added sodium bisulfite, and (b) instructions for processing an RNA sample. In some aspects, the solution contains 5% to 15% ammonium sulfite by weight. In some aspects, the solution comprises 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, or comprises up to 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, or comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of ammonium sulfite, or any range or value derivable therein. In some aspects, the kit further comprises an alkaline solution. In some aspects, the kit further comprises one or more buffer solutions. Any one or more of the above ingredients may be excluded from certain aspects.

[0020] In some aspects, the solution contains 50% to 70% by weight of ammonium bisulfite. In some aspects, the solution contains 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8% by weight. , 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight, 54.2% by weight, 54.3% by weight, 54. 4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight Amount%, 56 wt%, 56.1 wt%, 56.2 wt%, 56.3 wt%, 56.4 wt%, 56.5 wt%, 56.6 wt%, 56.7 wt%, 56.8 wt%, 56.9 wt%, 57 wt%, 57.1 wt%, 57.2 wt%, 57.3 wt%, 57.4 wt%, 5 7.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight, 58.3% by weight, 58.4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight Amount %, 59.1 wt%, 59.2 wt%, 59.3 wt%, 59.4 wt%, 59.5 wt%, 59.6 wt%, 59.7 wt%, 59.8 wt%, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt% , 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 6 3.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64.5% by weight, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65 .3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 6 6.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 67.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68 .5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite, or any range or value derivable therein, or at least 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 50.10%, 50.11%, 50.12%, 50.13%, 50.14%, 50.15%, 50.16%, 50.17%, 50.18%, 50.19%, 50.20%, 50.21%, 50.22%, 50.23%, 50.24%, 50.25%, 50.26%, 50.27%, 50.28%, 50.29%, 50.30%, 50.31%, 50.32%, 50.33%, 50.34%, 50.35%, 50.36%, 50.37%, 50.38%, 50.39%, 50.40%, 50.41%, 50.42%, 50.43%, 50.44%, 50.45%, 50.46%, 50.47%, 50.48%, 50.49%, 50.50%, 50.51%, 50.52%, 50.53%, 50.54%, 50.55%, 50.56%, 50.57%, 50.5 1% by weight, 51.1% by weight, 51.2% by weight, 51.3% by weight, 51.4% by weight, 51.5% by weight, 51.6% by weight, 51.7% by weight, 51.8% by weight, 51.9% by weight, 52% by weight, 52.1% by weight, 52.2% by weight, 52.3% by weight, 52.4% by weight, 52.5% by weight, 52 .6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight, 54.2 weight%, 54.3 weight%, 54.4 weight%, 54.5 weight%, 54.6 weight%, 54.7 weight%, 54.8 weight%, 54.9 weight%, 55 weight%, 55.1 weight%, 55.2 weight%, 55.3 weight%, 55.4 weight%, 55.5 weight%, 55.6 weight%, 55.7 weight%, 55.8 weight%, 55.9 weight%, 56 weight%, 56.1 weight%, 56.2 weight%, 56.3 weight%, 56.4 weight%, 56.5 weight%, 56.6 weight%, 56.7 weight%, 56.8 weight%, 56.9 weight%, 57 weight%, 57.1 weight%, 57.2 weight%, 57.3 weight%, 5 7.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58.4 wt%, 58.5 wt%, 58.6 wt%, 58.7 wt%, 58.8 wt%, 58.9 wt%, 59 wt%, 59.1 wt%, 59.2 wt%, 59.3 wt%, 59.4 wt%, 59.5 wt%, 59.6 wt%, 59.7 wt%, 59.8 wt%, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt% , 60.6 wt%, 60.7 wt%, 60.8 wt%, 60.9 wt%, 61 wt%, 61.1 wt%, 61.2 wt%, 61.3 wt%, 61.4 wt%, 61.5 wt%, 61.6 wt%, 61.7 wt%, 61.8 wt%, 61.9 wt%, 62 wt%, 62.1 wt%, 62.2 wt%, 62.3 wt%, 62.4 wt%, 62.5 wt%, 62.6 wt%, 62.7 wt%, 62.8 wt%, 62.9 wt%, 63 wt%, 63.1 wt%, 63.2 wt%, 63.3 wt%, 63.4 wt%, 63.5 wt%, 63.6 wt%, 63.7 wt %, 63.8 wt%, 63.9 wt%, 64 wt%, 64.1 wt%, 64.2 wt%, 64.3 wt%, 64.4 wt%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 wt%, 66.3 wt%, 66.4 wt%, 66.5 wt%, 66.6 wt%, 66.7 wt%, 66.8 wt%, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 67.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68.5% by weight, 68.6% by weight, 68.7% by weight, 68.8% by weight, 68.9% by weight, 69% by weight, 69.1% by weight, 69.2% by weight, 69.3% by weight, 69.4% by weight, 69.5% by weight, 69.6% by weight, 69.7% by weight, 69.8% by weight, 69.9% by weight, or 70% by weight ammonium bisulfite, or any range or value derivable therein, or up to 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5% by weight , 52.6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 54.1% by weight , 54.2% by weight, 54.3% by weight, 54.4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight Amount%, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56.8% by weight, 56.9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight Amount %, 57.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58.4 wt%, 58.5 wt%, 58.6 wt%, 58.7 wt%, 58.8 wt%, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight, 59.9% by weight, 60% by weight, 60.1% by weight, 60.2% by weight, 60.3% by weight, 60.4% by weight, 60.5% by weight, 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8 Weight%, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63 .3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64.5% by weight, 64.6% by weight, 64.7% by weight %, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 Weight%, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises 65% to 67% ammonium bisulfite by weight. In some aspects, the solution comprises about 66.7% ammonium bisulfite by weight.

[0021] In some aspects, the solution has a bisulfite concentration of 6.5M to 10M, or any range or value derivable therein. In some aspects, the solution has a bisulfite concentration of 8M to 10M. In some aspects, the solution has a bisulfite concentration of 9M to 10M. In some aspects, the solution has a bisulfite concentration of 6.5M to 7.5M. In some aspects, the solution is a solution with a bisulfite concentration of 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 10M, 10.1M, 10.2M, 10.3M, 10.4M, or 10.5M, or any range or value derivable therein. In some aspects, the solution is about 7.0 M bisulfite solution. In some aspects, the solution is about 9.5 M bisulfite solution.

[0022] In some aspects, the solution has a pH of 4.8 to 5.4. In some aspects, the pH of the solution is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9, or any range or value derivable therein, or is at least 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9, or any range or value derivable therein, or is at most 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9, or any range or value derivable therein. In some aspects, the pH of the solution is about 5.1.

[0023] In some aspects, the instructions include instructions to incubate the DNA sample with the solution at a temperature of, or at least 95°C, 96°C, 97°C, 98°C, 99°C, 99.5°C, 99.9°C, or any range or value derivable therein. In some aspects, the instructions include instructions to incubate the DNA sample with the solution at a temperature of at least 98°C. In some aspects, the instructions include instructions to incubate the DNA sample with the solution for up to 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, or 4 minutes, or any range or value derivable therein. In some aspects, the instructions include instructions to incubate the DNA sample with the solution for up to 10 minutes. In some aspects, the instructions include instructions to incubate the DNA sample with the solution for up to 8 minutes.

[0024] In some aspects, a method for 5-hydroxymethylcytosine analysis is provided, comprising the steps of: (a) incubating a first solution comprising a first DNA molecule and ammonium bisulfite at a temperature of at least 95° C. for a maximum of 12 minutes; (b) incubating a second solution comprising the second DNA molecule and ammonium bisulfite at a temperature of at least 95° C. for a maximum of 12 minutes; (c) subjecting the first DNA molecule to alkaline conditions; (d) subjecting the second DNA molecule to alkaline conditions; (e) treating the second DNA molecule with an APOBEC deaminase enzyme; (f) sequencing the first DNA molecule and the second DNA molecule. Also disclosed herein are methods, including: In some aspects, the first solution does not contain sodium bisulfite. In some aspects, the second solution does not contain sodium bisulfite. In some aspects, the first solution and the second solution are the same solution. In some aspects, the first solution and the second solution are different solutions. In some aspects, (a) and (b) are performed simultaneously. In some aspects, (c) and (d) are performed simultaneously. In some aspects, the first DNA molecule and the second DNA molecule have the same nucleotide sequence. In some aspects, the APOBEC deaminase enzyme is APOBEC3A.

[0025] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for any measuring or quantitating method.

[0026] The use of the words "a" and "an," when used in conjunction with the term "comprising," can mean "one," but is also consistent with the meanings "one or more," "at least one," and "one or more than one."

[0027] The phrase "and / or" means "and" or "or." By way of example, A, B, and / or C includes A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. In other words, "and / or" operates as an inclusive or.

[0028] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude further, unrecited elements or method steps.

[0029] The compositions and methods for using the compositions may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the components or steps disclosed limit the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the invention.

[0030] One of ordinary skill in the art will understand that a solution that does not contain a particular chemical (e.g., ammonium sulfite, sodium bisulfite, etc.) does not contain added amounts of that chemical. The term added means that the chemical is exogenously supplied, i.e., supplied in amounts greater than what would be considered trace or insignificant amounts.

[0031] It is specifically intended that any limitation discussed with respect to one aspect of the invention may also apply to any other aspect of the invention. Moreover, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or utilize any composition of the invention. Any aspect discussed with respect to one aspect of the disclosure may also apply to other aspects of the disclosure, and vice versa. For example, any step in a method described herein may apply to any other method. Furthermore, any method described herein may have the exclusion of any step or combination of steps. Aspects of the embodiments shown in the examples may also be implemented in the context of embodiments discussed elsewhere in different examples, or discussed elsewhere in this application, for example, in the summary, detailed description, claims, and brief description of the figures.

[0032] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various modifications and changes within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0034] [Figure 1] 1 shows a diagram or mechanism of a bisulfite sequencing reaction. [Diagram 2]Figure 2A shows matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (Maldi-TOF MS) monitoring the reaction of AGCGA (SEQ ID NO:1) with R-1G at 98°C. It shows that cytosine was completely converted to U-BS adduct within 3 min. Upon base treatment, U-BS adduct was quantitatively converted to U. Figure 2B shows Maldi-TOF MS monitoring the reaction of AGm5CGA (SEQ ID NO:2) with R-1G at 98°C. It shows that m5C did not react with R-1G even after 30 min of incubation. [Diagram 3] RNA fragment size distribution after treatment with R-1G for various times (min) at 95° C. or 98° C. In all cases the RNA fragments range from 150 to 300 bp. [Figure 4] Figure 1 shows the results of sequencing total RNA from A549 cells. The effect of reaction temperature and reaction time (x-axis) on the average mutation rate of known m5C sites (top, y-axis) and non-m5C sites (bottom, y-axis) is plotted. Conditions were identified that had suitably low levels of average mutations at non-m5C sites and suitably high levels of average mutation rates at known m5 sites. Conditions of 98°C for 9 minutes produced results characteristic of the improvements described in this disclosure. These conditions may be referred to as "optimal conditions" and / or "D.5" in certain portions of this disclosure. [Diagram 5] m5C detection levels at 28S rRNA sites are shown. The 28S rRNA m5C sites and background sites can serve as a benchmark for m5C detection assay sensitivity and background measurement. There are two fully modified m5C sites (marked with vertical lines) on 28S rRNA, and all other C sites are unmodified. As shown by the condition of 98 °C for 9 min, the non-conversion rate of the two known m5C sites was more than 95%, whereas the non-conversion rate of all C sites was less than 5%. [Figure 6]Figures 6A-6D show false positive sites in various BS sequencing methods. The x-axis represents the position in 28S human rRNA. The y-axis represents the detected C ratio. Red dots represent false positive sites and green dots represent known m5C sites (marked with vertical lines). Figure 6A shows results from a standard BS treatment (e.g., "Zymo kit"). Figure 6B shows results from the method of Yang et al.10. Figure 6C shows results from the method of Huang et al.15. Figure 6D shows results from the method of Zhang et al.21. [Figure 7A]Figures 7A-7F show the analysis of various treatment times and temperatures using the R-1G recipe and validation with 28S rRNA. Figure 7A shows the false positive rate on non-m5C sites under various conditions. A.1: 70°C for 40 min, B.1: 80°C for 30 min, B.2: 80°C for 60 min, B.3: 80°C for 120 min, C.1: 90°C for 20 min, C.2: 90°C for 30 min, C.3: 90°C for 45 min, C.4: 90°C for 60 min, D.1: 98°C for 5 min, D.2: 98°C for 6 min, D3: 98°C for 7 min, D.4: 98°C for 8 min, D.5: 98°C for 9 min, D.6: 98°C for 10 min, D.7: 98°C for 15 min. Figure 7B shows the detected methylation ratios on two known m5C sites under various times and temperatures. Figure 7C shows that the two known m5C sites showed high detection rates while the false positive rates were all less than 5% under condition D5. Figure 7D shows the sequence depth at various positions on the 28S rRNA. Figures 7E and 7F show the statistics of the false positive rate (FP) and the percentage of detected m5C sites in various mentioned methods (e.g., Zymo EZ RNA Methylation™ kit ("Zymo Kit"), Yang et al., 2017, Huang et al., 2019, or Zhang et al., 2021). Figure 7E shows a comparison of the false positive rate (using a cutoff of 10% or 5%) on non-m5C sites between various mentioned methods. While the reported methods showed false positives, no false positives were detected using the method described herein. Figure 7F shows a comparison of the percentage of m5C detected by various methods. The methods provided herein detected modification rates of over 95% for two known m5C sites similar to standard BS treatment (e.g., Zymo kit), whereas all other reported methods detected lower m5C rates, suggesting that these methods may produce false negatives. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 8] We show how the BS recipes and methods disclosed herein produce little bias in RNA degradation, and show more uniform coverage in highly structured regions when compared to various previously disclosed methods (e.g., Zymo EZ RNA Methylation™ kit ("Zymo Kit"), Yang et al., 2017, Huang et al., 2019, or Zhang et al., 2021). Using 28s rRNA as an example, we found that the compositions and methods of previous studies were biased in the degradation of high GC regions, especially with reduced coverage near m5C sites (green dashed vertical lines). This may result in bias in m5C stichometry. [Figure 9A] Figures 9A-9D show results from detection of m5C sites in tRNA. Figure 9A (from Figure 1 in AG Torres et al., 2014, Trends in Molecular Medicine.) shows canonical tRNA modifications. m5C modifications at sites 48, 49, and 50 are added by NSUN2, whereas m5C at site 38 is added by DNMT2. Figures 9B-9D show m5C sequencing analysis results that showed that the detected m5C fractions at sites 48, 49, and 50 were all sensitive to NSUN2 knockdown. In contrast, the m5C fraction at site 38 was unchanged. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 10]Figures 10A-10B show the results of detection and quantification of m5C sites in tRNA. Figure 10A shows the modification percentages at m5C sites detected in tRNA. Most of the m5C sites detected in tRNA showed high modification percentages. Figure 10B shows three m5C sites detected in tRNA GlyCCC. Two sites (49 and 50) showed very high m5C percentages, whereas one site (48) showed a relatively low percentage, whereas all other C sites showed very low background. [Figure 11] Figures 11A-11B show the distribution of m5C sites among multiple RNA species in total RNA of HeLa cells. Figure 11A shows the distribution of detected m5C sites among various RNA species, whereas Figure 11B shows the distribution of m5C sites among mRNAs. [Figure 12] Figures 12A-12B show m5C site detection in HeLa mRNA using the R-1G recipe under condition D.5 compared to that described in the literature. More m5C sites were detected by the immediate method (approximately 1,241 sites), and these sites covered the majority of sites reported in the literature. Figure 12A shows the overlap with Huang et al., 201915, while Figure 12B shows the overlap with Zhang et al., 202121. [Figure 13] 1 shows the distribution of modification levels of m5C sites in HeLa cell mRNA. For the m5C sites detected in HeLa cell mRNA, the modification ratios varied among the various sites, with approximately half of the sites showing a modification ratio of more than 10%. [Figure 14] Figures 14A-14B show the number of m5C sites detected per gene and their functional annotation based on Gene Ontology (GO). Figure 14A shows that the majority of the identified modified genes had only one m5C site. Figure 15B shows that genes modified by m5C were found to be involved in various gene functions, including glycoprotein metabolism, cytoskeleton organization, cellular localization, etc. [Figure 15]Figures 15A-15B show that m5C modification levels were consistent between various biological samples. Figure 15A shows that while the overall modification levels of m5C sites were consistent between HeLa (x-axis) and HEK293T (y-axis) cell lines, there were several differentially modified sites. Figure 15B shows that the m5C site motif in HeLa cells (top) was G-rich (e.g., CGGGG (SEQ ID NO:10), a signature associated with NSUN2, whereas the HEK293T (bottom) m5C site was CUCCA (SEQ ID NO:11) motif-rich, a signature for NSUN6. [Figure 16] m5C sites detected in NSUN2 (x-axis) or NSUN6 (y-axis) knockdown in HeLa cell line mRNA extracts are shown. The modification percentage was reduced by more than 90% in NSUN2 knockdown cell extracts. This result suggests that NSUN2 may play a major role in m5C modification in HeLa cells. [Figure 17] The distribution of m5C site positions in modified gene transcripts from HeLa and HEK293T cells is shown. The m5C modification was found to be enriched at the 5' end of the transcript (e.g., gene start and / or transcription (tx) start), indicating that the m5C modification may be associated with transcript translation. [Figure 18] Figures 18A-18B show that m5C modification at the 5' end of a transcript can modulate translation efficiency. Figure 18A shows that genes that showed methylation signals at the 5' end compared to unmethylated genes were also enriched in ribosome density signals in the 5'-UTR of the transcript (p-value = 1.05 x 10-6), whereas genes that showed methylation signals at the 3' end did not show significant enrichment signals for ribosome density signals (p = 0.37). Figure 18B shows that both 5'-end methylated and 3'-end methylated genes did not show ribosome density enrichment signals within the CDS region. [Figure 19]Figures 19A-19B show a comparison of the R-1G and A7 recipes using analysis of the DNA oligonucleotide AGCGA (SEQ ID NO:3). Figure 19A shows that treatment of a model DNA oligo with R-1G required 5 minutes at 98°C to completely convert C to U-BS. Subsequent alkaline treatment converted the U-BS adduct to U. Figure 19B shows that treatment of a model DNA oligo with A7 required only 3 minutes at 98°C to completely convert C to U-BS. [Figure 20] Maldi-TOF MS monitoring of the reaction of 5mC with BS for various times at 98° C. Minimal reaction was detected after 20 min of incubation. [Figure 21] Sanger sequencing of an 82mer synthetic DNA oligonucleotide (SEQ ID NO:8) containing both C and 5mC was shown. Sanger sequencing showed that at least 8 minutes of incubation was required to complete the C to U conversion, but even after 12 minutes of incubation, 5mC was still read as C. [Figure 22] 22A-22B show how the BS treatment disclosed herein (e.g., A7-BS) quantitatively deaminates 4mC in contrast to standard BS treatment (e.g., Zymo-BS treatment). FIG. 22A illustrates Maldi TOF MS results showing that the 4mC residue in (TA4mCTT (SEQ ID NO:9) was not deaminated by standard BS treatment, but the DNA BS treatment disclosed herein quantitatively deaminates 4mC. FIG. 22B illustrates Sanger sequencing data showing that two known sites of 4mC in a 100 bp synthetic oligonucleotide (SEQ ID NO:12) were only read as T when the DNA BS treatment disclosed herein was used, whereas, conversely, when standard BS treatment was used, both 4mC sites were partially read as C. The 5mC site was read as C in both conditions. [Diagram 23]Figures 23A-23B show a comparison of DNA damage caused using standard BS treatment (e.g., Zymo kit) and the disclosed BS recipe A7. Using fish gDNA (Figure 23A), it was found that in all cases using A7 for 4-12 min, the degradation of fish gDNA was less than that caused by using standard BS treatment. The lower bands represent small DNA fragments. Compared to A7, Zymo kit treatment resulted in more lower bands and fewer upper bands, suggesting that Zymo kit treatment caused more DNA damage. Using synthetic 164 bp DNA (Figure 23B), it was again found that Zymo kit caused more DNA degradation than A7 for 4-12 min. [Figure 24A]Figures 24A-24E show the bisulfite conversion rate of DNA using the recipes disclosed herein (e.g., A7) and various times compared to standard BS treatment (e.g., Zymo kit conditions). The results show that not only is the background of the disclosed protocol much lower than when using Zymo kit conditions, but the range of background is also much narrower. The use of recipe A7 and 10 minutes of incubation produced results characteristic of the improvements described in this disclosure. Figure 24A shows the average background noise ratio calculated from Figure 24B, which shows the raw background noise of various C sites along λDNA (SEQ ID NO:15). Figure 24C shows a comparison of the non-conversion ratio of λDNA treated with Zymo DNA methylation gold kit or the disclosed A7 recipe at various incubation times. The numbers in yellow (top) represent the median non-conversion rate, whereas the numbers in red (bottom) represent the average non-conversion rate. Figure 24D shows the bisulfite conversion efficiency of lambda DNA treated with Zymo DNA methylation gold kit or the disclosed A7 recipe for various incubation times. Figure 24E shows a comparison of background from lambda DNA treated with Zymo DNA methylation gold kit or the disclosed A7 recipe for various incubation times. The results demonstrate that the use of A7 results in a much lower background and a reduced background range compared to Zymo kit treatment. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 24E] See legend to Figure 24A. [Diagram 25]Figures 25A-25D show the efficacy of the recipe and protocol disclosed herein ("New BS") for 5mC analysis of low input DNA samples. The efficacy of the New BS protocol (e.g., 98°C for 10 minutes and Recipe A7) was tested using 10 ng and 3.3 ng of mESC starting gDNA. The background noise and 5mC detection signal after standard BS treatment (e.g., Zymo EZ DNA Methylation-Gold® Kit) or treatment with the disclosed protocol were confirmed using spike-in 164-mer dsDNA oligos (SEQ ID NO:13 and antisense SEQ ID NO:14). Figure 25A shows the background and detection 5mC signal using standard BS treatment with 10 ng and 3 ng of mES starting gDNA with spike-in oligos. Figure 25B illustrates a graphical analysis of the data shown in Figure 25A. This shows that the new BS treatment results in significantly lower background levels (% unconverted C) when compared to the standard BS treatment. Figure 26C shows the background and detected 5mC signal using the BS treatment protocol of the present disclosure (e.g., 98°C for 10 min and recipe A7) with 10 ng and 3 ng of mES starting gDNA with spike-in oligos. Figure 25D illustrates a graphical analysis of the data shown in Figure 25C. This shows that the new BS treatment results in undesired 5mC conversion similar to the standard BS treatment. As shown, the standard BS treatment caused high background noise in low-input DNA samples, and the noise increased as the input amount decreased. This noise may hinder the application of BS-seq in ultra-low input samples. [Figure 26] A comparison of methylation levels between standard BS treatment (y-axis) and the presently disclosed BS protocol ("New BS", x-axis) in mESC gDNA (e.g., as described in Figures 25A-25D) is shown. The methylation levels reported from the data from the standard BS treatment showed a higher ratio than the data reported from the presently disclosed New BS treatment. This result may be due to the relatively high level of background noise (e.g., insufficient conversion) associated with the standard BS treatment. [Figure 27] The standard BS treatment data (e.g., as shown in Figures 25A-25D) show that the standard BS treatment data reported more non-CpG sites than the BS protocol of the present disclosure. This observation may be due to the relative increase in background noise in the standard BS treatment compared to the BS protocol of the present disclosure. Background noise is random signals that often happen to be non-CpG sites, which can potentially cause problems in the study of non-CpG methylation and thus lead to erroneous conclusions in biological studies. [Figure 28] Figures 28A-28B show the coverage and conversion efficiency of the BS treatment disclosed herein for mESC genomic regions with diverse GC content. Figure 28A shows that the coverage of genomic regions with diverse GC content is similar between the currently disclosed BS treatment ("New BS" as described in Figures 25A-25D) and the standard BS treatment. Figure 28B shows that the non-converted C ratio increases as the GC% of the genomic region increases, but the BS treatment disclosed herein showed a lower background non-converted ratio in all GC content regions when compared to the standard BS treatment. [Figure 29] Figures 29A-29B show that the BS protocol disclosed herein showed more evenly distributed genome coverage in mESC gDNA when compared to standard BS treatment (e.g., as described in Figures 25A-25D). Figure 29A shows the relative coverage (Z-score) of different genome windows in a 100 kb overview. The distribution of the BS protocol disclosed herein was narrower than the standard BS treatment data as shown by the statistical data shown in the box plot. Interquartile range (IQR) was used to represent the statistical dispersion of the data. Comparison of the standard BS treatment compared to the BS protocol described herein showed a reduction in IQR values ​​of 7.5% and 9.9% for the 10 ng and 3.3 ng samples, respectively. Figure 29B shows the total raw genome coverage data of mESC chromosomes. [Diagram 30]A comparison of percent unconverted C (background) in lambda DNA spiked into gDNA from 1, 10, or 100 mESCs, where the DNA had been subjected to standard BS treatment or the BS treatment disclosed herein ("New BS"). [Diagram 31] A comparison of percent unconverted C (background) in mitochondrial DNA from gDNA extracts from 1, 10, or 100 mESCs is shown, where the DNA had been subjected to standard BS treatment or the BS treatment disclosed herein ("New BS"). [Diagram 32] Figure 32A shows Maldi-TOF MS demonstrating that 5hmC was converted to CMS within 1 minute using A7 treatment of oligonucleotide AG5hmCGA (SEQ ID NO:5) at 98° C. Figure 32B shows a diagram of the 5hmC to CMS conversion process. [Diagram 33] Figure 33A shows Maldi-TOF MS demonstrating that 5fC was converted to U-BS within 30 minutes at 98° C. using A7 treatment of oligonucleotide AG5fCGA (SEQ ID NO:6). Figure 33B shows a diagram of the 5fC to U conversion process. [Diagram 34] Figure 34A shows Maldi-TOF MS demonstrating that 5caC was converted to U-BS within 3 minutes at 98° C. Figure 34B shows a diagram of the 5caC to U conversion process. [Diagram 35] 13 shows Maldi-TOF MS results demonstrating that APOBEC3A efficiently deaminated 5mC to T, whereas CMS resisted deamination and remained unchanged upon APOBEC3A treatment. [Diagram 36] Shown are Sanger sequencing results demonstrating that 5mC was quantitatively converted to T and 5hmC was primarily converted to 5hmU and thus read as T, while a small fraction of 5hmC was not deaminated. In contrast, CMS resisted deamination upon APOBEC3A treatment and thus was still read as C. [Figure 37]Schematic diagram of workflow for sequencing 5mC and 5hmC in DNA using the disclosed method. Genomic DNA contains C and its derivatives, e.g., 5mC, 5hmC, 5fC, and 5caC. After treatment with the disclosed BS reagents and conditions, C, 5fC, and 5caC are converted to U, 5hmC is converted to CMS, and 5mC remains unchanged. Half of the sample (left) proceeds to sequencing where only 5mC and 5hmC sites are read as C. The other half of the sample (right) is treated with APOBEC3A to convert 5mC to T without changing CMS. After sequencing, only the original 5hmC sites are read as C, whereas all other C derivatives are read as T. Thus, the 5hmC sites are determined. Subtracting the two libraries gives the original 5mC sites. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Detailed Description Aspects of the present disclosure relate to compositions, methods, and kits for detecting and analyzing methylated DNA and methylated RNA. Certain aspects relate to compositions for bisulfite treatment of methylated DNA and methylated RNA, including sodium bisulfite-free bisulfite solutions. In some aspects, methods for bisulfite treatment of methylated DNA and methylated RNA are also disclosed, including methods that include short (e.g., <15 min) and high temperature (e.g., >95° C.) incubation with the disclosed bisulfite solutions. Kits including the disclosed compositions are also described herein, along with instructions for analysis of methylated DNA and / or methylated RNA. Aspects of the present disclosure provide bisulfite sequencing methods with rapid bisulfite treatment, low background noise, and high sensitivity, which allow the detection of m-DNA in RNA starting from low input biological RNA or DNA samples. 5 This allows for highly accurate sequencing of 5mC in C and DNA.

[0036] I. DNA Processing Methods Aspects of the present disclosure relate to compositions and methods for DNA treatment. Certain aspects relate to compositions comprising ammonium bisulfite and methods for using such compositions in the bisulfite treatment of DNA. Thus, in some aspects, a method for DNA treatment comprising: incubating a solution comprising DNA molecules and ammonium bisulfite under conditions sufficient to deaminate cytosine residues in the DNA molecules, said solution not containing sodium bisulfite or added sodium bisulfite; Disclosed herein is a method comprising: subjecting the DNA molecule to alkaline (i.e., basic) conditions. Such a method may further comprise subjecting the DNA molecule to alkaline (i.e., basic) conditions. As disclosed herein, incubation of one or more DNA molecules in the disclosed bisulfite solution under appropriate conditions results in extremely rapid deamination of cytosine with low DNA degradation while preserving 5-methylcytosine (5mC), thereby identifying methylated nucleotides with a very low false positive rate. In some aspects, the methods provided herein provide a BS treatment suitable for accurately distinguishing 5mC from N4-methylcytosine (4mC). In some aspects, the methods provided herein facilitate deamination of 4mC at a greater rate than standard BS treatment. In some aspects, the methods provided herein facilitate conversion of 4mC to uracil at a greater rate than standard BS treatment. In some aspects, the methods provided herein quantitatively deaminate 4mC. In some aspects, the methods provided herein facilitate deamination of 4mC with an efficiency greater than or equal to about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%, or any range derivable therein. In some aspects, the methods provided herein largely avoid BS treatment false positives caused by the presence of 4mC in the genome.

[0037] In some aspects, the DNA treatment method of the present disclosure includes incubating one or more DNA molecules in a bisulfite solution, the bisulfite solution including ammonium bisulfite and not including sodium bisulfite or added sodium bisulfite. In some aspects, the bisulfite solution includes 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or less, or up to 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 In some aspects, the solution contains sodium at a concentration of 10M, 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10-5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or less, or up to 10M, 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or less. In certain aspects, the solution does not contain ammonium sulfite or added ammonium sulfite.

[0038] In certain aspects, a solution (e.g., a bisulfite solution) of the present disclosure comprises 50% to 70% ammonium bisulfite by weight, including any range or value derivable therein. In some aspects, the solution comprises at least 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53 ... Weight%, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5% by weight, 53.6% by weight, 53.7% by weight, 53.8% by weight, 53.9% by weight, 54% by weight, 5 4.1% by weight, 54.2% by weight, 54.3% by weight, 54.4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5 Weight%, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56.8% by weight, 56.9% by weight %, 57 wt%, 57.1 wt%, 57.2 wt%, 57.3 wt%, 57.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58. 4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight %, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt%, 60.6 wt%, 60.7 wt%, 60.8 wt%, 60.9 wt%, 61 wt%, 61.1 wt%, 61.2 wt%, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62 .9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64. 5% by weight, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65.3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight, 66.1 wt%, 66.2 wt%, 66.3 wt%, 66.4 wt%, 66.5 wt%, 66.6 wt%, 66.7 wt%, 66.8 wt%, 66.9 wt%, 67 wt%, 67.1 wt%, 67.2 wt%, 67.3 wt%, 67.4 wt%, 67.5 wt%, 67.6 wt%, 67. 7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight of ammonium bisulfite, or any range or value derivable therein, up to 50%, 50.1%, 50.2%, 50. 3% by weight, 50.4% by weight, 50.5% by weight, 50.6% by weight, 50.7% by weight, 50.8% by weight, 50.9% by weight, 51% by weight, 51.1% by weight, 51.2% by weight, 51.3% by weight, 51.4% by weight, 51.5% by weight, 51.6% by weight, 51.7% by weight, 51.8% by weight, 51 .9% by weight, 52% by weight, 52.1% by weight, 52.2% by weight, 52.3% by weight, 52.4% by weight, 52.5% by weight, 52.6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4% ​​by weight, 53.5 wt%, 53.6 wt%, 53.7 wt%, 53.8 wt%, 53.9 wt%, 54 wt%, 54.1 wt%, 54.2 wt%, 54.3 wt%, 54.4 wt%, 54.5 wt%, 54.6 wt%, 54.7 wt%, 54.8 wt%, 54.9 wt%, 55 wt%, 55.1 wt%, 55.2 wt%, 55.3 wt%, 55.4 wt%, 55.5 wt%, 55.6 wt%, 55.7 wt%, 55.8 wt%, 55.9 wt%, 56 wt%, 56.1 wt%, 56.2 wt%, 56.3 wt%, 56.4 wt%, 56.5 wt%, 56.6 wt%, 56 .7 weight%, 56.8 weight, 56.9 weight, 57 weight, 57.1 weight, 57.2 weight, 57.3 weight, 57.4 weight, 57.5 weight, 57.6 weight, 57.7 weight, 57.8 weight, 57.9 weight, 58 weight, 58.1 weight, 58.2 weight, 58.3 weight, 58.4 weight, 58.5 weight, 58.6 weight, 58.7 weight, 58.8 weight, 58.9 weight, 59 weight, 59.1 weight, 59.2 weight, 59.3 weight, 59.4 weight, 59.5 weight, 59.6 weight, 59.7 weight, 59.8 weight, 59 .9 weight%, 60 weight%, 60.1 weight%, 60.2 weight%, 60.3 weight%, 60.4 weight%, 60.5 weight%, 60.6 weight%, 60.7 weight%, 60.8 weight%, 60.9 weight%, 61 weight%, 61.1 weight%, 61.2 weight%, 61.3 weight%, 61.4 weight%, 61.5 weight%, 61.6 weight%, 61.7 weight%, 61.8 weight%, 61.9 weight%, 62 weight%, 62.1 weight%, 62.2 weight%, 62.3 weight%, 62.4 weight%, 62.5 weight%, 62.6 weight%, 62.7 weight%, 62.8 weight%, 62.9 weight%, 63 weight%, 63. 1 wt%, 63.2 wt%, 63.3 wt%, 63.4 wt%, 63.5 wt%, 63.6 wt%, 63.7 wt%, 63.8 wt%, 63.9 wt%, 64 wt%, 64.1 wt%, 64.2 wt%, 64.3 wt%, 64.4 wt%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 wt%, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 6 7.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68.5% by weight, 68.6% by weight, 68.7% by weight, 68.8% by weight, 68.9% by weight, 69% by weight, 69.1% by weight, 69.2% by weight, 69.3% by weight, 69.4% by weight, 6 9.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite, or any range or value derivable therein, or about 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52% or 53% by weight. %, 52.1 wt%, 52.2 wt%, 52.3 wt%, 52.4 wt%, 52.5 wt%, 52.6 wt%, 52.7 wt%, 52.8 wt%, 52.9 wt%, 53 wt%, 53.1 wt%, 53.2 wt%, 53.3 wt%, 53.4 wt%, 53.5 wt%, 53.6 Weight%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55%, 55.1%, 55.2 Weight%, 55.3%, 55.4%, 55.5%, 55.6%, 55.7%, 55.8%, 55.9%, 56%, 56.1%, 56.2%, 56.3%, 56.4%, 56.5%, 56.6%, 56.7%, 56 .8% by weight, 56.9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight, 57.4% by weight, 57.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight, 58.3% by weight, 58.4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight, 59. 9% by weight, 60% by weight, 60.1% by weight, 60.2% by weight, 60.3% by weight, 60.4% by weight, 60.5% by weight, 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight %, 61.5 wt%, 61.6 wt%, 61.7 wt%, 61.8 wt%, 61.9 wt%, 62 wt%, 62.1 wt%, 62.2 wt%, 62.3 wt%, 62.4 wt%, 62.5 wt%, 62.6 wt%, 62.7 wt%, 62.8 wt%, 62.9 wt% , 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64 .5% by weight, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65.3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight Amount %, 66.1 wt%, 66.2 wt%, 66.3 wt%, 66.4 wt%, 66.5 wt%, 66.6 wt%, 66.7 wt%, 66.8 wt%, 66.9 wt%, 67 wt%, 67.1 wt%, 67.2 wt%, 67.3 wt%, 67.4 wt%, 67.5 wt. %, 67.6%, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight of ammonium bisulfite, or any range or value derivable therein. In some aspects, the solution comprises at least 66%, 66.01%, 66.02%, 66.03% by weight, 66.04% by weight, 66.05% by weight, 66.06% by weight, 66.07% by weight, 66.08% by weight, 66.09% by weight, 66.1% by weight, 66.11% by weight, 66.12% by weight, 66.13% by weight, 66.14% by weight, 66.15% by weight, 66.16% by weight, 66 .17% by weight, 66.18% by weight, 66.19% by weight, 66.2% by weight, 66.21% by weight, 66.22% by weight, 66.23% by weight, 66.24% by weight, 66.25% by weight, 66.26% by weight, 66.27% by weight, 66.28% by weight, 66.29% by weight, 66.3% by weight, 66 .31% by weight, 66.32% by weight, 66.33% by weight, 66.34% by weight, 66.35% by weight, 66.36% by weight, 66.37% by weight, 66.38% by weight, 66.39% by weight, 66.4% by weight, 66.41% by weight, 66.42% by weight, 66.43% by weight, 66.44% by weight, 6 6.45 wt%, 66.46 wt%, 66.47 wt%, 66.48 wt%, 66.49 wt%, 66.5 wt%, 66.51 wt%, 66.52 wt%, 66.53 wt%, 66.54 wt%, 66.55 wt%, 66.56 wt%, 66.57 wt%, 66.58 wt%, 6 6.59 wt%, 66.6 wt%, 66.61 wt%, 66.62 wt%, 66.63 wt%, 66.64 wt%, 66.65 wt%, 66.66 wt%, 66.67 wt%, 66.68 wt%, 66.69 wt%, 66.7 wt%, 66.71 wt%, 66.72 wt%, 6 6.73 weight%, 66.74 weight%, 66.75 weight%, 66.76 weight%, 66.77 weight%, 66.78 weight%, 66.79 weight%, 66.8 weight%, 66.81 weight%, 66.82 weight%, 66.83 weight%, 66.84 weight%, 66.85 weight%, 66.86 weight%, 66.87%, 66.88%, 66.89%, 66.9%, 66.9%, 66.91%, 66.92%, 66.93%, 66.94%, 66.95%, 66.96%, 66.97%, 66.98%, 66.99%, or 67% ammonium bisulfite, or any range or value derivable therein, or up to 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08%, 66.09 weight%, 66.1 weight%, 66.11 weight%, 66.12 weight%, 66.13 weight%, 66.14 weight%, 66.15 weight%, 66.16 weight%, 66.17 weight%, 66.18 weight%, 66.19 weight%, 66.2 weight%, 66.21 weight%, 66.22 weight%, 66.23 weight%, 66.24 weight%, 66.25 weight%, 66.26 weight%, 66.27 weight%, 66.28 weight%, 66.29 weight%, 66.3 weight%, 66.31 wt%, 66.32 wt%, 66.33 wt%, 66.34 wt%, 66.35 wt%, 66.36 wt%, 66.37 wt%, 66.38 wt%, 66.39 wt%, 66.4 wt%, 66.41 wt%, 66.42 wt%, 66.43 wt%, 66.44 wt%, 66.45 wt%, 66.46 wt%, 66.47 wt%, 66.48 wt%, 66.49 wt%, 66.5 wt%, 66.51 wt%, 66.52 wt %, 66.53 wt%, 66.54 wt%, 66.55 wt%, 66.56 wt%, 66.57 wt%, 66.58 wt%, 66.59 wt%, 66.6 wt%, 66.61 wt%, 66.62 wt%, 66.63 wt%, 66.64 wt%, 66.65 wt%, 66.66 wt%, 66.67 wt%, 66.68 wt%, 66.69 wt%, 66.7 wt%, 66.71 wt%, 66.72 wt%, 66.73 wt%, 66. 74 weight%, 66.75 weight%, 66.76 weight%, 66.77 weight%, 66.78 weight%, 66.79 weight%, 66.8 weight%, 66.81 weight%, 66.82 weight%, 66.83 weight%, 66.84 weight%, 66.85 weight%, 66.86 weight%, 66.87 weight%, 66.88 weight%, 66.89 weight%, 66.9 weight%, 66.91 weight%, 66.92 weight%, 66.93 weight%, 66.94 weight%, 66.95 weight%. , 66.96%, 66.97%, 66.98%, 66.99%, or 67% ammonium bisulfite by weight, or any range or value derivable therein, or about 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08%, 66.09%, 66.1%, 66.11%, 66.12%, 66.13%, 66.14%, 66.15%, 66.16%, 66. 17% by weight, 66.18% by weight, 66.19% by weight, 66.2% by weight, 66.21% by weight, 66.22% by weight, 66.23% by weight, 66.24% by weight, 66.25% by weight, 66.26% by weight, 66.27% by weight, 66.28% by weight, 66.29% by weight, 66.3% by weight, 66.31% by weight, 66.32% by weight, 66.33% by weight, 66.34% by weight, 66.35% by weight, 66.36% by weight, 66.37% by weight, 66.38% by weight, 66.39% by weight, 66.4% by weight, 66.41% by weight, 66.42% by weight, 66.43% by weight, 66.44% by weight Amount%, 66.45 wt%, 66.46 wt%, 66.47 wt%, 66.48 wt%, 66.49 wt%, 66.5 wt%, 66.51 wt%, 66.52 wt%, 66.53 wt%, 66.54 wt%, 66.55 wt%, 66.56 wt%, 66.57 wt%, 66 .58% by weight, 66.59% by weight, 66.6% by weight, 66.61% by weight, 66.62% by weight, 66.63% by weight, 66.64% by weight, 66.65% by weight, 66.66% by weight, 66.67% by weight, 66.68% by weight, 66.69% by weight, 66.7% by weight, 66.71% by weight, 66.72 wt%, 66.73 wt%, 66.74 wt%, 66.75 wt%, 66.76 wt%, 66.77 wt%, 66.78 wt%, 66.79 wt%, 66.8 wt%, 66.81 wt%, 66.82 wt%, 66.83 wt%, 66.84 wt%, 66.85 wt% Amount %, 66.86 wt%, 66.87 wt%, 66.88 wt%, 66.89 wt%, 66.9 wt%, 66.91 wt%, 66.92 wt%, 66.93 wt%, 66.94 wt%, 66.95 wt%, 66.96 wt%, 66.97 wt%, 66.98 wt%, 66.99% by weight, or 67% by weight ammonium bisulfite, or any range or value derivable therein. In some aspects, the solution contains about 66.67% by weight ammonium bisulfite. In some aspects, the bisulfite solution does not contain ammonium sulfite or added ammonium sulfite. In some aspects, the bisulfite solution contains ammonium sulfite.

[0039] In some aspects, the bisulfite solution is a solution with a bisulfite concentration of 6.5M to 10M, including any range or value derivable therebetween. In some aspects, the bisulfite solution is a solution with a bisulfite concentration of at least 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9 ...5M, 9.6M, 8.7M, 8.8M, 8.9M, , 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, up to 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9.1M, 9.2M, 9.3M ... 10M, 10M, 10M, 10M, 10M, 10M, 10M, 0.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, or about 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, The bisulfite solution may be a solution having a bisulfite concentration of 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein. In some aspects, the bisulfite solution is a solution having a bisulfite concentration of about 9.5M, and in some aspects, the bisulfite solution is a solution having a bisulfite concentration of 9.5M.

[0040] The bisulfite solution of the present disclosure can be made, for example, by mixing two ammonium bisulfite solutions having different weight percents of ammonium bisulfite. For example, the bisulfite solution of the present disclosure can be made by mixing a 70% ammonium bisulfite solution with a 50% ammonium bisulfite solution. In some aspects, the 70% ammonium bisulfite solution and the 50% ammonium bisulfite solution are mixed in a ratio of, for example, 10:0.1, 10:0.2, 10:0.3, 10:0.4, 10:0.5, 10:0.6, 10:0.7, 10:0.8, 10:0.9, 10:1, 10:1.1, 10:1.2, 10:1.3, 10:1.4, 10:1.5, 10:1.6, 10:1.7, 10:1.8, 10:1.9, or 10:2, or any range or value derivable therein. In some aspects, the 70% ammonium bisulfite solution and the 50% ammonium bisulfite solution are mixed in a 10:1 ratio.

[0041] In some aspects, the DNA treatment method includes incubating one or more DNA molecules in a bisulfite solution of the present disclosure (e.g., a solution containing ammonium bisulfite, e.g., 50%-70% ammonium bisulfite, without sodium bisulfite) at a temperature of at least 80° C. for up to 20 minutes. In some aspects, the method includes: cleaving one or more DNA molecules in a bisulfite solution at least 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 82.10°C, 82.12°C, 82.13°C, 82.14°C, 82.15°C, 82.16°C, 82.17°C, 82.18°C, 82.19°C, 82.20°C, 82.21°C, 82.22°C, 82.23°C, 82.24°C, 82.25°C, 82.26°C, 82.27°C, 82.28°C, 82.29°C, 82.30°C, 82.31°C, 82.32°C, 82.33°C, 82.34°C, 82.35°C, 82.36°C, 82.37°C, 82.38°C 2.9℃, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃, 85.2℃, 85.3℃, 85.4℃, 85.5℃, 85.6℃, 85.7℃, 85.8℃, 85.9℃, 86℃, 86.1℃, 86.2℃, 86.3℃, 86.4℃, 86.5℃, 86.6℃, 86.7℃, 86.8℃, 86.9℃, 87℃, 87.1℃, 87.2℃, 87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃, 89.5℃, 89.6℃, 89.7℃, 89.8℃, 89.9℃, 90℃, 90.1℃, 90.2℃ ,90.3℃,90.4℃,90.5℃,90.6℃,90.7℃,90.8℃,90.9℃,91℃,91.1℃,91.2℃,91.3℃,91.4℃,91.5℃,91.6℃,91.7℃,91.8℃,91.9℃,92℃,92.1℃,92.2℃,92.3℃,92.4℃,92.5℃,92.6℃,92.7℃,92.8℃,92.9℃,93℃,93.1℃,93.2℃,93.3℃,93.4℃,93.5℃,93.6℃,93.7℃,93.8℃,93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94.9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 95.8℃, 95.9℃, 96℃, 9 6.1℃, 96.2℃, 96.3℃, 96.4℃, 96.5℃, 96.6℃, 96.7℃, 96.8℃, 96.9℃, 97℃, 97.1℃, 97.2℃, 97.3℃, 97.4℃, 97.5℃, 97.6℃, 97.7℃, 97.8℃, 97.9℃, 98℃, 98.1℃, 98.2 ℃, 98.3℃, 98.4℃, 98.5℃, 98.6℃, 98.7℃, 98.8℃, 98.9℃, 99℃, 99.1℃, 99.2℃, 99.3℃, 99.4℃, 99.5℃, 99.6℃, 99.7℃, 99.8℃, 99.9℃ (or any range or value derivable therein), up to and including 80℃, 80.1℃, 80.2℃, 80.3℃, 80.4℃, 80.5℃, 80.6℃, 80.7℃, 80.8℃, 80.9℃, 81℃, 81.1℃, 81.2℃, 81.3℃, 81.4℃, 81.5℃, 81.6℃, 81.7℃, 81.8℃, 81.9℃, 82.0℃, 82.1℃, 82.2℃, 82.3℃, 82.4℃, 82.5℃, 82.6℃, 82.7℃, 82.8℃, 82.9 ... 2℃, 82.1℃, 82.2℃, 82.3℃, 82.4℃, 82.5℃, 82.6℃, 82.7℃, 82.8℃, 82.9℃, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃, 85.2℃, 85.3℃, 85.4℃, 85.5℃, 85.6℃, 85.7℃, 85.8℃, 85.9℃, 86℃, 86.1℃, 86.2℃, 86. 3℃, 86.4℃, 86.5℃, 86.6℃, 86.7℃, 86.8℃, 86.9℃, 87℃, 87.1℃, 87.2℃, 87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃, 89.5℃, 89.6℃, 89.7℃, 89.8℃, 89.9℃, 90℃, 90.1℃, 90.2℃, 90.3℃, 90.4℃, 90.5℃, 90.6℃, 90.7℃, 90.8℃, 90.9℃, 91℃, 91.1℃, 91.2℃, 91.3℃, 91.4℃, 91.5℃, 91.6℃, 91.7℃, 91.8℃, 91.9℃, 92℃, 92.1℃, 92.2℃, 92.3℃, 92.4℃, 92.5℃, 92.6℃, 92.7℃, 92.8℃, 92.9℃, 93℃, 93.1℃, 93.2℃, 93.3℃, ​​93.4℃, 93.5℃, 93.6℃, ​​93.7℃, 93.8℃, 93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94. 9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 95.8℃, 95.9℃, 96℃, 96.1℃, 96.2℃, 96.3℃, 96.4℃, 96.5℃, 96.6℃, 96.7℃, 96.8℃, 96.9℃, 97℃, 97 .1℃, 97.2℃, 97.3℃, 97.4℃, 97.5℃, 97.6℃, 97.7℃, 97.8℃, 97.9℃, 98℃, 98.1℃, 98.2℃, 98.3℃, 98.4℃, 98.5℃, 98.6℃, 98.7℃, 98.8℃, 98.9℃, 99℃, 99.1℃, 99.2℃ , 99.3°C, 99.4°C, 99.5°C, 99.6°C, 99.7°C, 99.8°C, 99.9°C (or any range or value derivable therein), or at about 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃ ,85.2℃,85.3℃,85.4℃,85.5℃,85.6℃,85.7℃,85.8℃,85.9℃,86℃,86.1℃,86.2℃,86.3℃,86.4℃,86.5℃,86.6℃,86.7℃,86.8℃,86.9℃,87℃,87.1℃,87.2℃,87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃ ,89.5℃,89.6℃,89.7℃,89.8℃,89.9℃,90℃,90.1℃,90.2℃,90.3℃,90.4℃,90.5℃,90.6℃,90.7℃,90.8℃,90.9℃,91℃,91.1℃,91.2℃,91.3℃,91.4℃,91.5℃,91 .6℃, 91.7℃, 91.8℃, 91.9℃, 92℃, 92.1℃, 92.2℃, 92.3℃, 92.4℃, 92.5℃, 92.6℃, 92.7℃, 92.8℃, 92.9℃, 93℃, 93.1℃, 93.2℃, 93.3℃, ​​93.4℃, 93.5℃, 93.6℃, ​​93.7℃ ,93.8℃,93.9℃,94℃,94.1℃,94.2℃,94.3℃,94.4℃,94.5℃,94.6℃,94.7℃,94.8℃,94.9℃,95℃,95.1℃,95.2℃,95.3℃,95.4℃,95.5℃,95.6℃,95.7℃,95.8℃,95 .9°C, 96°C, 96.1°C, 96.2°C, 96.3°C, 96.4°C, 96.5°C, 96.6°C, 96.7°C, 96.8°C, 96.9°C, 97°C, 97.1°C, 97.2°C, 97.3°C, 97.4°C, 97.5°C, 97.6°C, 97.7°C, 97.8°C, 97.9°C, 98°C, 98.1°C, 98.2°C, 98.3°C, 98.4°C, 98.5°C, 98.6°C, 98.7°C, 98.8°C, 98.9°C, 99°C, 99.1°C, 99.2°C, 99.3°C, 99.4°C, 99.5°C, 99.6°C, 99.7°C, 99.8°C, 99.9°C (or any derivable therein) range or value) for up to 15 minutes, 14.9 minutes, 14.8 minutes, 14.7 minutes, 14.6 minutes, 14.5 minutes, 14.4 minutes, 14.3 minutes, 14.2 minutes, 14.1 minutes, 14 minutes, 13.9 minutes, 13.8 minutes, 13.7 minutes, 13.6 minutes, 13.5 minutes, 13.4 minutes, 13.3 minutes, 13.2 minutes, 13.1 minutes, 13 minutes, 12.9 minutes, 12.8 minutes, 12.7 minutes, 12.6 minutes, 12.5 minutes, 12.4 minutes, 12.3 minutes, 12.2 minutes, 12.1 minutes, 12 minutes, 11.9 minutes, 11.8 minutes, 11.7 minutes, 11.6 minutes, 11.5 minutes, 11.4 minutes, 11.3 minutes, 11.2 minutes, 11.1 minutes, 11 minutes, 10.9 minutes, 10.8 minutes, 10.7 minutes, 10.6 minutes, 10.5 minutes, 10.4 minutes, 10.3 minutes, 10.2 minutes, 10.1 minutes, 10 minutes, 9.9 minutes, 9.8 minutes, 9.7 minutes, 9.6 minutes, 9.5 minutes, 9.4 minutes, 9.3 minutes, 9.2 minutes, 9.1 minutes, 9 minutes, 8.9 minutes, 8.8 minutes, 8.7 minutes, 8.6 minutes, 8.5 minutes, 8.4 minutes, 8.3 minutes, 8.2 minutes, 8.1 minutes, 8 minutes, 7.9 minutes, 7.8 minutes, 7.7 minutes, 7.6 minutes, 7.5 minutes, 7.4 minutes, 7.3 minutes, 7.2 minutes, 7.1 minutes, 7 minutes, 6.9 minutes, 6.8 minutes, 6.7 minutes, 6.6 minutes, 6.5 minutes, 6.4 minutes, 6.3 minutes, 6.2 minutes, 6.1 minutes, 6 minutes, 5.9 minutes, 5.8 minutes, 5.7 minutes, 5.6 minutes, 5.5 minutes, 5.4 minutes, 5.3 minutes, 5.2 minutes, 5.1 minutes, 5 minutes, 4.9 minutes, 4.8 minutes, 4.7 minutes, 4.6 minutes, 4.5 minutes, 4.4 minutes, 4.3 minutes, 4.2 minutes, 4.1 minutes, 4 minutes, 3.9 minutes, 3.8 minutes, 3.7 minutes, 3.6 minutes, 3.5 minutes, 3.4 minutes, 3. 3 minutes, 3.2 minutes, 3.1 minutes, 3 minutes, 2.9 minutes, 2.8 minutes, 2.7 minutes, 2.6 minutes, 2.5 minutes, 2.4 minutes, 2.3 minutes, 2.2 minutes, 2.1 minutes, 2 minutes, 1.9 minutes, 1.8 minutes, 1.7 minutes, 1.6 minutes, 1.5 minutes, 1.4 minutes, 1.3 minutes, 1.2 minutes, 1.1 minutes, or 1 minute (or any range or value derivable therein), or about 15 minutes, 14.9 minutes, 14.8 minutes, 14.7 minutes, 14.6 minutes, 14.5 minutes, 14.4 minutes, 14.3 minutes, 14.2 minutes, 14.1 minutes, 14 minutes, 13.9 minutes, 13.8 minutes, 13.7 minutes, 13.6 minutes, 13.5 minutes, 13.4 minutes, 13.3 minutes, 13.2 minutes, 13.1 minutes, 13 minutes, 12.9 minutes, 12.8 minutes, 12.7 minutes, 12.6 minutes, 12.5 minutes, 12.4 minutes, 12.3 minutes, 12.2 minutes, 12.1 minutes, 12 minutes, 11.9 minutes, 11.8 minutes, 11.7 minutes, 11.6 minutes, 11.5 minutes, 11.4 minutes, 11.3 minutes, 11.2 minutes, 11.1 minutes, 11 minutes, 10.9 minutes, 10.8 minutes, 10.7 minutes, 10.6 minutes, 10.5 minutes, 10.4 minutes, 10.3 minutes, 10.2 minutes, 10.1 minutes, 10 minutes, 9.9 minutes, 9.8 minutes, 9.7 minutes, 9.6 minutes, 9.5 minutes, 9.4 minutes, 9.3 minutes, 9.2 minutes, 9.1 minutes, 9 minutes, 8.9 minutes, 8.8 minutes, 8.7 minutes, 8.6 minutes, 8.5 minutes, 8.4 minutes, 8.3 minutes, 8.2 minutes, 8.1 minutes, 8 minutes, 7.9 minutes, 7.8 minutes, 7.7 minutes, 7.6 minutes, 7.5 minutes, 7.4 minutes, 7.3 minutes, 7.2 minutes, 7.1 minutes, 7 minutes, 6.9 minutes, 6.8 minutes, 6.7 minutes, 6.6 minutes, 6.5 minutes, 6.4 minutes, 6.3 minutes, 6.2 minutes, 6.1 minutes, 6 minutes, 5.9 minutes, 5.8 minutes, 5.7 minutes, 5.6 minutes, 5.5 minutes, 5.4 minutes, 5.3 minutes, Incubating for 5.2 minutes, 5.1 minutes, 5 minutes, 4.9 minutes, 4.8 minutes, 4.7 minutes, 4.6 minutes, 4.5 minutes, 4.4 minutes, 4.3 minutes, 4.2 minutes, 4.1 minutes, 4 minutes, 3.9 minutes, 3.8 minutes, 3.7 minutes, 3.6 minutes, 3.5 minutes, 3.4 minutes, 3.3 minutes, 3.2 minutes, 3.1 minutes, 3 minutes, 2.9 minutes, 2.8 minutes, 2.7 minutes, 2.6 minutes, 2.5 minutes, 2.4 minutes, 2.3 minutes, 2.2 minutes, 2.1 minutes, 2 minutes, 1.9 minutes, 1.8 minutes, 1.7 minutes, 1.6 minutes, 1.5 minutes, 1.4 minutes, 1.3 minutes, 1.2 minutes, 1.1 minutes, or 1 minute (or any range or value derivable therein). Any combination of the aforementioned incubation times and temperatures can be used in the DNA processing methods of the present disclosure. .

[0042] In some aspects, the DNA treatment method comprises incubating one or more DNA molecules in a bisulfite solution of the present disclosure at a temperature of at least 95° C. for up to 12 minutes, at a temperature of at least 96° C. for up to 12 minutes, at a temperature of at least 97° C. for up to 12 minutes, at a temperature of at least 98° C. for up to 12 minutes, at a temperature of at least 99° C. for up to 11 minutes, at a temperature of at least 95° C. for up to 11 minutes, at a temperature of at least 96° C. for up to 11 minutes, at a temperature of at least 97° C. for up to 11 minutes, at least at least 98°C for a maximum of 11 minutes, at least 99°C for a maximum of 11 minutes, at least 95°C for a maximum of 10 minutes, at least 96°C for a maximum of 10 minutes, at least 97°C for a maximum of 10 minutes, at least 98°C for a maximum of 10 minutes, at least 99°C for a maximum of 10 minutes, at least 95°C for a maximum of 9 minutes, at least 96°C for a maximum of 9 minutes, at least 97°C for a maximum of 9 minutes, at least 98°C for a maximum of 9 minutes 30 minutes, at a temperature of at least 99°C for up to 9 minutes, at a temperature of at least 95°C for up to 8 minutes, at a temperature of at least 96°C for up to 8 minutes, at a temperature of at least 97°C for up to 8 minutes, at a temperature of at least 98°C for up to 8 minutes, at a temperature of at least 99°C for up to 7 minutes, at a temperature of at least 95°C for up to 7 minutes, at a temperature of at least 96°C for up to 7 minutes, at a temperature of at least 97°C for up to 7 minutes, at a temperature of at least 98°C for up to 7 minutes, at a temperature of at least 99°C for up to 6 minutes, at a temperature of at least 96°C for up to 6 minutes, at a temperature of at least 97°C for up to 6 minutes, at a temperature of at least 98°C for up to 6 minutes, at a temperature of at least 99°C for up to 5 minutes, at a temperature of at least 96°C for up to 5 minutes, at a temperature of at least 97°C for up to 5 minutes, at a temperature of at least 98°C for up to 5 minutes, or at least 99°C for up to 5 minutes.

[0043] As disclosed herein, incubating a DNA molecule with a bisulfite solution of the present disclosure (e.g., a solution containing ammonium bisulfite, e.g., 50%-70% ammonium bisulfite, without sodium bisulfite or added sodium bisulfite) under appropriate conditions (e.g., at a temperature of at least 95°C for up to 12 minutes) is sufficient to deaminate the majority of cytosine residues in the DNA molecule. In some aspects, after incubating the DNA molecule with a bisulfite solution of the present disclosure under appropriate conditions, more than 90% of the DNA molecules do not contain cytosine residues. In some aspects, 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93.0%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9 ... .9%, 93%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96. 1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 9 Greater than 9.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any range or value derivable therein), or 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92.8%, 91.9%, 92%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94% ,94.1%,94.2%,94.3%,94.4%,94.5%,94.6%,94.7%,94.8%,94.9%,95%,95.1%,95.2%,95.3%,95.4%,95.5%,95.6%,95.7%,95.8%,95.9%,96%,96.1%,96.2% , 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any range or value derivable therein) of free cytosine residues. In some aspects, greater than 99% of DNA molecules do not contain cytosine residues.

[0044] The disclosed DNA processing methods can be useful, for example, for preparing DNA molecules for sequencing to detect, quantify, and / or analyze DNA cytosine methylation. In some aspects, the disclosed DNA processing methods provide DNA molecules for sequencing analysis that result in reduced levels of false positives, increased levels of true positives, reduced levels of false negatives, and / or increased levels of true negatives compared to standard BS processing.

[0045] II. RNA Processing Methods Aspects of the present disclosure relate to compositions and methods for RNA processing. Certain aspects relate to compositions comprising ammonium bisulfite and methods for using such compositions in the bisulfite treatment of RNA. Thus, in some aspects, a method for RNA processing comprises: incubating a solution containing the RNA molecule, ammonium bisulfite, and ammonium sulfite under conditions sufficient to deaminate cytosine residues in the RNA molecule, said solution not containing sodium bisulfite or added sodium bisulfite; Disclosed herein is a method comprising: (a) subjecting RNA molecules to alkaline (i.e., basic) conditions; (b) subjecting RNA molecules to alkaline (i.e., basic) conditions; (c) subjecting RNA molecules to alkaline (i.e., basic) conditions; (d) subjecting RNA molecules to alkaline (i.e., basic) conditions; (e) subjecting RNA molecules to alkaline (i.e., basic) conditions; (f) subjecting RNA molecules to alkaline (i.e., alkaline) conditions; (g) subjecting RNA molecules to alkaline (i.e., alkaline) conditions; (h) subjecting RNA molecules to alkaline (i.e., alkaline) conditions; ...

[0046] In some aspects, the RNA processing methods of the present disclosure include: incubating one or more RNA molecules in a bisulfite solution, the bisulfite solution comprising ammonium bisulfite and ammonium sulfite, the bisulfite solution not comprising sodium bisulfite or added sodium bisulfite; In some aspects, the bisulfite solution includes 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20M or less, or at 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or less. In some aspects, the bisulfite solution is sodium-free.

[0047] In certain aspects, a solution (e.g., a bisulfite solution) of the present disclosure comprises 50% to 70% ammonium bisulfite by weight, including any range or value derivable therein. In some aspects, the solution comprises at least 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55.8%, 55.9%, 56.1%, 56.2%, 56.3%, 56.4%, 56.5%, 56.6%, 56.7%, 56.8%, 56.9%, 57.1%, 57.2%, 57.3%, 57.4%, 57.5%, 57.6%, 57.7%, 57.8%, 57.9%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.6%, 58.7%, 58.8%, 58.9%, 59.8%, 59.9%, 59.9%, 59.9%, 59.9%, 59.8%, 59.9%, 59. %, 52.7 wt%, 52.8 wt%, 52.9 wt%, 53 wt%, 53.1 wt%, 53.2 wt%, 53.3 wt%, 53.4 wt%, 53.5 wt%, 53.6 wt%, 53.7 wt%, 53.8 wt%, 53.9 wt%, 54 wt%, 54.1 Weight%, 54.2% by weight, 54.3% by weight, 54.4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56.8% by weight, 56.9% by weight, 57% by weight Amount %, 57.1 wt%, 57.2 wt%, 57.3 wt%, 57.4 wt%, 57.5 wt%, 57.6 wt%, 57.7 wt%, 57.8 wt%, 57.9 wt%, 58 wt%, 58.1 wt%, 58.2 wt%, 58.3 wt%, 58.4 wt%, 5 8.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight, 59.9% by weight Amount %, 60% by weight, 60.1% by weight, 60.2% by weight, 60.3% by weight, 60.4% by weight, 60.5% by weight, 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63 Weight%, 63.1%, 63.2%, 63.3%, 63.4%, 63.5%, 63.6%, 63.7%, 63.8%, 63.9%, 64%, 64.1%, 64.2%, 64.3%, 64.4%, 64.5%, 64. 6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65.3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight, 66.1% by weight, 66.2 Weight%, 66.3%, 66.4%, 66.5%, 66.6%, 66.7%, 66.8%, 66.9%, 67%, 67.1%, 67.2%, 67.3%, 67.4%, 67.5%, 67.6%, 67.7%, 67. 8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite by weight, or any range or value derivable therein, up to 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, or 70% ammonium bisulfite by weight. 4% by weight, 50.5% by weight, 50.6% by weight, 50.7% by weight, 50.8% by weight, 50.9% by weight, 51% by weight, 51.1% by weight, 51.2% by weight, 51.3% by weight, 51.4% by weight, 51.5% by weight, 51.6% by weight, 51.7% by weight, 51.8% by weight, 51.9% by weight, 52 Weight%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6 wt%, 53.7 wt%, 53.8 wt%, 53.9 wt%, 54 wt%, 54.1 wt%, 54.2 wt%, 54.3 wt%, 54.4 wt%, 54.5 wt%, 54.6 wt%, 54.7 wt%, 54.8 wt%, 54.9 wt%, 55 wt%, 55.1 wt%, 55.2 wt%, 55.3 wt%, 55.4 wt%, 55.5 wt%, 55.6 wt%, 55.7 wt%, 55.8 wt%, 55.9 wt%, 56 wt%, 56.1 wt%, 56.2 wt%, 56.3 wt%, 56.4 wt%, 56.5 wt%, 56.6 wt%, 56.7 wt%, 56 .8 weight%, 56.9 weight%, 57 weight%, 57.1 weight%, 57.2 weight%, 57.3 weight%, 57.4 weight%, 57.5 weight%, 57.6 weight%, 57.7 weight%, 57.8 weight%, 57.9 weight%, 58 weight%, 58.1 weight%, 58.2 weight%, 58.3 weight%, 58.4 weight%, 58.5 weight%, 58.6 weight%, 58.7 weight%, 58.8 weight%, 58.9 weight%, 59 weight%, 59.1 weight%, 59.2 weight%, 59.3 weight%, 59.4 weight%, 59.5 weight%, 59.6 weight%, 59.7 weight%, 59.8 weight%, 59.9 weight%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt%, 60.6 wt%, 60.7 wt%, 60.8 wt%, 60.9 wt%, 61 wt%, 61.1 wt%, 61.2 wt%, 61.3 wt%, 61.4 wt%, 61.5 wt%, 61.6 wt%, 61.7 wt%, 61.8 wt%, 61.9 wt%, 62 wt%, 62.1 wt%, 62.2 wt%, 62.3 wt%, 62.4 wt%, 62.5 wt%, 62.6 wt%, 62.7 wt%, 62.8 wt%, 62.9 wt%, 63 wt%, 63.1 wt%, 63. 2 wt%, 63.3 wt%, 63.4 wt%, 63.5 wt%, 63.6 wt%, 63.7 wt%, 63.8 wt%, 63.9 wt%, 64 wt%, 64.1 wt%, 64.2 wt%, 64.3 wt%, 64.4 wt%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1 wt%, 66.2 wt%, 66.3 wt%, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 67.7% by weight, 67.8% by weight, 67.9% by weight, 6 8% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68.5% by weight, 68.6% by weight, 68.7% by weight, 68.8% by weight, 68.9% by weight, 69% by weight, 69.1% by weight, 69.2% by weight, 69.3% by weight, 69.4% by weight, 69.5% by weight, 6 9.6%, 69.7%, 69.8%, 69.9%, or 70% ammonium bisulfite, or any range or value derivable therein, or about 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53.0%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55%, 55.8%, 55.9%, 56%, 56.1%, 56.2%, 56.3%, 56.4%, 56.5%, 56.6%, 56.7%, 56.8%, 56.9%, 57%, 57.8%, 57.9%, 57.9%, 58%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.6%, 58.7%, 58.8%, 58.9%, 59%, 60%, 60%, 60%, %, 52.2 wt%, 52.3 wt%, 52.4 wt%, 52.5 wt%, 52.6 wt%, 52.7 wt%, 52.8 wt%, 52.9 wt%, 53 wt%, 53.1 wt%, 53.2 wt%, 53.3 wt%, 53.4 wt%, 53.5 wt%, 53.6 wt%, 53.7 Weight%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55%, 55.1%, 55.2%, 55.3 Weight%, 55.4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56.8% by weight, 56 .9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight, 57.4% by weight, 57.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight, 58.3% by weight, 58.4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight, 59.8% by weight, 59.9% by weight, 60 Weight%, 60.1%, 60.2%, 60.3%, 60.4%, 60.5%, 60.6%, 60.7%, 60.8%, 60.9%, 61%, 61.1%, 61.2%, 61.3%, 61.4%, 61.5 Weight%, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight %, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight, 64.5% by weight %, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65.3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65.9% by weight, 66% by weight, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6%, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight of ammonium bisulfite, or any range or value derivable therein. In some aspects, the solution comprises at least 66%, 66.01%, 66.02%, 66.03%, 66.04% by weight, 66.05% by weight, 66.06% by weight, 66.07% by weight, 66.08% by weight, 66.09% by weight, 66.1% by weight, 66.11% by weight, 66.12% by weight, 66.13% by weight, 66.14% by weight, 66.15% by weight, 66.16% by weight, 66.17% by weight, 66 .18% by weight, 66.19% by weight, 66.2% by weight, 66.21% by weight, 66.22% by weight, 66.23% by weight, 66.24% by weight, 66.25% by weight, 66.26% by weight, 66.27% by weight, 66.28% by weight, 66.29% by weight, 66.3% by weight, 66.31% by weight, 66. 32% by weight, 66.33% by weight, 66.34% by weight, 66.35% by weight, 66.36% by weight, 66.37% by weight, 66.38% by weight, 66.39% by weight, 66.4% by weight, 66.41% by weight, 66.42% by weight, 66.43% by weight, 66.44% by weight, 66.45% by weight, 66. 46% by weight, 66.47% by weight, 66.48% by weight, 66.49% by weight, 66.5% by weight, 66.51% by weight, 66.52% by weight, 66.53% by weight, 66.54% by weight, 66.55% by weight, 66.56% by weight, 66.57% by weight, 66.58% by weight, 66.59% by weight, 66. 6% by weight, 66.61% by weight, 66.62% by weight, 66.63% by weight, 66.64% by weight, 66.65% by weight, 66.66% by weight, 66.67% by weight, 66.68% by weight, 66.69% by weight, 66.7% by weight, 66.71% by weight, 66.72% by weight, 66.73% by weight, 66. 74% by weight, 66.75% by weight, 66.76% by weight, 66.77% by weight, 66.78% by weight, 66.79% by weight, 66.8% by weight, 66.81% by weight, 66.82% by weight, 66.83% by weight, 66.84% by weight, 66.85% by weight, 66.86% by weight, 66.87% by weight, 66. 88%, 66.89%, 66.9%, 66.91%, 66.92%, 66.93%, 66.94%, 66.95%, 66.96%, 66.97%, 66.98%, 66.99%, or 67% ammonium bisulfite, or any range or value derivable therein, up to 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08%, 66.09%, 66.1%, 66.11 wt%, 66.12 wt%, 66.13 wt%, 66.14 wt%, 66.15 wt%, 66.16 wt%, 66.17 wt%, 66.18 wt%, 66.19 wt%, 66.2 wt%, 66.21 wt%, 66.22 wt%, 66.23 wt%, 66.24 wt%, 66.25 wt%, 66.26 wt%, 66.27 wt%, 66.28 wt%, 66.29 wt%, 66.3 wt%, 66.31 wt%, 66.32 wt %, 66.33 wt%, 66.34 wt%, 66.35 wt%, 66.36 wt%, 66.37 wt%, 66.38 wt%, 66.39 wt%, 66.4 wt%, 66.41 wt%, 66.42 wt%, 66.43 wt%, 66.44 wt%, 66.45 wt%, 66.46 wt%, 66.47 wt%, 66.48 wt%, 66.49 wt%, 66.5 wt%, 66.51 wt%, 66.52 wt%, 66.53 wt%, 66.54 wt%, 66.55 wt%, 66.56 wt%, 66.57 wt%, 66.58 wt%, 66.59 wt%, 66.6 wt%, 66.61 wt%, 66.62 wt%, 66.63 wt%, 66.64 wt%, 66.65 wt%, 66.66 wt%, 66.67 wt%, 66.68 wt%, 66.69 wt%, 66.7 wt%, 66.71 wt%, 66.72 wt%, 66.73 wt%, 66.74 wt%, 66. 75 wt%, 66.76 wt%, 66.77 wt%, 66.78 wt%, 66.79 wt%, 66.8 wt%, 66.81 wt%, 66.82 wt%, 66.83 wt%, 66.84 wt%, 66.85 wt%, 66.86 wt%, 66.87 wt%, 66.88 wt%, 66.89 wt%, 66.9 wt%, 66.91 wt%, 66.92 wt%, 66.93 wt%, 66.94 wt%, 66.95 wt%, 66.96 wt. %, 66.97%, 66.98%, 66.99%, or 67% by weight of ammonium bisulfite, or any range or value derivable therein, or about 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08%, 66.09%, 66.1%, 66.11%, 66.12%, 66.13%, 66.14%, 66.15%, 66.16%, 66.17%, 66.18%, 66.19%, 66.20%, 66.21%, 66.22%, 66.23%, 66.24%, 66.25%, 66.26%, 66.27%, 66.28%, 66.29%, 66.30%, 66.31%, 66.32%, 66.33%, 66.34%, 66.35%, 66.36%, 66.37%, 66.38%, 66.39%, 66.40%, 66.41%, 66.42%, 66.43%, 66.44%, 66.45%, 66.46%, 66.47%, 66.48%, 66.49%, 67. .18wt%, 66.19wt%, 66.2wt%, 66.21wt%, 66.22wt%, 66.23wt%, 66.24wt%, 66.25wt%, 66.26wt%, 66.27wt%, 66.28wt%, 66.29wt%, 66.3wt%, 66.31wt %, 66.32 wt%, 66.33 wt%, 66.34 wt%, 66.35 wt%, 66.36 wt%, 66.37 wt%, 66.38 wt%, 66.39 wt%, 66.4 wt%, 66.41 wt%, 66.42 wt%, 66.43 wt%, 66.44 wt%, 66. 45% by weight, 66.46% by weight, 66.47% by weight, 66.48% by weight, 66.49% by weight, 66.5% by weight, 66.51% by weight, 66.52% by weight, 66.53% by weight, 66.54% by weight, 66.55% by weight, 66.56% by weight, 66.57% by weight, 66.58% by weight %, 66.59 wt%, 66.6 wt%, 66.61 wt%, 66.62 wt%, 66.63 wt%, 66.64 wt%, 66.65 wt%, 66.66 wt%, 66.67 wt%, 66.68 wt%, 66.69 wt%, 66.7 wt%, 66.71 wt%, 66. 72% by weight, 66.73% by weight, 66.74% by weight, 66.75% by weight, 66.76% by weight, 66.77% by weight, 66.78% by weight, 66.79% by weight, 66.8% by weight, 66.81% by weight, 66.82% by weight, 66.83% by weight, 66.84% by weight, 66.85% by weight %, 66.86 wt%, 66.87 wt%, 66.88 wt%, 66.89 wt%, 66.9 wt%, 66.91 wt%, 66.92 wt%, 66.93 wt%, 66.94 wt%, 66.95 wt%, 66.96 wt%, 66.97 wt%, 66.98 wt%, 66.99% by weight, or 67% by weight ammonium bisulfite, or any range or value derivable therein.

[0048] In some aspects, the bisulfite solution is a solution with a bisulfite concentration of 6.5M to 10M, including any range or value derivable therebetween. In some aspects, the bisulfite solution is a solution with a bisulfite concentration of at least 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8.0M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9.0M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9 ... M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, up to 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8.0M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9.0M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, or about 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7.0M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, The bisulfite solution may be a solution with a bisulfite concentration of 7.6M, 7.7M, 7.8M, 7.9M, 8.0M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9.0M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein. In some aspects, the bisulfite solution is a solution with a bisulfite concentration of about 7.0M. In some aspects, the bisulfite solution is a solution with a bisulfite concentration of 7.0M.

[0049] In some aspects, the disclosed bisulfite solutions used to treat RNA contain 5% to 15% ammonium sulfite by weight, or any range or value derivable therein. In some aspects, the solutions contain at least 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 85%, 86%, 87%, 88%, 89%, 90 .1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight , 9.8% by weight, 9.9% by weight, 10% by weight, 10.1% by weight, 10.2% by weight, 10.3% by weight, 10.4% by weight, 10.5% by weight, 10.6% by weight, 10.7% by weight, 10.8% by weight, 10.9% by weight, 11% by weight, 11.1% by weight, 11.2% by weight, 11.3% by weight, 11.4% by weight, 11.5% by weight, 11.6% by weight, 11.7% by weight, 11.8% by weight, 11.9% by weight, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 12.7% by weight Amount %, 12.8 wt%, 12.9 wt%, 13 wt%, 13.1 wt%, 13.2 wt%, 13.3 wt%, 13.4 wt%, 13.5 wt%, 13.6 wt%, 13.7 wt%, 13.8 wt%, 13.9 wt%, 14 wt%, 14.1 wt%, 14.2 %, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, up to 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9 ...5%, 7.6%, 7.7%, 7.8%, 7.9%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.5%, 7.6%, 7.7%, 7.8%, 7.8%, 7.9%, 7.1%, 7.2%,8% by weight, 6.9% by weight, 7% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight, 7.7% by weight, 7.8% by weight, 7.9% by weight, 8% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6 Weight%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 1 0.4% by weight, 10.5% by weight, 10.6% by weight, 10.7% by weight, 10.8% by weight, 10.9% by weight, 11% by weight, 11.1% by weight, 11.2% by weight, 11.3% by weight, 11.4% by weight, 11.5% by weight, 11.6% by weight, 11.7% by weight, 11.8% by weight, 11.9% by weight %, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 12.7% by weight, 12.8% by weight, 12.9% by weight, 13% by weight, 13.1% by weight, 13.2% by weight, 13.3% by weight, 13.4% by weight, 13.5% by weight %, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, or about 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3% by weight, 6.4% by weight, 6.5% by weight, 6.6% by weight, 6.7% by weight, 6.8% by weight, 6.9% by weight, 7% by weight, 7.1% by weight, 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight, 7.7% by weight, 7.8% by weight, 7.9% by weight, 8% by weight, 8. 1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, 10% by weight, 10.1% by weight, 10.2% by weight, 10.3% by weight, 10.4% by weight, 10.5% by weight, 10.6% by weight, 10.7% by weight, 10.8% by weight, 10.9% by weight, 11% by weight, 11.1% by weight, 11.2% by weight, 11.3% by weight Amount%, 11.4% by weight, 11.5% by weight, 11.6% by weight, 11.7% by weight, 11.8% by weight, 11.9% by weight, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 12.7% by weight %, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, or 15% ammonium sulfite by weight, or any range or value derivable therein. In some aspects, the bisulfite solution comprises 8% to 12% ammonium sulfite by weight. In some aspects, the bisulfite solution comprises about 10% ammonium sulfite by weight. In some aspects, the bisulfite solution is made by mixing an ammonium bisulfite solution (e.g., 50%-70% ammonium bisulfite) with ammonium sulfite (e.g., ammonium sulfite monohydrate solid).

[0050] In some aspects, the RNA processing method includes incubating one or more RNA molecules in a bisulfite solution of the present disclosure (e.g., a solution containing ammonium bisulfite and ammonium sulfite, without sodium bisulfite or added sodium bisulfite) at a temperature of at least 80° C. for up to 20 minutes. In some aspects, the method includes heating one or more RNA molecules in a bisulfite solution at at least 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 82.10°C, 82.12°C, 82.13°C, 82.14°C, 82.15°C, 82.16°C, 82.17°C, 82.18°C, 82.19°C, 82.20°C, 82.21°C, 82.22°C, 82.23°C, 82.24°C, 82.25°C, 82.26°C, 82.27°C, 82.28°C, 82.29°C, 82.30°C, 82.31°C, 82.32°C, 82.33°C, 82.34°C, 82.35°C, 82.36°C, 82.37°C, 82.38°C, 8 2.9℃, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃, 85.2℃, 85.3℃, 85.4℃, 85.5℃, 85.6℃, 85.7℃, 85.8℃, 85.9℃, 86℃, 86.1℃, 86.2℃, 86.3℃, 86.4℃, 86.5℃, 86.6℃, 86.7℃, 86.8℃, 86.9℃, 87℃, 87.1℃, 87.2℃, 87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃, 89.5℃, 89.6℃, 89.7℃, 89.8℃, 89.9℃, 90℃, 90.1℃, 90.2℃ ,90.3℃,90.4℃,90.5℃,90.6℃,90.7℃,90.8℃,90.9℃,91℃,91.1℃,91.2℃,91.3℃,91.4℃,91.5℃,91.6℃,91.7℃,91.8℃,91.9℃,92℃,92.1℃,92.2℃,92.3℃,92.4℃,92.5℃,92.6℃,92.7℃,92.8℃,92.9℃,93℃,93.1℃,93.2℃,93.3℃,93.4℃,93.5℃,93.6℃,93.7℃,93.8℃,93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94.9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 95.8℃, 95.9℃, 96℃, 9 6.1℃, 96.2℃, 96.3℃, 96.4℃, 96.5℃, 96.6℃, 96.7℃, 96.8℃, 96.9℃, 97℃, 97.1℃, 97.2℃, 97.3℃, 97.4℃, 97.5℃, 97.6℃, 97.7℃, 97.8℃, 97.9℃, 98℃, 98.1℃, 98.2 °C, 98.3 °C, 98.4 °C, 98.5 °C, 98.6 °C, 98.7 °C, 98.8 °C, 98.9 °C, 99 °C, 99.1 °C, 99.2 °C, 99.3 °C, 99.4 °C, 99.5 °C, 99.6 °C, 99.7 °C, 99.8 °C, or 99.9 °C (or any range or value derivable therein), up to 80 °C, 80.1 °C, 80.2 °C, 80.3 °C, 80.4 °C, 80.5 °C, 80.6 °C, 80.7 °C, 80.8 °C, 80.9 °C, 81 °C, 81.1 °C, 81.2 °C, 81.3 °C, 81.4 °C, 81.5 °C, 81.6 °C, 81.7 °C, 81.8 °C, 81.9 ... 2℃, 82.1℃, 82.2℃, 82.3℃, 82.4℃, 82.5℃, 82.6℃, 82.7℃, 82.8℃, 82.9℃, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃, 85.2℃, 85.3℃, 85.4℃, 85.5℃, 85.6℃, 85.7℃, 85.8℃, 85.9℃, 86℃, 86.1℃, 86.2℃, 86. 3℃, 86.4℃, 86.5℃, 86.6℃, 86.7℃, 86.8℃, 86.9℃, 87℃, 87.1℃, 87.2℃, 87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃, 89.5℃, 89.6℃, 89.7℃, 89.8℃, 89.9℃, 90℃, 90.1℃, 90.2℃, 90.3℃, 90.4℃, 90.5℃, 90.6℃, 90.7℃, 90.8℃, 90.9℃, 91℃, 91.1℃, 91.2℃, 91.3℃, 91.4℃, 91.5℃, 91.6℃, 91.7℃, 91.8℃, 91.9℃, 92℃, 92.1℃, 92.2℃, 92.3℃, 92.4℃, 92.5℃, 92.6℃, 92.7℃, 92.8℃, 92.9℃, 93℃, 93.1℃, 93.2℃, 93.3℃, ​​93.4℃, 93.5℃, 93.6℃, ​​93.7℃, 93.8℃, 93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94. 9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 95.8℃, 95.9℃, 96℃, 96.1℃, 96.2℃, 96.3℃, 96.4℃, 96.5℃, 96.6℃, 96.7℃, 96.8℃, 96.9℃, 97℃, 97 .1℃, 97.2℃, 97.3℃, 97.4℃, 97.5℃, 97.6℃, 97.7℃, 97.8℃, 97.9℃, 98℃, 98.1℃, 98.2℃, 98.3℃, 98.4℃, 98.5℃, 98.6℃, 98.7℃, 98.8℃, 98.9℃, 99℃, 99.1℃, 99.2℃ , 99.3°C, 99.4°C, 99.5°C, 99.6°C, 99.7°C, 99.8°C, or 99.9°C (or any range or value derivable therein), or about 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 83℃, 83.1℃, 83.2℃, 83.3℃, ​​83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃ ,85.2℃,85.3℃,85.4℃,85.5℃,85.6℃,85.7℃,85.8℃,85.9℃,86℃,86.1℃,86.2℃,86.3℃,86.4℃,86.5℃,86.6℃,86.7℃,86.8℃,86.9℃,87℃,87.1℃,87.2℃,87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃, 89.4℃ ,89.5℃,89.6℃,89.7℃,89.8℃,89.9℃,90℃,90.1℃,90.2℃,90.3℃,90.4℃,90.5℃,90.6℃,90.7℃,90.8℃,90.9℃,91℃,91.1℃,91.2℃,91.3℃,91.4℃,91.5℃,91 .6℃, 91.7℃, 91.8℃, 91.9℃, 92℃, 92.1℃, 92.2℃, 92.3℃, 92.4℃, 92.5℃, 92.6℃, 92.7℃, 92.8℃, 92.9℃, 93℃, 93.1℃, 93.2℃, 93.3℃, ​​93.4℃, 93.5℃, 93.6℃, ​​93.7 ℃, 93.8℃, 93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94.9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 95.8℃, 9 5.9°C, 96°C, 96.1°C, 96.2°C, 96.3°C, 96.4°C, 96.5°C, 96.6°C, 96.7°C, 96.8°C, 96.9°C, 97°C, 97.1°C, 97.2°C, 97.3°C, 97.4°C, 97.5°C, 97.6°C, 97.7°C, 97.8°C, 97.9°C, 98°C, 98.1°C, 98.2°C, 98.3°C, 98.4°C, 98.5°C, 98.6°C, 98.7°C, 98.8°C, 98.9°C, 99°C, 99.1°C, 99.2°C, 99.3°C, 99.4°C, 99.5°C, 99.6°C, 99.7°C, 99.8°C, or 99.9°C (or any derivative thereof) 15 minutes, 14.9 minutes, 14.8 minutes, 14.7 minutes, 14.6 minutes, 14.5 minutes, 14.4 minutes, 14.3 minutes, 14.2 minutes, 14.1 minutes, 14 minutes, 13.9 minutes, 13.8 minutes, 13.7 minutes, 13.6 minutes, 13.5 minutes, 13.4 minutes, 13.3 minutes, 13.2 minutes, 13.1 minutes, 13 minutes, 12.9 minutes, 12.8 minutes, 12.7 minutes, 12.6 minutes, 12.5 minutes, 12.4 minutes, 12.3 minutes, 12.2 minutes, 12.1 minutes, 12 minutes, 11.9 minutes, 11.8 minutes, 11.7 minutes, 11.6 minutes, 11.5 minutes, 11.4 minutes, 11.3 minutes, 11.2 minutes, 11.1 minutes, 11 minutes, 10.9 minutes, 10.8 minutes, 10.7 minutes, 10.6 minutes, 10.5 minutes, 10.4 minutes, 10.3 minutes, 10.2 minutes, 10.1 minutes, 10 minutes, 9.9 minutes, 9.8 minutes, 9.7 minutes, 9.6 minutes, 9.5 minutes, 9.4 minutes, 9.3 minutes, 9.2 minutes, 9.1 minutes, 9 minutes, 8.9 minutes, 8.8 minutes, 8.7 minutes, 8.6 minutes, 8.5 minutes, 8.4 minutes, 8.3 minutes, 8.2 minutes, 8.1 minutes, 8 minutes, 7.9 minutes, 7.8 minutes, 7.7 minutes, 7.6 minutes, 7.5 minutes, 7.4 minutes, 7.3 minutes, 7.2 minutes, 7.1 minutes, 7 minutes, 6.9 minutes, 6.8 minutes, 6.7 minutes, 6.6 minutes, 6.5 minutes, 6.4 minutes, 6.3 minutes, 6.2 minutes, 6.1 minutes, 6 minutes, 5.9 minutes, 5.8 minutes, 5.7 minutes, 5.6 minutes, 5.5 minutes, 5.4 minutes, 5.3 minutes, 5.2 minutes, 5.1 minutes, 5 minutes, 4.9 minutes, 4.8 minutes, 4.7 minutes, 4.6 minutes, 4.5 minutes, 4.4 minutes, 4.3 minutes, 4.2 minutes, 4.1 minutes, 4 minutes, 3.9 minutes, 3.8 minutes, 3.7 minutes, 3.6 minutes, 3.5 minutes, 3.4 minutes, 3.3 minutes, 3.2 minutes, 3.1 minutes, 3 minutes, 2.9 minutes, 2.8 minutes, 2.7 minutes, 2.6 minutes, 2.5 minutes, 2.4 minutes, 2.3 minutes, 2.2 minutes, 2.1 minutes, 2 minutes, 1.9 minutes, 1.8 minutes, 1.7 minutes, 1.6 minutes, 1.5 minutes, 1.4 minutes, 1.3 minutes, 1.2 minutes, 1.1 minutes, or 1 minute (or over any range or value derivable therein), or about 15 minutes, 14.9 minutes, 14.8 minutes, 14.7 minutes, 14.6 minutes, 14.5 minutes, 14.4 minutes, 14.3 minutes, 14.2 minutes, 14.1 minutes, 14 minutes Between, 13.9 minutes, 13.8 minutes, 13.7 minutes, 13.6 minutes, 13.5 minutes, 13.4 minutes, 13.3 minutes, 13.2 minutes, 13.1 minutes, 13 minutes, 12.9 minutes, 12.8 minutes, 12.7 minutes, 12.6 minutes, 12.5 minutes, 12.4 minutes, 12.3 minutes, 12.2 minutes, 12.1 minutes, 12 minutes, 11.9 minutes, 11.8 minutes, 11.7 minutes, 11.6 minutes, 11.5 minutes, 11.4 minutes, 11.3 minutes, 11.2 minutes, 11.1 minutes, 11 minutes, 10.9 minutes, 10.8 minutes, 10.7 minutes, 10.6 minutes, 10.5 minutes, 10.4 minutes, 10.3 minutes, 10.2 minutes, 10.1 minutes, 10 minutes, 9.9 minutes, 9.8 minutes, 9.7 minutes, 9.6 minutes, 9.5 minutes, 9.4 minutes, 9.3 minutes, 9.2 minutes, 9.1 minutes, 9 minutes, 8.9 minutes, 8.8 minutes, 8.7 minutes, 8.6 minutes, 8.5 minutes, 8.4 minutes, 8.3 minutes, 8.2 minutes, 8.1 minutes, 8 minutes, 7.9 minutes, 7.8 minutes, 7.7 minutes, 7.6 minutes, 7.5 minutes, 7.4 minutes, 7.3 minutes, 7.2 minutes, 7.1 minutes, 7 minutes, 6.9 minutes, 6.8 minutes, 6.7 minutes, 6.6 minutes, 6.5 minutes, 6.4 minutes, 6.3 minutes, 6.2 minutes, 6.1 minutes, 6 minutes, 5.9 minutes, 5.8 minutes, 5.7 minutes, 5.6 minutes, 5.5 minutes, 5.4 minutes, 5.3 minutes , 5.2 minutes, 5.1 minutes, 5 minutes, 4.9 minutes, 4.8 minutes, 4.7 minutes, 4.6 minutes, 4.5 minutes, 4.4 minutes, 4.3 minutes, 4.2 minutes, 4.1 minutes, 4 minutes, 3.9 minutes, 3.8 minutes, 3.7 minutes, 3.6 minutes, 3.5 minutes, 3.4 minutes, 3.3 minutes, 3.2 minutes, 3.1 minutes, 3 minutes, 2.9 minutes, 2.8 minutes, 2.7 minutes, 2.6 minutes, 2.5 minutes, 2.4 minutes, 2.3 minutes, 2.2 minutes, 2.1 minutes, 2 minutes, 1.9 minutes, 1.8 minutes, 1.7 minutes, 1.6 minutes, 1.5 minutes, 1.4 minutes, 1.3 minutes, 1.2 minutes, 1.1 minutes, or 1 minute (or over any range or value derivable therein). Any combination of the aforementioned incubation times and temperatures can be used in the RNA processing methods of the present disclosure. .

[0051] In some aspects, the RNA treatment method comprises treating one or more RNA molecules in a bisulfite solution of the disclosure at a temperature of at least 95° C. for up to 12 minutes, at a temperature of at least 96° C. for up to 12 minutes, at a temperature of at least 97° C. for up to 12 minutes, at a temperature of at least 98° C. for up to 12 minutes, at a temperature of at least 99° C. for up to 11 minutes, at a temperature of at least 95° C. for up to 11 minutes, at a temperature of at least 96° C. for up to 11 minutes, at a temperature of at least 97° C. for up to 11 minutes, at least at least 98°C for a maximum of 11 minutes, at least 99°C for a maximum of 11 minutes, at least 95°C for a maximum of 10 minutes, at least 96°C for a maximum of 10 minutes, at least 97°C for a maximum of 10 minutes, at least 98°C for a maximum of 10 minutes, at least 99°C for a maximum of 10 minutes, at least 95°C for a maximum of 9 minutes, at least 96°C for a maximum of 9 minutes, at least 97°C for a maximum of 9 minutes, at least 98°C for a maximum of 9 minutes 30 minutes, at a temperature of at least 99°C for up to 9 minutes, at a temperature of at least 95°C for up to 8 minutes, at a temperature of at least 96°C for up to 8 minutes, at a temperature of at least 97°C for up to 8 minutes, at a temperature of at least 98°C for up to 8 minutes, at a temperature of at least 99°C for up to 7 minutes, at a temperature of at least 95°C for up to 7 minutes, at a temperature of at least 96°C for up to 7 minutes, at a temperature of at least 97°C for up to 7 minutes, at a temperature of at least 98°C for up to 7 minutes, at a temperature of at least 99°C for up to 6 minutes, at a temperature of at least 96°C for up to 6 minutes, at a temperature of at least 97°C for up to 6 minutes, at a temperature of at least 98°C for up to 6 minutes, at a temperature of at least 99°C for up to 5 minutes, at a temperature of at least 96°C for up to 5 minutes, at a temperature of at least 97°C for up to 5 minutes, at a temperature of at least 98°C for up to 5 minutes, or at least 99°C for up to 5 minutes.

[0052] As disclosed herein, incubating an RNA molecule with a bisulfite solution of the present disclosure (e.g., a solution containing ammonium bisulfite and ammonium sulfite, without sodium bisulfite or added sodium bisulfite) under appropriate conditions (e.g., at a temperature of at least 95°C for up to 12 minutes) is sufficient to deaminate the majority of cytosine residues in the RNA molecule. In some aspects, after incubating an RNA molecule with a bisulfite solution of the present disclosure under appropriate conditions, more than 90% of the RNA molecules do not contain cytosine residues. In some aspects, 90%, 90.1%, 90.2%, 90.3%, 90.4%, 90.5%, 90.6%, 90.7%, 90.8%, 90.9%, 91%, 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, 91.6%, 91.7%, 91.8%, 91.9%, 92%, 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, 92.6%, 92.7%, 92.8%, 92.9%, 92.10%, 92.11%, 92.12%, 92.13%, 92.14%, 92.15%, 92.16%, 92.17%, 92.18%, 92.19%, 92.20%, 92.21%, 92.22%, 92.23%, 92.24%, 92.25%, 92.26%, 92.27%, 92.28%, 92.29%, 92.30%, 92.31%, 92.32%, 92.33%, 92.34%, 92.35%, 92.36%, 92.37%, 92.38%, 92.39 ... 2.5%, 92.6%, 92.7%, 92.8%, 92.9%, 93%, 93.1%, 93.2%, 93.3%, 93.4%, 93.5%, 93.6%, 93.7%, 93.8%, 93.9%, 94%, 94.1%, 94.2%, 94.3%, 94.4%, 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95%, 95.1%, 95.2 %, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9% , 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any range or value derivable therein) do not contain cytosine residues. In some aspects, more than 99% of the RNA molecules do not contain cytosine residues.

[0053] The RNA processing methods of the present disclosure can be useful, for example, to prepare RNA molecules for sequencing to detect, quantitate, and / or analyze RNA cytosine methylation.

[0054] III.5hmC analysis method Aspects of the present disclosure relate to compositions and methods for detecting, quantifying, and analyzing 5-hydroxymethylcytosine (5hmC) in DNA. As described herein, the disclosed DNA processing methods are useful for the rapid deamination of cytosine and the rapid spontaneous conversion of 5hmC to cytosine methylene sulfonate (CMS). APOBEC3A has been reported to have high deamination reactivity for C and 5mC. 28 Thus, in certain aspects, disclosed herein are methods for 5hmC analysis that include incubating DNA molecules under sufficient conditions (e.g., at a temperature of at least 95°C for up to 12 minutes) in a bisulfite solution of the present disclosure (e.g., sodium bisulfite, or ammonium bisulfite without added sodium bisulfite, e.g., a solution containing 50%-70% ammonium bisulfite), followed by subjecting the DNA molecules to alkaline conditions, thereby converting C to U and 5hmC to CMS. This is then optionally followed by treating a portion of the DNA molecule with an APOBEC deaminase enzyme (e.g., APOBEC3A under appropriate conditions, such as those disclosed in Schutsky, E., DeNizio, et al. Nat Biotechnol 36, 1083-1090 (2018), which is incorporated herein by reference in its entirety), thus converting 5mC to U. The DNA molecules are then all subjected to sequencing and the sequences are compared to identify the 5hmC residues that were present on the original DNA molecule. A schematic diagram of one example of the disclosed 5hmC analysis method is shown in FIG.

[0055] IV. General Assay Methods A. Detection and Analysis of Methylated Nucleic Acids Aspects of the methods include assaying a nucleic acid (e.g., DNA, RNA) to determine its expression level and / or methylation level. Certain exemplary methods for detecting and analyzing nucleic acid methylation are described herein.

[0056] In certain aspects, the method provided herein facilitates the creation of BS-treated sequencing libraries with low DNA input and / or ultra-low DNA input.In certain aspects, the method provided herein facilitates the creation of BS-treated sequencing libraries with low RNA input and / or ultra-low RNA input.In some aspects, the method provided herein reduces background levels in assays that include low DNA input and / or ultra-low DNA input compared to standard BS treatment.In some aspects, the method provided herein reduces background levels in assays that include low RNA input and / or ultra-low RNA input compared to standard BS treatment.In some aspects, the methods provided herein reduce the false positive rate to about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 times, or any of them 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 101, 104 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 times, or any range derivable therein.In some aspects, the methods provided herein provide a true positive detection rate of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 10 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent, or any combination thereof or approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent, or any range derivable therein.

[0057] In some aspects, the methods provided herein reduce the rate of non-converted C in high GC% regions compared to standard BS treatment. In some aspects, the methods provided herein reduce the rate of non-converted C in high GC% regions by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 1 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent, or any range derivable therein, or about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent, or any range derivable therein.

[0058] 1. HPLC-UV The HPLC-UV (High Performance Liquid Chromatography-Ultraviolet) technique was developed by Kuo and coworkers in 1980 (further described in Kuo KC et al., Nucleic Acids Res. 1980;8:4763-4776, incorporated herein by reference) and can be used to quantify the amount of deoxycytidine (dC) and methylated cytosine (5mC) present in a hydrolyzed DNA sample. The method involves hydrolyzing DNA into its constituent nucleoside bases, where the 5mC and dC bases are separated by chromatography and then the ratios are measured. The 5mC / dC ratio can then be calculated for each sample, which can be compared between experimental and control samples.

[0059] 2.LC-MS / MS Liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) is a highly sensitive approach to HPLC-UV that requires extremely small amounts of hydrolyzed DNA sample. For mammalian DNA, where approximately 2%-5% of all cytosine residues are methylated, LC-MS / MS has been validated for detection of methylation levels at the 0.05%-10% level and can confidently detect sample-to-sample differences as small as 0.25% of total cytosine residues, corresponding to approximately 5% differences in global DNA methylation. The procedure routinely requires 50-100 ng of DNA sample, although much smaller amounts (as little as 5 ng) have been successfully characterized.

[0060] 3. ELISA-based Methods There are several commercially available kits that allow for the rapid assessment of DNA methylation status, all based on enzyme-linked immunosorbent assays (ELISAs). These assays include the Global DNA Methylation ELISA available from Cell Biolabs; the Imprint Methylated DNA Quantification kit (sandwich ELISA) available from Sigma-Aldrich; the EpiSeeker methylated DNA Quantification Kit available from abcam; the Global DNA Methylation Assay-LINE-1 available from Active Motif; the 5-mC DNA ELISA Kit available from Zymo Research; the MethylFlash Methylated DNA 5-mC Quantification Kit and the MethylFlash Methylated DNA 5-mC Quantification Kit available from Epigentek.

[0061] Briefly, DNA samples are captured on an ELISA plate and methylated cytosines are detected by sequential incubation steps with (1) a primary antibody raised against 5Mc; (2) a labeled secondary antibody, followed by (3) a colorimetric / fluorescent detection reagent.

[0062] The Global DNA Methylation Assay-LINE-1 specifically determines the methylation levels of LINE-1 (long interspersed nuclear elements-1) retrotransposons, which comprise approximately 17% of the human genome. They are well established as a surrogate for global DNA methylation. Briefly, fragmented DNA is hybridized to a biotinylated LINE-1 probe and then subsequently immobilized on a streptavidin-coated plate. After washing and blocking steps, methylated cytosines are quantified using an anti-5mC antibody, an HRP-conjugated secondary antibody, and a chemiluminescent detection reagent. Samples are quantified against a standard curve generated from standards with known LINE-1 methylation levels.

[0063] 4. LINE-1 Pyrosequencing Alternatively, the level of LINE-1 methylation can be assessed by another method involving bisulfite conversion of DNA followed by PCR amplification of LINE-1 conserved sequences. The methylation status of the amplified fragments is then quantified by pyrosequencing, which is able to resolve DNA sample differences as low as about 5%. Although this technique evaluates LINE-1 elements and therefore relatively few CpG sites, it has been shown to reflect global DNA methylation changes very well. This method is particularly well suited for high-throughput analysis of cancer samples, where hypomethylation is very well associated with poor prognosis. The method is particularly suitable for human DNA, but there are also versions adapted for the rat and mouse genomes.

[0064] 5. AFLP and RFLP Detection of differentially methylated fragments could be accomplished by conventional PCR-based protocols using amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), or a combination of both.

[0065] 6. LUMA The LUMA (luminometric methylation assay) method utilizes a combination of two DNA restriction digestion reactions performed in parallel and a subsequent pyrosequencing reaction to fill in the protruding ends of digested DNA strands. One digestion reaction is performed with the CpG methylation-sensitive enzyme HpaII. In contrast, a parallel reaction uses the methylation-insensitive enzyme MspI, which cleaves at all CCGG sites. The enzyme EcoRI is included in both reactions as an internal standard. Both MspI and HpaII generate 5'-CG overhangs after DNA cleavage, whereas EcoRI generates 5'-AATT overhangs, which are then filled in by a subsequent pyrosequencing-based extension assay. In essence, the light signal measurement, calculated as the HpaII / MspI ratio, is proportional to the amount of unmethylated DNA present in the sample. Because the sequence of nucleotides added to the pyrosequencing reaction is known, the specificity of this method is very high and the variability is low. This is essential for detecting subtle changes in global methylation. LUMA requires relatively small amounts of DNA (250-500 ng), shows little variability, and has the advantage of an internal standard to account for variability in DNA input amounts.

[0066] 7. Bisulfite Sequencing Bisulfite treatment of DNA mediates the deamination of cytosine to uracil, and these converted residues are read as thymine when confirmed by PCR amplification and subsequent Sanger sequencing analysis. However, 5-methylcytosine (5mC) residues are resistant to this conversion and therefore still read as cytosine. Thus, comparing Sanger sequencing read data from an untreated DNA sample with the same sample after bisulfite treatment allows for the detection of methylated cytosines. With the advent of next-generation sequencing (NGS) technologies, this approach can be extended to whole-genome DNA methylation analysis. Controls may be incorporated into the bisulfite reaction to ensure complete conversion of unmethylated cytosines.

[0067] Whole-genome bisulfite sequencing (WGBS) is almost identical to whole-genome sequencing, except for the additional step of bisulfite conversion. Sequencing of genomic fractions enriched in 5mC is not only an inexpensive approach, but also allows for increased sequencing coverage and therefore greater accuracy in revealing differentially methylated regions. Sequencing could be performed using any existing NGS platform. Both Illumina™ and Life Technologies™ provide kits for such analysis.

[0068] Bisulfite sequencing methods include reduced representation bisulfite sequencing (RRBS), in which only a portion of the genome is sequenced. In RRBS, CpG-rich regions are enriched by isolating short fragments after MspI digestion, which recognizes CCGG sites (it can cleave both methylated and unmethylated sites). This ensures that approximately 85% of the CpG islands in the human genome are isolated. This is followed by the same bisulfite conversion and library preparation as for WGBS. Typically, the RRBS procedure requires approximately 100 ng to 1 μg of DNA.

[0069] 8. Methods excluding bisulfite conversion In some aspects, direct detection of modified bases without bisulfite conversion may be used to detect methylation.Pacific Biosciences has developed a method to directly detect methylated bases by monitoring the kinetics of polymerase during single molecule sequencing, and provides a commercial product for such sequencing (as described further in Flusberg BA, et al., Nat. Methods. 2010;7:461-465, which is incorporated herein by reference).Other methods include nanopore-based single-molecule real-time sequencing technology (SMRT), which can directly detect modified bases (as described in Laszlo AH et al., Proc. Natl. Acad. Sci. USA. 2013 and Schreiber J., et al., Proc. Natl. Acad. Sci. USA. 2013, which is incorporated herein by reference).

[0070] 9. Array or Bead Hybridization Typically, the methylated DNA portion of the genome could be obtained by immunoprecipitation and used for hybridization with a microarray. Currently available examples of such arrays include the Human CpG Island Microarray Kit (Agilent®), the GeneChip Human Promoter 1.0R Array, and the GeneChip Human Tiling 2.0R Array Set (Affymetrix®).

[0071] Searching for differentially methylated regions using bisulfite converted DNA could be done using a variety of techniques. Some of these techniques are easier to implement and analyze than others because only a portion of the genome is used. The most prominent functional effects of DNA methylation occur within gene promoter regions, enhancer regulatory elements, and 3' untranslated regions (3'UTRs). Assays focused on these specific regions, such as the Infinium HumanMethylation450 Bead Chip arrays by Illumina™, can be used. These arrays can be used to detect the methylation status of genes, including miRNA promoters, 5'UTRs, 3'UTRs, coding regions (about 17 CpG per gene), and island shores (regions about 2 kb upstream of CpG islands).

[0072] Briefly, bisulfite-treated genomic DNA is mixed with assay oligos, one of which is complementary to the uracil (converted from the original unmethylated cytosine) and another complementary to the cytosine at the methylated (and thus protected from conversion) site. After hybridization, the primer is extended and ligated with the locus-specific oligo to create a template for universal PCR. Finally, labeled PCR primers are used to create detectable products that are immobilized on bar-coded beads and the signal is measured. The ratio between the two types of beads for each locus (individual CpG) is an indication of its methylation level.

[0073] Kits that utilize methylation-specific primer extension for validation studies are available for purchase. The VeraCode Methylation assay from Illumina™ assays 96 or 384 user-specified CpG loci with the GoldenGate® Assay for Methylation. Unlike the BeadChip assay, the VeraCode assay requires a BeadXpress® Reader for scanning.

[0074] 10. Methyl-Sensitive Cut Counting: Endonuclease Digestion Followed by Sequencing Instead of sequencing significant amounts of methylated (or unmethylated) DNA, snippets could be generated from these regions and mapped to the genome after sequencing. Furthermore, the coverage in NGS might be good enough to quantify the methylation levels of specific loci. The technique of serial analysis of gene expression (SAGE) has been adapted for this purpose, also known as methylation-specific digital karyotyping, as well as a similar technique called methyl-sensitive cut counting (MSCC).

[0075] In summary, in all of these methods, a methylation-sensitive endonuclease, such as HpaII, is used to first digest genomic DNA at unmethylated sites, followed by the ligation of an adapter that contains a site for another digestive enzyme, such as EcoP15I or MmeI, that cuts outside its recognition site. These methods generate small fragments that are located in close proximity to the original HpaII site. NGS and mapping to the genome are then performed. The number of reads at each HpaII site correlates with its methylation level.

[0076] Recently, a number of restriction enzymes (methylation-dependent endonucleases) have been discovered that use methylated DNA as a substrate. These include, for example, BisI, BlsI, GlaI, GluI, KroI, MteI, PcsI, and PkrI. The unique ability of these enzymes to cleave only methylated sites has been exploited in methods to perform selective amplification of methylated DNA. Three methylation-dependent endonucleases available from New England Biolabs (FspEI, MspJI, and LpnPI) are type IIS enzymes that cleave outside of their recognition sites and thus can generate 32-bp snippets around fully methylated recognition sites that contain CpG. These short fragments could be sequenced and aligned to a reference genome. The number of reads obtained for each particular 32-bp fragment could be an indication of its methylation level. Similarly, short fragments could be generated from methylated CpG islands by the Escherichia coli methyl-specific endonuclease McrBC. The E. coli methyl-specific endonuclease McrBC cleaves DNA between two (G / A)mC half-sites, located within 50 bp to 3000 bp from each other.

[0077] B. Sequencing 1.DNA Sequencing In some aspects, DNA may be analyzed by sequencing.DNA can be prepared for sequencing by any method known in the art, such as library preparation, hybrid capture, sample quality control, product-based ligation-based library preparation, or combinations thereof.DNA can be prepared for any sequencing method.In some aspects, the unique genetic readout information of each sample can be generated by genotyping one or more highly polymorphic SNPs. In some aspects, sequencing, e.g., base pair and / or paired end sequencing, may be performed to cover about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more percent of the target oligonucleotides with 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage (or any range derivable therein), or with greater than 20x, 25x, 30x, 35x, 40x, 45x, 50x, or greater than 50x coverage (or any range derivable therein). In certain aspects, mutations, SNPS, indels, copy number alterations (somatic and / or germline), or other genetic differences may be identified from sequencing using at least one bioinformatics tool, including, but not limited to, VarScan2, any R package (including CopywriteR), and / or Annovar.

[0078] 2. RNA Sequencing In some aspects, RNA may be determined by sequencing. RNA may be prepared for sequencing by any method known in the art, including, but not limited to, poly-A selection, cDNA synthesis, strand-specific or non-strand-specific library preparation, or a combination thereof. RNA may be prepared for any type of RNA sequencing method, including, but not limited to, stranded specific RNA sequencing. In some aspects, sequencing may be performed to generate about 10M, 15M, 20M, 25M, 30M, 35M, 40M or more reads, including paired reads. In some aspects, sequencing may be performed with read lengths of about 50bp, 55bp, 60bp, 65bp, 70bp, 75bp, 80bp, 85bp, 90bp, 95bp, 100bp, 105bp, 110bp, or more (or any range derivable therein). In some aspects, raw sequencing data may be converted to estimated read count (RSEM), fragments per kilobase of transcript per million mapped reads (FPKM), and / or reads per kilobase of transcript per million mapped reads (RPKM).

[0079] 3. Example Sequencing Methods DNA (including bisulfite converted DNA) and / or RNA (including bisulfite converted RNA) may be used to amplify and subsequently sequence one or more regions of interest. Thus, aspects of the present disclosure may include sequencing nucleic acids to detect and / or quantify methylation of nucleic acid biomarkers. In some aspects, the methods of the present disclosure include sequencing methods. Sequencing may be excluded from certain methods of the present disclosure. Exemplary sequencing methods include, but are not limited to, the sequencing methods described below.

[0080] a. Massively parallel signature sequencing (MPSS). The first of the next-generation sequencing technologies, Massively Parallel Signature Sequencing (or MPSS), was developed at Lynx Therapeutics in the 1990s. MPSS was a bead-based method that used a hybrid approach of adapter ligation followed by adapter decoding and sequence readout in 4-nucleotide increments. This method provided high sensitivity to sequence-specific bias or loss of certain sequences.

[0081] b. Polony sequencing Polony sequencing, developed in the laboratory of George M. Church at Harvard, was among the first next-generation sequencing systems used to sequence a complete genome in 2005. It combined in vitro paired-tag libraries with emulsion PCR, automated microscopy, and ligation-based sequencing chemistry to sequence the E. coli genome with >99.9999% accuracy and approximately one-ninth the cost of Sanger sequencing.

[0082] c.454 Pyrosequencing(TM) A parallelized version of pyrosequencing was developed by 454 Life Sciences™, which was subsequently acquired by Roche Diagnostics™. The method amplifies DNA inside droplets in an oil solution (emulsion PCR), with each droplet containing one DNA template attached to one primer-coated bead that later forms a clonal colony. The sequencing machine has many picoliter-volume wells, each containing one bead and a sequencing enzyme. Pyrosequencing uses luciferase to generate light for the detection of individual nucleotides added to the nascent DNA, and the combined data is used to create a sequence readout. The technology offers intermediate read lengths and prices per base compared to Sanger sequencing on the one hand, and Solexa and SOLiD™ on the other.

[0083] d. Illumina™ (Solexa) Sequencing Solexa developed a sequencing method based on reversible dye terminator technology and an engineered polymerase developed in-house by Solexa. The terminated chemistry was developed in-house at Solexa and the concept of the Solexa system was conceived by Balasubramanian and Klennerman at Cambridge University's chemistry department. In 2004, Solexa acquired Manteia Predictive Medicine to acquire a massively parallel sequencing technology based on "DNA clusters" with clonal amplification of DNA on a surface. The cluster technology was acquired in conjunction with Lynx Therapeutics of California. Solexa Ltd. later merged with Lynx to form Solexa Inc.

[0084] In this method, DNA molecules and primers are first mounted on a slide and amplified with polymerase to form localized clonal DNA colonies, later named "DNA clusters". Four types of reversible terminator bases (RT-bases) are added to determine the sequence, and unincorporated nucleotides are washed away. A camera takes an image of the fluorescently labeled nucleotides, and then the dye is chemically removed from the DNA along with the terminal 3' blocker, allowing the next cycle to begin. Unlike pyrosequencing, the DNA strand is extended one nucleotide at a time, and image capture can be performed with a delay. This allows very large arrays of DNA colonies to be captured by successive images taken from a single camera.

[0085] Decoupling the enzymatic reaction from the image capture allows optimal throughput and theoretically unlimited sequencing capacity. Thus, in an optimal configuration, the final achievable instrument throughput is determined only by the analog-to-digital conversion rate of the cameras multiplied by the number of cameras divided by the number of pixels per DNA colony required for optimal visualization (~10 pixels / colony). In 2012, with cameras operating at A / D conversion rates of over 10 MHz and with the available optics, fluidics, and enzymatics, throughput can be as much as 1 million nucleotides / sec. This roughly corresponds to one human genome equivalent of 1x coverage / hour / instrument, and one human genome sequenced in one day (~30x) on one instrument (with one camera).

[0086] e.SOLiD™ Sequencing The SOLiD™ technology uses sequencing by ligation, where a pool of all possible oligonucleotides of a certain length is labeled according to the position to be sequenced. The oligonucleotides are annealed and ligated. Preferential ligation by DNA ligase to matching sequences results in an informative signal for the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting beads, each containing one copy of the same DNA molecule, are deposited on a glass slide. The result is sequences of comparable quantity and length to Illumina™ sequencing.

[0087] f.Ion Torrent™ Semiconductor Sequencing Ion Torrent™ Systems Inc. has developed a system based on the use of standard sequencing chemistry, but with a novel semiconductor-based detection system. This sequencing method is based on the detection of hydrogen ions released during DNA polymerization, in contrast to the optical methods used in other sequencing systems. A microwell containing the template DNA strand to be sequenced is filled with one type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide, it is incorporated into the growing complementary strand. This releases hydrogen ions that trigger an ultrasensitive ion sensor, indicating that a reaction has occurred. If homopolymeric repeats are present in the template sequence, multiple nucleotides are incorporated in one cycle. This causes a corresponding number of hydrogen releases, resulting in a proportionally high electronic signal.

[0088] g. DNA Nanoballs™ Sequencing DNA Nanoballs™ sequencing is a type of high-throughput sequencing technology used to determine an organism's entire genome sequence. CompleteGenomics® uses this technology to sequence samples submitted by independent researchers. The method uses rolling circle amplification to amplify small fragments of genomic DNA into DNA nanoballs. The nucleotide sequence is then determined using unchained sequencing by ligation. This DNA sequencing method allows for sequencing of many DNA nanoballs per run with less reagent cost compared to other next generation sequencing platforms. However, only a short DNA sequence is determined from each DNA nanoball, which can make mapping short reads to a reference genome difficult. This technology has been used in several genome sequencing projects.

[0089] h. Heliscope single molecule sequencing Heliscope sequencing is a method of single molecule sequencing developed by Helicos Biosciences. It uses DNA fragments and added polyA tail adapters attached to the surface of a flow cell. The next step involves extension-based sequencing with fluorescently labeled nucleotides and cyclic washing of the flow cell (one nucleotide type at a time, similar to the Sanger method). The reads are generated by the Heliscope sequencer. The reads are short, up to 55 bases per run, but recent improvements allow for more precise reading of segments of one type of nucleotide. This sequencing method and instrumentation have been used to sequence the genome of the M13 bacteriophage.

[0090] i. Single molecule real-time (SMRT) sequencing SMRT sequencing is based on a sequencing by synthesis approach. DNA is synthesized in a zero-mode waveguide (ZMW) - a small well-like container with a capture tool located at the bottom of the well. Sequencing is performed using an unmodified polymerase (attached to the bottom of the ZMW) and fluorescently labeled nucleotides flowing freely in solution. The wells are constructed in such a way that only the fluorescence generated near the bottom of the well is detected. When a nucleotide is incorporated into the DNA strand, the fluorescent label is cleaved from the nucleotide, leaving an unmodified DNA strand. According to Pacific Biosciences, the developer of the SMRT technology, this method makes it possible to detect nucleotide modifications (e.g., cytosine methylation). This occurs through the observation of polymerase kinetics. Using this approach, it is possible to read more than 20,000 nucleotides with an average read length of 5 kilobases.

[0091] C. Further Assay Methods In some aspects, the method involves amplifying and / or sequencing one or more target genomic regions using at least one pair of primers specific to the target genomic region.In certain aspects, the primer is a heptamer.In certain aspects, an enzyme such as primase or primase / polymerase combination enzyme is added to the amplification step to synthesize primer.

[0092] In some aspects, the nucleic acid of the present disclosure can be detected using an array. An array comprises a solid support on which a nucleic acid probe is attached. An array typically comprises a plurality of different nucleic acid probes attached to different known locations on the surface of a support layer. These arrays are also called "microarrays" or colloquially "chips" and are generally described in the art, for example, in U.S. Patent Nos. 5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186, and Fodor et al., 1991), each of which is incorporated by reference in its entirety for all purposes. Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261, which is incorporated by reference in its entirety for all purposes. Although in certain aspects a planar array surface is used, the array may be fabricated on a surface of virtually any shape, and even on multiple surfaces. The array may be nucleic acid on beads, gels, polymeric surfaces, fibers, such as optical fibers, glass, or any other support layer. See also U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193, and 5,800,992, which are incorporated herein in their entirety for all purposes.

[0093] In addition to the use of arrays and microarrays, it is contemplated that a number of different assays can be used to analyze nucleic acids. Such assays include nucleic acid amplification, polymerase chain reaction, quantitative PCR, RT-PCR, in situ hybridization, digital PCR, ddPCR (droplet digital PCR), nCounter® (nanoString®), BEAMing (beads, emulsion, amplification, and magnetics) (Inostics), ARMS (Amplification Refractory Mutation Systems), RNA-Seq, TAm-Seg (tagged amplicon deep sequencing), PAP (pyrophosphorolysis-activation polymerization), next-generation RNA sequencing, northern hybridization, hybridization protection assay (HPA) (GenProbe), branched DNA (bDNA) assay (Chiron), rolling circle amplification (RCA), single molecule hybridization detection (US Genomics), Invader assay (ThirdWave Technologies), and / or Bridge Litigation Assay (BLA). Examples of suitable antibodies include, but are not limited to, the IgG1 Assay (Genaco).

[0094] Amplification primers or hybridization probes can be prepared to be complementary to the genomic regions, biomarkers, probes, or oligos described herein. The term "primer" as used herein is intended to encompass any nucleic acid capable of priming nascent nucleic acid synthesis in a template-dependent process and / or pairing with a single strand or portion of an oligo of the present disclosure. Typically, primers are oligonucleotides from 10-20 and / or 30 nucleic acids in length, although longer sequences can be used. Primers may be provided in double-stranded and / or single-stranded form, although single-stranded form is preferred.

[0095] The use of primers of 13-100 nucleotides, particularly 17-100 nucleotides, or in some aspects up to 1-2 kilobases or more in length allows for the formation of stable and selective duplex molecules. Molecules having complementary sequences over a contiguous region of more than 20 bases in length may be used to increase the stability and / or selectivity of the resulting hybrid molecules. Nucleic acid molecules for hybridization may be designed with one or more complementary sequences of 20-30 nucleotides, or even longer if desired. Such fragments may be readily prepared, for example, by directly synthesizing the fragments by chemical means or by introducing selected sequences into a recombinant vector for recombinant production.

[0096] In some aspects, each probe / primer comprises at least 15 nucleotides. For example, each probe may comprise at least 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein), or up to 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400 or more nucleotides (or any range derivable therein). Probe / primer may have these lengths and may have identical or complementary sequences to genes described herein. In particular, each probe / primer has a relatively high sequence complexity and does not have any ambiguous residues (undetermined "n" residues). The probe / primer can hybridize to the target gene, including its RNA transcript, under stringent or highly stringent conditions. It is contemplated that the probe or primer may have inosine or other design implementations that accommodate the recognition of multiple human sequences for a particular biomarker.

[0097] For applications requiring high selectivity, it is typically desirable to use relatively high stringency conditions to form hybrids. For example, relatively low salt and / or high temperature conditions, such as those provided by about 0.02M to about 0.10M NaCl at a temperature of about 50°C to about 70°C. Such high stringency conditions are less tolerant of mismatches between the probe or primer and the template or target strand, and would be particularly suitable for isolating a specific gene or detecting a specific mRNA transcript. It is generally accepted that conditions can be made more stringent by adding increasing amounts of formamide.

[0098] In some aspects, quantitative RT-PCR (including but not limited to TaqMan™, ABI) is used to detect and compare the levels or abundance of nucleic acids in samples. The concentration of target DNA in the linear portion of the PCR process is proportional to the starting concentration of the target before PCR begins. By determining the concentration of the PCR product of the target DNA in PCR reactions that are completed in the same number of cycles and are in the linear range, the relative concentration of a particular target sequence in the initial DNA mixture can be determined. This direct proportional relationship between PCR product concentration and relative abundance in the starting material applies in the linear range portion of the PCR reaction. The final concentration of target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mixture and is independent of the initial concentration of target DNA. Thus, sampling and quantification of amplified PCR products can be performed when the PCR reaction is in the linear portion of its curve. Furthermore, the relative concentration of amplifiable DNA can be normalized to some independent standard / control. The standard / control can be based on the DNA species present internally or on the DNA species introduced externally. The abundance of a particular DNA species may also be determined relative to the average abundance of all DNA species in a sample.

[0099] In some aspects, PCR amplification utilizes one or more PCR internal standards.Internal standards can be abundant housekeeping genes in cells, specifically GAPDH, GUSB and β-2 microglobulin.These standards can be used to normalize expression levels so that the expression levels of different gene products can be directly compared.Those skilled in the art will know how to use internal standards to normalize expression levels.

[0100] An inherent problem with some samples is the variable quantity and / or quality of the samples. This problem can be overcome if RT-PCR is performed as relative quantitative RT-PCR using an internal standard. In this case, the internal standard is an amplifiable DNA fragment similar to or larger than the target DNA fragment, and the abundance of DNA corresponding to the internal standard is approximately 5-100 times that of DNA corresponding to the target nucleic acid region.

[0101] In some aspects, relative quantitative RT-PCR uses an external standard protocol, under which PCR products are sampled in the linear portion of their amplification curves. The optimal number of PCR cycles for sampling can be empirically determined for each target DNA fragment. Furthermore, nucleic acids isolated from different samples can be normalized to equal concentrations of amplifiable DNA.

[0102] A nucleic acid array may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more different polynucleotide probes that can hybridize to different biomarkers and / or the same biomarker. Multiple probes for the same gene can be used in one nucleic acid array. Probes for other disease genes can also be included in the nucleic acid array. The density of probes on the array can be in any range. In some aspects, the density can be 50, 100, 200, 300, 400, 500, or more probes / cm. 2(or any range derivable therein), and may be at least 50, 100, 200, 300, 400, 500, or more probes / cm 2 (or any range that can be derived therein).

[0103] Particularly contemplated are chip-based nucleic acid technologies, such as those described in Hacia et al. (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for rapid and accurate analysis of a large number of genes. By tagging genes with oligonucleotides or using a certain array of probes, chip technology can be used to separate target molecules as high-density arrays and screen these molecules based on hybridization (see also Pease et al., 1994; and Fodor et al, 1991). It is contemplated that this technology may be used in conjunction with the evaluation of the expression level of one or more cancer biomarkers for diagnostic, prognostic, and treatment methods.

[0104] Certain aspects may involve the use of arrays or data generated from arrays. The data may be readily available. Additionally, arrays may be prepared to generate data, which may then be used in correlation studies.

[0105] V. How to use A. Identification of DNA methylation variants Recently, the field of DNA methylation analysis has developed with the identification of several cytosine variants. Traditional DNA methylation involves the transfer of a methyl group to the carbon 5 position of cytosine to produce 5-methylcytosine (5mC). However, studies have demonstrated that the Tet family of cytosine oxygenase enzymes are involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).

[0106] 5-Formylcytosine (5fC) is one of the DNA variants generated when Tet enzymes act on 5-hydroxymethylcytosine. Further oxidation of 5-formylcytosine by Tet enzymes results in its conversion to 5-carboxylcytosine. Oxidation of 5-methylcytosine through various DNA methylation variants is a mechanism of DNA demethylation, and this demethylation pathway is thought to have a function during development and germ cell programming. 5-Formylcytosine is present in mouse embryonic stem (ES) cells and major mouse organs. This DNA modification also appears in the paternal pronucleus after fertilization, and this is accompanied by the disappearance of 5-methylcytosine. This suggests that 5-formylcytosine is involved in the DNA demethylation process.

[0107] 5-Carboxylcytosine (5caC) has been identified as one of the DNA methylation variants generated when Tet enzymes oxidize 5-hydroxymethylcytosine and subsequently 5-formylcytosine. The oxidation of 5-methylcytosine to 5-carboxylcytosine is a DNA demethylation mechanism, and this demethylation pathway is thought to have a function during development and germ cell programming. It has been suggested that 5caC is excised from genomic DNA by thymine DNA glycosylase (TDG), which returns the cytosine residue to its unmodified state. 5-Carboxylcytosine has been identified in mouse embryonic stem (ES) cells. This DNA modification appears in the paternal pronucleus after fertilization, which coincides with the disappearance of 5-methylcytosine, supporting the variant being part of the DNA demethylation pathway.

[0108] 5-Methylcytosine (5mC) is a DNA modification resulting from the transfer of a methyl group from S-adenosylmethionine (also known as AdoMet or SAM) to the carbon 5 position of cytosine residues. This transfer is catalyzed by DNA methyltransferase enzymes (DNMTs). 5-Methylcytosine is the most common and widely studied type of DNA methylation. 5-Methylcytosine usually occurs within CpG dinucleotide motifs, although non-CpG methylation has been identified in embryonic stem cells.

[0109] 5-Hydroxymethylcytosine (5hmC) is a DNA methylation modification that results from the enzymatic oxidation of 5-methylcytosine (5mC) by the Tet family of iron-dependent deoxygenases. 3 5-Hydroxymethylcytosine can be found in high amounts in certain mammalian tissues, such as mouse Purkinje cells and granule neurons. Alternatively, 5hmC can be generated by the addition of formaldehyde to DNA cytosine by DNMT proteins.

[0110] Other methods are provided for distinguishing epigenetic modifications. It is intended that this method can be applied and combined with other methods disclosed in the art. Examples of methods disclosed in the art include U.S. Patent No. 8,741,567, U.S. Patent No. 9,611,510, PCT Application Publication WO201416577, U.S. Patent No. 11,130,991, and U.S. Patent Application Publication No. 2021 / 0310062. Each of these is incorporated herein by reference in its entirety. In some aspects, this method may include or exclude the steps described in the patents and patent application publications referenced above.

[0111] B. Clinical and Diagnostic Uses The method of the present disclosure may be useful for evaluating DNA and / or RNA for clinical and / or diagnostic purposes.Certain aspects relate to the method for evaluating DNA.Certain aspects relate to the method for evaluating RNA.Certain aspects relate to the method for evaluating the sample that comprises DNA molecules and / or RNA molecules.This evaluation may be the detection or determination of specific cytosine modification, or the differential detection or determination of specific modification.

[0112] The sample may be derived from a biopsy, such as a fine needle aspiration, core needle biopsy, aspiration biopsy, incision biopsy, excision biopsy, punch biopsy, shave biopsy, or skin biopsy. In certain aspects, the sample is obtained from a biopsy from cancer tissue by any of the biopsy methods previously described. In certain aspects, the sample may be obtained from any tissue provided herein, including but not limited to gallbladder, skin, heart, lung, breast, pancreas, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source, including but not limited to blood, sweat, hair follicle, cheek tissue, tears, menstrual secretions, feces, or saliva. In certain aspects, the sample is obtained from cyst fluid or body fluid from a tumor or neoplasm. In certain aspects, the cyst, tumor, or neoplasm is a colorectal cyst, tumor, or neoplasm. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, can obtain the biological sample for testing. Still further, in certain aspects, the biological sample can be obtained without the assistance of a medical professional.

[0113] The sample may include, but is not limited to, tissue, cells, or biological material from or derived from a subject's cells. In some aspects, the sample includes cell-free DNA. In some aspects, the sample includes fertilized eggs, zygotes, blastocysts, or blastomeres. The biological sample may be a heterogeneous or homogeneous population of cells or tissues. The biological sample may be obtained using any method known to those skilled in the art that can provide a sample suitable for the analytical methods described herein. The sample may be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, buccal swabbing, saliva collection, urine collection, fecal collection, menstrual secretions, tears, or semen collection.

[0114] In some aspects, the methods of the disclosure can be used in the discovery of novel biomarkers for disease or illness. In some aspects, the methods of the disclosure can be performed on a sample from a patient to indicate a prognosis for a certain disease or condition in the patient. In some aspects, the methods of the disclosure can be performed on a sample from a patient to predict the patient's response to a certain therapy. In some aspects, the disease includes cancer. For example, cancer includes pancreatic cancer, colon cancer, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, pediatric cerebellar or cerebral basal cell carcinoma, cholangiocarcinoma, extrahepatic bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma brain tumor, cerebral astrocytoma / malignant glioma brain tumor, ependymoma brain tumor, medulloblastoma brain tumor, supratentorial primitive neuroectodermal ... brain tumors, optic tract and hypothalamic gliomas, breast cancer, lymphomas, bronchial adenoma / carcinoid, tracheal cancer, Burkitt's lymphoma, carcinoid tumors, childhood carcinoid tumors, gastrointestinal cancer of unknown primary, central nervous system lymphoma, primary cerebellar astrocytoma, childhood cerebral astrocytoma / malignant glioma, childhood cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's, childhood extragonadal germ cell tumor tumor), extrahepatic bile duct cancer, eye cancer, eye cancer that is intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumors: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, brain stem glioma, glioma, childhood cerebral astrocytoma, childhood optic tract and hypothalamic glioma, gastric carcinoid, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and optic tract glioma, childhood intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia, acute lymphoblastic (also called acute lymphocytic leukemia) leukemia,Acute myeloid (also called acute myeloid leukemia) leukemia, chronic lymphocytic (also called chronic lymphocytic) leukemia, chronic myelogenous (also called chronic myeloid leukemia) leukemia, hairy cell lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin (an older classification of all lymphomas except Hodgkin lymphoma) lymphoma Breast cancer, primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, childhood medulloblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, adult malignant mesothelioma, childhood mesothelioma, metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, chronic myeloid leukemia, adult acute myeloid leukemia, childhood acute myeloid leukemia, multiple Myeloma, chronic myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant, fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumors), ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, islet cell paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, childhood pituitary adenoma, plasma cell neoplasm Biology / Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Transitional cell carcinoma of the renal pelvis and ureter, Retinoblastoma, Rhabdomyosarcoma, Childhood salivary gland carcinosarcoma, Ewing family of tumors, Kaposi's sarcoma, Soft tissue sarcoma, Uterine Sézary syndrome sarcoma, Skin cancer (non-melanoma), Skin cancer (melanoma), Skin cancer, Merkel cell small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma, Squamous cell carcinoma of unknown primary site (squamous The cancer may be a tumor of the thymus, a tumor of the thymus gland ...

[0115] In some aspects, the cancer comprises ovarian cancer, prostate cancer, colon cancer or lung cancer. In some aspects, the method is for determining novel biomarkers of ovarian cancer, prostate cancer, colon cancer or lung cancer by evaluating cell-free DNA using the method of the present disclosure. In some embodiments, the method of the present disclosure may be used on fetal DNA isolated from pregnant females. In some aspects, the method of the present disclosure may be used for prenatal diagnosis using fetal DNA isolated from pregnant females. In some aspects, the method of the present disclosure may be used for evaluation of fertilized embryos such as zygotes or blastocysts to determine embryo quality or the presence or absence of certain disease markers.

[0116] In some aspects, the methods disclosed herein are performed on low input concentrations of DNA and / or RNA. In some aspects, the low input DNA and / or RNA concentration is about 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15 nanograms, or any derivative thereof In some embodiments, the range is about 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or 15 nanograms, or any range derivable therein. In some aspects, the low input DNA and / or RNA concentration is about 1-10 ng, 5-10 ng, 10-50 ng, or 10-100 ng of total DNA and / or RNA.In some aspects, a low input concentration of DNA and / or RNA is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or is obtained from 500 cells, or from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or less than 500 cells.

[0117] VI. Sample Preparation In certain aspects, the method involves obtaining a sample (also "biological sample") from a subject. The obtaining method provided herein may include a biopsy method, such as fine needle aspiration, core needle biopsy, aspiration biopsy, incision biopsy, excision biopsy, punch biopsy, shave biopsy, liquid biopsy, or skin biopsy. In certain aspects, the sample is obtained from a biopsy material derived from a tissue by any of the biopsy methods previously described. In certain aspects, the sample can be obtained from any of the tissues provided herein, including but not limited to non-cancerous or cancerous tissues, and non-cancerous or cancerous tissues from serum, gallbladder, mucosa, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample can be obtained from any other source, including but not limited to blood, sweat, hair follicle, cheek tissue, tears, menstrual secretions, feces, or saliva. In certain aspects of the method, the biological sample can be obtained for testing by any medical professional, such as a doctor, nurse, or medical technician. Still further, the biological sample can be obtained without the assistance of a medical professional.

[0118] Biological samples may include, but are not limited to, subject tissues, cells, or biological materials from or derived from cells. In some aspects, biological samples include extracellular vesicles such as exosomes. Biological samples may be heterogeneous or homogeneous populations of cells or tissues. Biological samples may be acellular samples. Biological samples may be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. Samples may be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, cheek swabbing, saliva collection, cerebrospinal fluid collection, urine collection, fecal collection, menstrual secretions, tears, or semen collection.

[0119] The sample can be obtained by methods known in the art. In certain aspects, the sample is obtained by biopsy. In certain aspects, the sample is obtained by swabbing, endoscopy, scraping, phlebotomy, or any other method known in the art. In some cases, the sample may be obtained, stored, or transported using the components of the kit of the method. In some cases, multiple samples may be obtained by the methods described herein for diagnosis. In other cases, multiple samples, such as one or more samples from one tissue and one or more samples from another specimen (e.g., serum), may be obtained by the methods for diagnosis. In some cases, multiple samples, such as one or more samples from one tissue type and one or more samples from another specimen (e.g., serum), may be obtained at the same time or at different times. The samples may be obtained at different times and stored and / or analyzed by different methods. For example, the samples may be obtained and analyzed by routine staining methods or any other cytological analysis methods.

[0120] In some aspects, the biological sample may be obtained by other medical professionals, such as a doctor, nurse, medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or pulmonologist. The medical professional may indicate the appropriate test or assay to perform on the sample. In certain aspects, the molecular profiling business may consult on which assay or test is most appropriately indicated. In further aspects of the method, the patient or subject may obtain the biological sample for testing without the assistance of a medical professional, such as obtaining a whole blood sample, a urine sample, a fecal sample, a buccal sample, or a saliva sample.

[0121] In other cases, samples are obtained by invasive procedures, including, but not limited to, biopsy, needle aspiration, endoscopy, or venisection. Methods of needle aspiration may further include fine needle aspiration, core needle biopsy, aspiration biopsy, or large core biopsy. In some aspects, multiple samples may be obtained by the methods herein to ensure a sufficient amount of biological material.

[0122] The general method for obtaining biological samples is also known in the art. Publications such as Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001, the entirety of which is incorporated herein by reference, describe the general method for biopsy and cytological methods. In some aspects, the sample is a fine needle aspirate of tissue or suspected tumor or neoplasm. In some cases, the fine needle aspirate sampling procedure may be guided by the use of ultrasound, X-ray, or other imaging devices.

[0123] In some aspects of the method, the molecular profiling business may obtain the biological sample directly from the subject, from a healthcare professional, from a third party, or from a kit provided by the molecular profiling business or a third party. In some cases, the biological sample may be obtained by the molecular profiling business after the subject, healthcare professional, or third party obtains the biological sample and ships it to the molecular profiling business. In some cases, the molecular profiling business may provide suitable containers and excipients for storing the biological sample and for transporting the biological sample to the molecular profiling business.

[0124] In some aspects of the methods described herein, a medical professional need not be involved in the initial diagnosis or sample acquisition. Instead, an individual may obtain a sample using an over the counter (OTC) kit. The OTC kit may include a means for obtaining the sample as described herein, a means for storing the sample for testing, and instructions for proper use of the kit. In some cases, the molecular profiling service is included in the price of kit purchase. In other cases, a separate invoice is sent for the molecular profiling service. A sample suitable for use by a molecular profiling business may be any material that contains tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, or gene expression product fragments of an individual to be tested. Methods are provided for determining the suitability and / or appropriateness of a sample.

[0125] In some aspects, the subject may be referred to a specialist, such as an oncologist, surgeon, or endocrinologist. The specialist may also obtain the biological sample for testing and may refer the individual to a testing center or laboratory to submit the biological sample. In some cases, a medical professional may refer the subject to a testing center or laboratory to submit the biological sample. In other cases, the subject may provide the sample. In some cases, a molecular profiling business may obtain the sample.

[0126] VII. Kit Also disclosed herein are kits that may be useful in practicing the methods of the present disclosure. The contents of the kit may include one or more of the reagents described throughout this disclosure and / or one or more of the reagents known in the art for practicing one or more of the steps described throughout this disclosure. For example, the kit may include the following: bisulfite, ammonium bisulfite, ammonium sulfite, ammonium sulfite monohydrate, sodium bisulfite, bisulfite solution containing ammonium bisulfite, bisulfite solution containing ammonium bisulfite and ammonium sulfite, 70% ammonium bisulfite solution, 50% ammonium bisulfite solution, 50%-70% ammonium bisulfite solution, APOBEC deaminase enzyme, APOBEC3A, nuclease-free water, one or more primers, polyethylene glycol, magnetic beads, DNA polymerase, taq polymerase, DNA ligase, RNA ligase, reverse transcriptase, dNTPs, DNA polymerase buffer, RNA polymerase, DTT, redox reagent, Mg 2+ , K + The kit may comprise one or more of the following: an adapter, a DNA adapter, a DNA containing an RNA promoter, a protease, an alkaline solution, a sodium hydroxide solution, and an NTP. Any one or more of the above components may be excluded from the kit in certain aspects of the present disclosure. In some aspects, the kit of the present disclosure does not include sodium bisulfite or added sodium bisulfite. In some aspects, the kit of the present disclosure does not include ammonium sulfite or added ammonium sulfite.

[0127] In certain aspects, the kits of the present disclosure include a solution comprising ammonium bisulfite, hi some aspects, the solution comprises 50% to 70% ammonium bisulfite by weight, including any range or value derivable therein. In some aspects, the kits of the present disclosure comprise at least 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55.8%, 55.9%, 56.1%, 56.2%, 56.3%, 56.4%, 56.5%, 56.6%, 56.7%, 56.8%, 56.9%, 57.1%, 57.2%, 57.3%, 57.4%, 57.5%, 57.6%, 57.7%, 57.8%, 57.9%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.5%, 58.6%, 58.7%, 58.8%, 58.9%, 59.8%, 59.9%, 59.9%, 59.9%, 59.9%, 59.9 %, 52.6 wt%, 52.7 wt%, 52.8 wt%, 52.9 wt%, 53 wt%, 53.1 wt%, 53.2 wt%, 53.3 wt%, 53.4 wt%, 53.5 wt%, 53.6 wt%, 53.7 wt%, 53.8 wt%, 53.9 wt% , 54% by weight, 54.1% by weight, 54.2% by weight, 54.3% by weight, 54.4% by weight, 54.5% by weight, 54.6% by weight, 54.7% by weight, 54.8% by weight, 54.9% by weight, 55% by weight, 55.1% by weight, 55.2% by weight, 55.3% by weight, 55 .4% by weight, 55.5% by weight, 55.6% by weight, 55.7% by weight, 55.8% by weight, 55.9% by weight, 56% by weight, 56.1% by weight, 56.2% by weight, 56.3% by weight, 56.4% by weight, 56.5% by weight, 56.6% by weight, 56.7% by weight, 56. 8% by weight, 56.9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight, 57.4% by weight, 57.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight %, 58.3 wt%, 58.4 wt%, 58.5 wt%, 58.6 wt%, 58.7 wt%, 58.8 wt%, 58.9 wt%, 59 wt%, 59.1 wt%, 59.2 wt%, 59.3 wt%, 59.4 wt%, 59.5 wt%, 59.6 wt% , 59.7% by weight, 59.8% by weight, 59.9% by weight, 60% by weight, 60.1% by weight, 60.2% by weight, 60.3% by weight, 60.4% by weight, 60.5% by weight, 60.6% by weight, 60.7% by weight, 60.8% by weight, 60.9% by weight, 61% by weight, 61.1% by weight, 61.2% by weight, 61.3% by weight, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62 .7% by weight, 62.8% by weight, 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64. 3% by weight, 64.4% by weight, 64.5% by weight, 64.6% by weight, 64.7% by weight, 64.8% by weight, 64.9% by weight, 65% by weight, 65.1% by weight, 65.2% by weight, 65.3% by weight, 65.4% by weight, 65.5% by weight, 65.6% by weight, 65.7% by weight, 65.8% by weight, 65 .9% by weight, 66% by weight, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67. 5%, 67.6%, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight of ammonium bisulfite, or any range or value derivable therein, up to 50%, 50. 1% by weight, 50.2% by weight, 50.3% by weight, 50.4% by weight, 50.5% by weight, 50.6% by weight, 50.7% by weight, 50.8% by weight, 50.9% by weight, 51% by weight, 51.1% by weight, 51.2% by weight, 51.3% by weight, 51.4% by weight, 51.5% by weight, 51.6% by weight, 51 .7% by weight, 51.8% by weight, 51.9% by weight, 52% by weight, 52.1% by weight, 52.2% by weight, 52.3% by weight, 52.4% by weight, 52.5% by weight, 52.6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3 wt%, 53.4 wt%, 53.5 wt%, 53.6 wt%, 53.7 wt%, 53.8 wt%, 53.9 wt%, 54 wt%, 54.1 wt%, 54.2 wt%, 54.3 wt%, 54.4 wt%, 54.5 wt%, 54.6 wt%, 54.7 wt%, 54.8 wt%, 54.9 wt%, 55 wt%, 55.1 wt%, 55.2 wt%, 55.3 wt%, 55.4 wt%, 55.5 wt%, 55.6 wt%, 55.7 wt%, 55.8 wt%, 55.9 wt%, 56 wt%, 56.1 wt%, 56.2 wt%, 56.3 wt%, 56.4 wt%, 56 .5 weight%, 56.6 weight%, 56.7 weight%, 56.8 weight%, 56.9 weight%, 57 weight%, 57.1 weight%, 57.2 weight%, 57.3 weight%, 57.4 weight%, 57.5 weight%, 57.6 weight%, 57.7 weight%, 57.8 weight%, 57.9 weight%, 58 weight%, 58.1 weight%, 58.2 weight%, 58.3 weight%, 58.4 weight%, 58.5 weight%, 58.6 weight%, 58.7 weight%, 58.8 weight%, 58.9 weight%, 59 weight%, 59.1 weight%, 59.2 weight%, 59.3 weight%, 59.4 weight%, 59.5 weight%, 59.6 weight%, 59 .7 weight%, 59.8 weight%, 59.9 weight%, 60 weight%, 60.1 weight%, 60.2 weight%, 60.3 weight%, 60.4 weight%, 60.5 weight%, 60.6 weight%, 60.7 weight%, 60.8 weight%, 60.9 weight%, 61 weight%, 61.1 weight%, 61.2 weight%, 61.3 weight%, 61.4 weight%, 61.5 weight%, 61.6 weight%, 61.7 weight%, 61.8 weight%, 61.9 weight%, 62 weight%, 62.1 weight%, 62.2 weight%, 62.3 weight%, 62.4 weight%, 62.5 weight%, 62.6 weight%, 62.7 weight%, 62.8 weight%, 6 2.9 wt%, 63 wt%, 63.1 wt%, 63.2 wt%, 63.3 wt%, 63.4 wt%, 63.5 wt%, 63.6 wt%, 63.7 wt%, 63.8 wt%, 63.9 wt%, 64 wt%, 64.1 wt%, 64.2 wt%, 64.3 wt%, 64.4 wt%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66 wt%, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight, 67.6% by weight, 6 7.7% by weight, 67.8% by weight, 67.9% by weight, 68% by weight, 68.1% by weight, 68.2% by weight, 68.3% by weight, 68.4% by weight, 68.5% by weight, 68.6% by weight, 68.7% by weight, 68.8% by weight, 68.9% by weight, 69% by weight, 69.1% by weight, 69.2% by weight, 6 9.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight of ammonium bisulfite, or any range or value derivable therein, or about 50%, 50.1%, 50.2%, 50.3%, 50.4%, 50.5%, 50.6%, 50.7%, 50.8%, 50.9%, 51%, 51.1%, 51.2%, 51.3%, 51.4%, 51.5%, 51.6%, 51.7%, 51.8%, 51.9%, 52.0%, 52.1%, 52.2%, 52.3%, 52.4%, 52.5%, 52.6%, 52.7%, 52.8%, 52.9%, 53.0%, 53.1%, 53.2%, 53.3%, 53.4%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9 ... Weight%, 51.9% by weight, 52% by weight, 52.1% by weight, 52.2% by weight, 52.3% by weight, 52.4% by weight, 52.5% by weight, 52.6% by weight, 52.7% by weight, 52.8% by weight, 52.9% by weight, 53% by weight, 53.1% by weight, 53.2% by weight, 53.3% by weight, 53.4 Weight%, 53.5%, 53.6%, 53.7%, 53.8%, 53.9%, 54%, 54.1%, 54.2%, 54.3%, 54.4%, 54.5%, 54.6%, 54.7%, 54.8%, 54.9%, 55 Weight%, 55.1%, 55.2%, 55.3%, 55.4%, 55.5%, 55.6%, 55.7%, 55.8%, 55.9%, 56%, 56.1%, 56.2%, 56.3%, 56.4%, 56.5%, 56 .6% by weight, 56.7% by weight, 56.8% by weight, 56.9% by weight, 57% by weight, 57.1% by weight, 57.2% by weight, 57.3% by weight, 57.4% by weight, 57.5% by weight, 57.6% by weight, 57.7% by weight, 57.8% by weight, 57.9% by weight, 58% by weight, 58.1% by weight, 58.2% by weight, 58.3% by weight, 58.4% by weight, 58.5% by weight, 58.6% by weight, 58.7% by weight, 58.8% by weight, 58.9% by weight, 59% by weight, 59.1% by weight, 59.2% by weight, 59.3% by weight, 59.4% by weight, 59.5% by weight, 59.6% by weight, 59.7% by weight %, 59.8 wt%, 59.9 wt%, 60 wt%, 60.1 wt%, 60.2 wt%, 60.3 wt%, 60.4 wt%, 60.5 wt%, 60.6 wt%, 60.7 wt%, 60.8 wt%, 60.9 wt%, 61 wt%, 61.1 wt%, 61.2 wt%, 61.3 Weight%, 61.4% by weight, 61.5% by weight, 61.6% by weight, 61.7% by weight, 61.8% by weight, 61.9% by weight, 62% by weight, 62.1% by weight, 62.2% by weight, 62.3% by weight, 62.4% by weight, 62.5% by weight, 62.6% by weight, 62.7% by weight, 62.8% by weight , 62.9% by weight, 63% by weight, 63.1% by weight, 63.2% by weight, 63.3% by weight, 63.4% by weight, 63.5% by weight, 63.6% by weight, 63.7% by weight, 63.8% by weight, 63.9% by weight, 64% by weight, 64.1% by weight, 64.2% by weight, 64.3% by weight, 64.4% by weight Amount%, 64.5 wt%, 64.6 wt%, 64.7 wt%, 64.8 wt%, 64.9 wt%, 65 wt%, 65.1 wt%, 65.2 wt%, 65.3 wt%, 65.4 wt%, 65.5 wt%, 65.6 wt%, 65.7 wt%, 65.8 wt%, 65.9 wt%, 66% by weight, 66.1% by weight, 66.2% by weight, 66.3% by weight, 66.4% by weight, 66.5% by weight, 66.6% by weight, 66.7% by weight, 66.8% by weight, 66.9% by weight, 67% by weight, 67.1% by weight, 67.2% by weight, 67.3% by weight, 67.4% by weight, 67.5% by weight %, 67.6%, 67.7%, 67.8%, 67.9%, 68%, 68.1%, 68.2%, 68.3%, 68.4%, 68.5%, 68.6%, 68.7%, 68.8%, 68.9%, 69%, 69.1%, 69.2%, 69.3%, 69.4%, 69.5%, 69.6%, 69.7%, 69.8%, 69.9%, or 70% by weight ammonium bisulfite, or any range or value derivable therein. In some aspects, the solution comprises at least 66%, 66.01% by weight, 66.02% by weight, 66.03% by weight, 66.04% by weight, 66.05% by weight, 66.06% by weight, 66.07% by weight, 66.08% by weight, 66.09% by weight, 66.1% by weight, 66.11% by weight, 66.12% by weight, 66.13% by weight, 66.14% by weight, 66 .15% by weight, 66.16% by weight, 66.17% by weight, 66.18% by weight, 66.19% by weight, 66.2% by weight, 66.21% by weight, 66.22% by weight, 66.23% by weight, 66.24% by weight, 66.25% by weight, 66.26% by weight, 66.27% by weight, 66.28% by weight, 66 .29% by weight, 66.3% by weight, 66.31% by weight, 66.32% by weight, 66.33% by weight, 66.34% by weight, 66.35% by weight, 66.36% by weight, 66.37% by weight, 66.38% by weight, 66.39% by weight, 66.4% by weight, 66.41% by weight, 66.42% by weight, 66. 43% by weight, 66.44% by weight, 66.45% by weight, 66.46% by weight, 66.47% by weight, 66.48% by weight, 66.49% by weight, 66.5% by weight, 66.51% by weight, 66.52% by weight, 66.53% by weight, 66.54% by weight, 66.55% by weight, 66.56% by weight, 66. 57% by weight, 66.58% by weight, 66.59% by weight, 66.6% by weight, 66.61% by weight, 66.62% by weight, 66.63% by weight, 66.64% by weight, 66.65% by weight, 66.66% by weight, 66.67% by weight, 66.68% by weight, 66.69% by weight, 66.7% by weight, 66.7 1% by weight, 66.72% by weight, 66.73% by weight, 66.74% by weight, 66.75% by weight, 66.76% by weight, 66.77% by weight, 66.78% by weight, 66.79% by weight, 66.8% by weight, 66.81% by weight, 66.82% by weight, 66.83% by weight, 66.84% by weight, 66.8 5%, 66.86%, 66.87%, 66.88%, 66.89%, 66.9%, 66.91%, 66.92%, 66.93%, 66.94%, 66.95%, 66.96%, 66.97%, 66.98%, 66.99%, or 67% by weight of ammonium bisulfite, or any range or value derivable therein, up to 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08 wt%, 66.09 wt%, 66.1 wt%, 66.11 wt%, 66.12 wt%, 66.13 wt%, 66.14 wt%, 66.15 wt%, 66.16 wt%, 66.17 wt%, 66.18 wt%, 66.19 wt%, 66.2 wt%, 66.21 wt%, 66.22 wt%, 66.23 wt%, 66.24 wt%, 66.25 wt%, 66.26 wt%, 66.27 wt%, 66.28 wt%, 66 .29 wt%, 66.3 wt%, 66.31 wt%, 66.32 wt%, 66.33 wt%, 66.34 wt%, 66.35 wt%, 66.36 wt%, 66.37 wt%, 66.38 wt%, 66.39 wt%, 66.4 wt%, 66.41 wt%, 66.42 wt%, 66.43 wt%, 66.44 wt%, 66.45 wt%, 66.46 wt%, 66.47 wt%, 66.48 wt%, 66.49 wt%, 6 6.5 wt%, 66.51 wt%, 66.52 wt%, 66.53 wt%, 66.54 wt%, 66.55 wt%, 66.56 wt%, 66.57 wt%, 66.58 wt%, 66.59 wt%, 66.6 wt%, 66.61 wt%, 66.62 wt%, 66.63 wt%, 66.64 wt%, 66.65 wt%, 66.66 wt%, 66.67 wt%, 66.68 wt%, 66.69 wt%, 66.7 wt%, 6 6.71 wt%, 66.72 wt%, 66.73 wt%, 66.74 wt%, 66.75 wt%, 66.76 wt%, 66.77 wt%, 66.78 wt%, 66.79 wt%, 66.8 wt%, 66.81 wt%, 66.82 wt%, 66.83 wt%, 66.84 wt%, 66.85 wt%, 66.86 wt%, 66.87 wt%, 66.88 wt%, 66.89 wt%, 66.9 wt%, 66.91 wt%,. 66.92%, 66.93%, 66.94%, 66.95%, 66.96%, 66.97%, 66.98%, 66.99%, or 67% ammonium bisulfite, or any range or value derivable therein, or about 66%, 66.01%, 66.02%, 66.03%, 66.04%, 66.05%, 66.06%, 66.07%, 66.08%, 66.09%, 66.1%, 66.11%, 66.12%, 66.13%, 66.14%, 66.15%, 66.16%, 66.17%, 66.18%, 66.19%, 66.20%, 66.21%, 66.22%, 66.23%, 66.24%, 66.25%, 66.26%, 66.27%, 66.28%, 66.29%, 66.30%, 66.31%, 66.32%, 66.33%, 66.34%, 66.35%, 66.36%, 66.37%, 66.38%, 66.39%, 66.40%, 66.41%, 66.42%, 66.43%, 66.44%, 66.45%, 66.46%, 66.47%, 66.48%, 66.49%, 66.50%, 66.51%, 66.52%, 66.53%, 66.54%, 66.55%, 66.56%, 66.57%, 66.58%, 66.59%, 66.60%, 66.61%, 4% by weight, 66.15% by weight, 66.16% by weight, 66.17% by weight, 66.18% by weight, 66.19% by weight, 66.2% by weight, 66.21% by weight, 66.22% by weight, 66.23% by weight, 66.24% by weight, 66.25% by weight, 66.26% by weight, 66.27% by weight, 66. 28% by weight, 66.29% by weight, 66.3% by weight, 66.31% by weight, 66.32% by weight, 66.33% by weight, 66.34% by weight, 66.35% by weight, 66.36% by weight, 66.37% by weight, 66.38% by weight, 66.39% by weight, 66.4% by weight, 66.41% by weight, 66.4 2% by weight, 66.43% by weight, 66.44% by weight, 66.45% by weight, 66.46% by weight, 66.47% by weight, 66.48% by weight, 66.49% by weight, 66.5% by weight, 66.51% by weight, 66.52% by weight, 66.53% by weight, 66.54% by weight, 66.55% by weight, 66. 56% by weight, 66.57% by weight, 66.58% by weight, 66.59% by weight, 66.6% by weight, 66.61% by weight, 66.62% by weight, 66.63% by weight, 66.64% by weight, 66.65% by weight, 66.66% by weight, 66.67% by weight, 66.68% by weight, 66.69% by weight, 66. 7% by weight, 66.71% by weight, 66.72% by weight, 66.73% by weight, 66.74% by weight, 66.75% by weight, 66.76% by weight, 66.77% by weight, 66.78% by weight, 66.79% by weight, 66.8% by weight, 66.81% by weight, 66.82% by weight, 66.83% by weight, 66. 84% by weight, 66.85% by weight, 66.86% by weight, 66.87% by weight, 66.88% by weight, 66.89% by weight, 66.9% by weight, 66.91% by weight, 66.92% by weight, 66.93% by weight, 66.94% by weight, 66.95% by weight, 66.96% by weight, 66.97% by weight, 66.98%, 66.99%, or 67% by weight ammonium bisulfite, or any range or value derivable therein. In some aspects, the solution comprises about 66.67% by weight ammonium bisulfite.

[0128] In some aspects, the solution includes ammonium sulfite. In some aspects, the solution includes at least 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 8.1%, 8.2%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 8.1%, 8.2%, 8.3 ... .3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, 10% by weight , 10.1 wt%, 10.2 wt%, 10.3 wt%, 10.4 wt%, 10.5 wt%, 10.6 wt%, 10.7 wt%, 10.8 wt%, 10.9 wt%, 11 wt%, 11.1 wt%, 11.2 wt%, 11.3 wt%, 11.4 wt%, 11.5 wt%, 11. 6% by weight, 11.7% by weight, 11.8% by weight, 11.9% by weight, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 12.7% by weight, 12.8% by weight, 12.9% by weight, 13% by weight, 13.1% by weight, 13.2% by weight, 13.3% by weight, 13.4% by weight, 13.5% by weight, 13.6% by weight, 13.7% by weight, 13.8% by weight, 13.9% by weight, 14% by weight, 14.1% by weight, 14.2% by weight, 14.3% by weight, 14.4% by weight, 14.5% by weight, 14.6% by weight, 14.7 %, 14.8%, 14.9%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, up to 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6% by weight, 7.7% by weight, 7.8% by weight, 7.9% by weight, 8% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight Amount %, 9.5 wt%, 9.6 wt%, 9.7 wt%, 9.8 wt%, 9.9 wt%, 10 wt%, 10.1 wt%, 10.2 wt%, 10.3 wt%, 10.4 wt%, 10.5 wt%, 10.6 wt%, 10.7 wt%, 10.8 wt%, 10.9 wt%, 11 wt%, 11. 1% by weight, 11.2% by weight, 11.3% by weight, 11.4% by weight, 11.5% by weight, 11.6% by weight, 11.7% by weight, 11.8% by weight, 11.9% by weight, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 1 2.7% by weight, 12.8% by weight, 12.9% by weight, 13% by weight, 13.1% by weight, 13.2% by weight, 13.3% by weight, 13.4% by weight, 13.5% by weight, 13.6% by weight, 13.7% by weight, 13.8% by weight, 13.9% by weight, 14% by weight, 14.1% by weight, 14.2% by weight, 14 0.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, or 15% by weight of ammonium sulfite, or any range or value derivable therein, or about 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1% by weight , 7.2% by weight, 7.3% by weight, 7.4% by weight, 7.5% by weight, 7.6% by weight, 7.7% by weight, 7.8% by weight, 7.9% by weight, 8% by weight, 8.1% by weight, 8.2% by weight, 8.3% by weight, 8.4% by weight, 8.5% by weight, 8.6% by weight, 8.7% by weight, 8.8% by weight, 8.9% by weight, 9% by weight, 9.1% by weight, 9.2% by weight, 9.3% by weight, 9.4% by weight, 9.5% by weight, 9.6% by weight, 9.7% by weight, 9.8% by weight, 9.9% by weight, 10% by weight, 10.1% by weight, 10.2% by weight, 10.3% by weight, 10.4% by weight, 10.5% by weight, 10.6% by weight, 10.7% by weight, 10.8% by weight, 10.9% by weight, 11% by weight, 11.1% by weight, 11.2% by weight, 11.3% by weight, 11.4% by weight, 11.5% by weight, 11.6% by weight, 11.7% by weight, 11.8% by weight, 11. 9% by weight, 12% by weight, 12.1% by weight, 12.2% by weight, 12.3% by weight, 12.4% by weight, 12.5% ​​by weight, 12.6% by weight, 12.7% by weight, 12.8% by weight, 12.9% by weight, 13% by weight, 13.1% by weight %, 13.2%, 13.3%, 13.4%, 13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, or 15% by weight of ammonium sulfite, or any range or value derivable therein. In some aspects, the solution comprises 0.1M, 0.01M, 1x10. -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or less, or at 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M or less. In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less ammonium bisulfite by weight. In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less ammonium bisulfite by weight. In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less ammonium bisulfite by weight. In certain aspects, the solution does not contain ammonium sulfite or added ammonium sulfite.

[0129] In some aspects, the solution does not contain ammonium sulfite or added ammonium sulfite. In some aspects, the solution is 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or lower, or at 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 Contains ammonium sulfite at a concentration of M or less.

[0130] In some aspects, the solution is a solution with a bisulfite concentration of 6.5M to 10M, including any range or value derivable therebetween. In some aspects, the solution is a solution with a bisulfite concentration of at least 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, 9.1M, 9.2M, 9.3M, 9.4 ... 4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, up to 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5 M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, or about 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, 7.6M, 7.7M, 7.8M, 7.9M, 8.9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein, or about 6.5M, 6.6M, 6.7M, 6.8M, 6.9M, 7M, 7.1M, 7.2M, 7.3M, 7.4M, 7.5M, The solution may be a solution having a bisulfite concentration of about 0.6M, 7.7M, 7.8M, 7.9M, 8M, 8.1M, 8.2M, 8.3M, 8.4M, 8.5M, 8.6M, 8.7M, 8.8M, 8.9M, 9M, 9.1M, 9.2M, 9.3M, 9.4M, 9.5M, 9.6M, 9.7M, 9.8M, 9.9M, or 10M, or any range or value derivable therein. In some aspects, the solution is a solution having a bisulfite concentration of about 7.0M. In some aspects, the solution is a solution having a bisulfite concentration of about 7.0M. In some aspects, the solution is a solution having a bisulfite concentration of about 9.5M, and in some aspects, the solution is a solution having a bisulfite concentration of about 9.5M. In some aspects, the solution has a pH of 4.8 to 5.4, including any range or value derivable therein.In some aspects, the pH of the solution is at least 4.8, 4.9, 5, 5.1, 5.2, 5.3, or 5.4, and up to or about 4.8, 4.9, 5, 5.1, 5.2, 5.3, or 5.4. In some aspects, the pH of the solution is about 5.1.

[0131] In some aspects, the solution does not contain sodium bisulfite or added sodium bisulfite. In some aspects, the solution is 1M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or less. In some aspects, the solution is free of sodium.

[0132] In some aspects, the solution does not contain sodium bisulfite or added sodium bisulfite. In some aspects, the solution is 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10-13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 M or lower, or at 1M, 0.9M, 0.8M, 0.7M, 0.6M, 0.5M, 0.4M, 0.3M, 0.2M, 0.1M, 0.01M, 1x10 -3 M, 1x10 -4 M, 1x10 -5 M, 1x10 -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M, 1x10 -13 M, 1x10 -14 M, 1x10 -15 M, 1x10 -16 M, 1x10 -17 M, 1x10 -18 M, 1x10 -19 M, 1x10 -20 In some aspects, the solution contains less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% or less sodium bisulfite by weight.

[0133] In certain aspects, the kit of the present disclosure comprises instructions for processing nucleic acid samples, such as DNA samples or RNA samples.The instructions may comprise instructions for using one or more components of the kit in the methods disclosed herein.For example, the instructions may comprise one or more of instructions for incubating nucleic acid samples with bisulfite solution, instructions for mixing nucleic acid samples with bisulfite solution, instructions for bisulfite treatment of nucleic acid, instructions for isolating nucleic acid from samples, instructions for nucleic acid amplification, and instructions for preparing samples for sequencing. The instructions for incubating a nucleic acid sample with a bisulfite solution may include instructions to incubate the sample with the solution for 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute, or less, or any range or value derivable therein, or up to 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute, or less, or any range or value derivable therein. The instructions for incubating the nucleic acid sample with the bisulfite solution include: 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 83°C, 83.1°C, 83.2°C, 83.3°C, 83.4°C, 83.5°C, 83.6°C, 83.7°C, 83.8°C, 83.9 ... ℃, 83.4℃, 83.5℃, 83.6℃, ​​83.7℃, 83.8℃, 83.9℃, 84℃, 84.1℃, 84.2℃, 84.3℃, 84.4℃, 84.5℃, 84.6℃, 84.7℃, 84.8℃, 84.9℃, 85℃, 85.1℃, 85.2℃, 85.3℃, 85.4℃, 85.5℃, 85.6℃, 85.7℃, 85.8℃, 85.9℃, 86℃, 86.1℃, 86.2℃, 86.3℃, 86.4℃, 86.5℃, 86.6℃, 86.7℃, 86.8℃, 86.9℃, 87℃, 87.1℃, 87.2℃, 87.3℃, 87.4℃, 87.5℃, 87.6℃, 87.7℃, 87.8℃, 87.9℃, 88℃, 88.1℃, 88.2℃, 88.3℃, 88.4℃, 88.5℃, 88.6℃, 88.7℃, 88.8℃, 88.9℃, 89℃, 89.1℃, 89.2℃, 89.3℃ ,89.4℃,89.5℃,89.6℃,89.7℃,89.8℃,89.9℃,90℃,90.1℃,90.2℃,90.3℃,90.4℃,90.5℃,90.6℃,90.7℃,90.8℃,90.9℃,91℃,91.1℃,91.2℃,91.3℃,91.4℃,91 .5℃, 91.6℃, 91.7℃, 91.8℃, 91.9℃, 92℃, 92.1℃, 92.2℃, 92.3℃, 92.4℃, 92.5℃, 92.6℃, 92.7℃, 92.8℃, 92.9℃, 93℃, 93.1℃, 93.2℃, 93.3℃, ​​93.4℃, 93.5℃, 93.6 ℃, 93.7℃, 93.8℃, 93.9℃, 94℃, 94.1℃, 94.2℃, 94.3℃, 94.4℃, 94.5℃, 94.6℃, 94.7℃, 94.8℃, 94.9℃, 95℃, 95.1℃, 95.2℃, 95.3℃, 95.4℃, 95.5℃, 95.6℃, 95.7℃, 9 5.8℃, 95.9℃, 96℃, 96.1℃, 96.2℃, 96.3℃, 96.4℃, 96.5℃, 96.6℃, 96.7℃, 96.8℃, 96.9℃, 97℃, 97.1℃, 97.2℃, 97.3℃, 97.4℃, 97.5℃, 97.6℃, 97.7℃, 97.8℃, 97. 9°C, 98°C, 98.1°C, 98.2°C, 98.3°C, 98.4°C, 98.5°C, 98.6°C, 98.7°C, 98.8°C, 98.9°C, 99°C, 99.1°C, 99.2°C, 99.3°C, 99.4°C, 99.5°C, 99.6°C, 99.7°C, 99.8°C, 99.9°C or any of the above. At a temperature of at least 80°C, 80.1°C, 80.2°C, 80.3°C, 80.4°C, 80.5°C, 80.6°C, 80.7°C, 80.8°C, 80.9°C, 81°C, 81.1°C, 81.2°C, 81.3°C, 81.4°C, 81.5°C, 81.6°C, 81.7°C, 81.8°C, 81.9°C, 82°C, 82.1°C, 82.2°C, 82.3°C, 82.4°C, 82.5°C, 82.6°C, 82.7°C, 82.8°C, 82.9°C, 83°C, 83.1°C, 83.2°C, 83.3°C, 83.4°C, 83.5°C, 83.6°C, 83.7°C, 83.8°C, 83.9 ...1°C, 83.2°C, 83.3°C, 83.4°C, 83.5°C,6℃、83.7℃、83.8℃、83.9℃、84℃、84.1℃、84.2℃、84.3℃、84.4℃、84.5℃、84.6℃、84.7℃、84.8℃、84.9℃、85℃、85.1℃、85.2℃、85.3℃、85.4℃、85.5℃、85.6℃、85.7℃、85.8℃、85.9℃、86℃、86.1℃、86.2℃、86.3℃、86.4℃、86.5℃、86.6℃、86.7℃、86.8℃、86.9℃、87℃、87.1℃、87.2℃、87.3℃、87.4℃、87.5℃、87.6℃、87.7℃、87.8℃、87.9℃、88℃、88.1℃、88.2℃、88.3℃、88.4℃、88.5℃、88.6℃、88.7℃、88.8℃、88.9℃、89℃、89.1℃、89.2℃、89.3℃、89.4℃、89.5℃、89.6℃、89.7℃、89.8℃、89.9℃、90℃、90.1℃、90.2℃、90.3℃、90.4℃、90.5℃、90.6℃、90.7℃、90.8℃、90.9℃、91℃、91.1℃、91.2℃、91.3℃、91.4℃、91.5℃、91.6℃、91.7℃、91.8℃、91.9℃、92℃、92.1℃、92.2℃、92.3℃、92.4℃、92.5℃、92.6℃、92.7℃、92.8℃、92.9℃、93℃、93.1℃、93.2℃、93.3℃、93.4℃、93.5℃、93.6℃、93.7℃、93.8℃、93.9℃、94℃、94.1℃、94.2℃、94.3℃、94.4℃、94.5℃、94.6℃、94.7℃、94.8℃、94.9℃、95℃、95.1℃、95.2℃、95.3℃、95.4℃、95.5℃、95.6℃、95.7℃、95.8℃、95.9℃、96℃、96.1℃、96.2℃、96.3℃、96.4℃、96.5℃、96.6℃、96.7℃、96.8℃、96.9℃、97℃、97.1℃、97.2℃、97.3℃、97.4℃、97.5℃、97.6℃、97.7℃、97.8℃、97.9℃、98℃、98.1℃、98.2℃、98.3℃、98.4℃、98.5℃、98.6℃、98.7℃、98.8℃、98.9℃、99℃、99.1℃、99.2℃、99.3℃、99.4℃、99.5℃、99.6℃、99.7℃、99.8℃、99.The instructions may include instructions to incubate the sample and the solution at a temperature of about 98° C. or above, or any range or value derivable therein. In some aspects, the instructions include instructions to incubate the sample at about 98° C. In some aspects, the instructions include instructions to incubate the sample at 98° C.

[0134] The one or more reagents are preferably provided in a solid form or in a liquid buffer suitable for storage in inventory and for subsequent addition to the reaction medium when the method using the reagent is carried out. Suitable packaging is provided. The kit may provide additional components useful in the procedure. These additional components may include buffers, capture reagents, color reagents, labels, reaction surfaces, means for detection, control samples, instructions, and explanatory information.

[0135] Any component of the kits described herein can be used in the methods disclosed herein. Additionally, components described in the context of the disclosed methods may be provided in the kits of the present disclosure.

[0136] VIII. Aspect The following non-limiting aspects are included to demonstrate certain features of the invention disclosed herein.

[0137] Aspect 1. 1. A method for DNA processing, comprising the steps of: (a) incubating a solution comprising DNA molecules and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, the solution not containing added sodium bisulfite; and (b) subjecting the DNA molecule to alkaline conditions.

[0138] Aspect 2. 2. The method of aspect 1, wherein the solution does not contain added ammonium sulfite.

[0139] Aspect 3. Aspect 3. The method of aspect 1 or 2, wherein the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0140] Aspect 4. Aspect 4. The method of any of aspects 1-3, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0141] Aspect 5. Aspect 5. The method of any one of Aspects 1 to 4, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0142] Aspect 6. Aspect 6. The method of any one of Aspects 1 to 5, wherein the solution has a bisulfite concentration of 8 M to 10 M.

[0143] Aspect 7. Aspect 7. The method of any one of Aspects 1 to 6, wherein the solution has a bisulfite concentration of 9 M to 10 M.

[0144] Aspect 8. Aspect 8. The method of any of aspects 1 to 7, wherein the solution is a solution with a bisulfite concentration of about 9.5 M.

[0145] Aspect 9. Aspect 9. The method of any of aspects 1 to 8, wherein the solution comprises 50% to 70% by weight ammonium bisulfite.

[0146] Aspect 10. Aspect 10. The method of any one of aspects 1 to 9, wherein the solution comprises 60% to 70% by weight ammonium bisulfite.

[0147] Aspect 11. Aspect 11. The method of any one of aspects 1 to 10, wherein the solution comprises 65% to 68% by weight ammonium bisulfite.

[0148] Aspect 12. Aspect 12. The method of any of aspects 1-11, wherein the solution comprises about 66.7% by weight ammonium bisulfite.

[0149] Aspect 13. Aspect 13. The method of any of aspects 1 to 12, wherein the solution has a pH of 4.8 to 5.4.

[0150] Aspect 14. Aspect 14. The method of any of aspects 1-13, wherein the solution has a pH of about 5.1.

[0151] Aspect 15. Aspect 15. The method of any of aspects 1-14, wherein (a) comprises incubating the solution at a temperature of about 98° C.

[0152] Aspect 16. 16. The method of any of aspects 1-15, wherein (a) comprises incubating the solution for up to 10 minutes.

[0153] Aspect 17. 17. The method of any of aspects 1-16, wherein (a) comprises incubating the solution for up to 8 minutes.

[0154] Aspect 18. Aspect 18. The method of any of aspects 1-17, wherein the DNA molecule comprises 4mC and after incubation, more than 50% of the 4mC is deaminated.

[0155] Aspect 19. Aspect 19. The method of any of aspects 1-18, wherein after incubation, more than 75% of 4mC is deaminated.

[0156] Aspect 20. Aspect 20. The method of any of aspects 1-19, wherein after incubation, substantially all of the 4mC is deaminated.

[0157] Aspect 21. 1. A method for DNA processing, comprising the steps of: (a) creating a solution comprising DNA molecules and ammonium bisulfite, the solution not containing added sodium bisulfite; (b) incubating the solution at a temperature of at least 95° C.; and (c) after up to 12 minutes of (a), removing the DNA molecules from the solution.

[0158] Aspect 22. 22. The method of aspect 21, wherein the solution does not contain added ammonium sulfite.

[0159] Aspect 23. Aspect 23. The method of aspect 21 or 22, wherein the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0160] Aspect 24. Aspect 24. The method of any of aspects 21-23, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0161] Aspect 25. Aspect 25. The method of any one of Aspects 21 to 24, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0162] Aspect 26. Aspect 26. The method of any one of Aspects 21 to 25, wherein the solution has a bisulfite concentration of 8 M to 10 M.

[0163] Aspect 27. Aspect 27. The method of any one of Aspects 21 to 26, wherein the solution has a bisulfite concentration of 9 M to 10 M.

[0164] Aspect 28. 28. The method of any of Aspects 21 to 27, wherein the solution is a solution with a bisulfite concentration of about 9.5 M.

[0165] Aspect 29. Aspect 29. The method of any of aspects 21 to 28, wherein the solution comprises 50% to 70% by weight ammonium bisulfite.

[0166] Aspect 30. Aspect 30. The method of any one of aspects 21 to 29, wherein the solution comprises 60% to 70% by weight ammonium bisulfite.

[0167] Aspect 31. Aspect 31. The method of any of aspects 21 to 30, wherein the solution comprises 65% to 68% ammonium bisulfite by weight.

[0168] Aspect 32. Aspect 32. The method of any of aspects 21-31, wherein the solution comprises about 66.7% by weight ammonium bisulfite.

[0169] Aspect 33. Aspect 33. The method of any of aspects 21 to 32, wherein the solution has a pH of 4.8 to 5.4.

[0170] Aspect 34. Aspect 34. The method of any of aspects 21-33, wherein the solution has a pH of about 5.1.

[0171] Aspect 35. Aspect 35. The method of any of Aspects 21-34, wherein (b) comprises incubating the solution at a temperature of about 98° C.

[0172] Aspect 36. Aspect 36. The method of any of Aspects 21-35, wherein (c) comprises removing the DNA molecules from the solution up to 10 minutes after (a).

[0173] Aspect 37. Aspect 37. The method of any of Aspects 21-36, wherein (c) comprises removing the DNA molecules from the solution up to 8 minutes after (a).

[0174] Aspect 38. Aspect 38. The method of any of aspects 21-37, wherein (a) comprises mixing a 70% ammonium bisulfite solution with a 50% bisulfite solution.

[0175] Aspect 39. Aspect 39. The method of any of aspects 21 to 38, wherein the DNA molecule comprises 4mC and after incubation, more than 50% of the 4mC is deaminated.

[0176] Aspect 40. Aspect 40. The method of any of aspects 21 to 39, wherein after incubation, more than 75% of 4mC is deaminated.

[0177] Aspect 41. Aspect 41. The method of any of Aspects 21 to 40, wherein after incubation, substantially all of the 4mC is deaminated.

[0178] Aspect 42. 1. A method for processing a nucleic acid sample, comprising the steps of: Incubating a solution comprising DNA molecules and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes, wherein the solution does not contain added sodium bisulfite, each of the DNA molecules comprises one or more cytosine residues, and after incubating the solution, more than 99% of the DNA molecules do not comprise a cytosine residue.

[0179] Aspect 43. Aspect 43. The method of aspect 42, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0180] Aspect 44. The method of aspect 42 or 43, further comprising subjecting the plurality of DNA molecules to alkaline conditions.

[0181] Aspect 45. Aspect 45. The method of any one of aspects 42 to 44, wherein the solution comprises 50% to 70% ammonium bisulfite by weight.

[0182] Aspect 46. Aspect 46. The method of any one of aspects 42 to 45, wherein the solution comprises 60% to 70% by weight ammonium bisulfite.

[0183] Aspect 47. Aspect 47. The method of any one of aspects 42 to 46, wherein the solution comprises 65% to 68% ammonium bisulfite by weight.

[0184] Aspect 48. Aspect 48. The method of any of aspects 42-47, wherein the solution comprises about 66.7% ammonium bisulfite by weight.

[0185] Aspect 49. Aspect 49. The method of any of aspects 42-48, wherein the solution does not contain added ammonium sulfite.

[0186] Aspect 50. Aspect 50. The method of any of aspects 42-49, wherein the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0187] Aspect 51. Aspect 51. The method of any one of Aspects 42 to 50, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0188] Aspect 52. Aspect 52. The method of any one of Aspects 42 to 51, wherein the solution has a bisulfite concentration of 8 M to 10 M.

[0189] Aspect 53. Aspect 53. The method of any one of Aspects 42 to 52, wherein the solution has a bisulfite concentration of 9 M to 10 M.

[0190] Aspect 54. Aspect 54. The method of any of Aspects 42 to 53, wherein the solution is a solution with a bisulfite concentration of about 9.5 M.

[0191] Aspect 55. Aspect 55. The method of any of aspects 42 to 54, wherein the solution has a pH of 4.8 to 5.4.

[0192] Aspect 56. 56. The method of any of aspects 42-55, wherein the DNA molecule comprises 4mC and after incubation, more than 50% of the 4mC is deaminated.

[0193] Aspect 57. 57. The method of any of aspects 42-56, wherein after incubation, more than 75% of 4mC is deaminated.

[0194] Aspect 58. 58. The method of any of Aspects 42 to 57, wherein substantially all of the 4mC is deaminated after incubation.

[0195] Aspect 59. below: (a) a solution containing ammonium bisulfite having a bisulfite concentration of 6.5 M to 10 M, the solution not containing sodium bisulfite; (b) instructions for processing the DNA sample; A DNA processing kit comprising:

[0196] Aspect 60. 60. The kit of aspect 59, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0197] Aspect 61. 61. The kit of aspect 59 or 60, wherein the solution has a bisulfite concentration of 8 M to 10 M.

[0198] Aspect 62. 62. The kit of any one of Aspects 59 to 61, wherein the solution has a bisulfite concentration of 9 M to 10 M.

[0199] Aspect 63. 63. The kit of any of Aspects 59 to 62, wherein the solution has a bisulfite concentration of about 9.5 M.

[0200] Aspect 64. Aspect 64. The kit of any one of Aspects 59 to 63, wherein the solution contains 50% to 70% ammonium bisulfite by weight.

[0201] Aspect 65. 65. The kit of any one of Aspects 59 to 64, wherein the solution contains 60% to 70% ammonium bisulfite by weight.

[0202] Aspect 66. 66. The kit of any one of aspects 59 to 65, wherein the solution contains 65% to 68% ammonium bisulfite by weight.

[0203] Aspect 67. 67. The kit of any of aspects 59 to 66, wherein the solution comprises about 66.7% ammonium bisulfite by weight.

[0204] Aspect 68. 68. The kit of any of aspects 59 to 67, wherein the solution has a pH of 4.8 to 5.4.

[0205] Aspect 69. 69. The kit of any of aspects 59 to 68, wherein the solution has a pH of about 5.1.

[0206] Aspect 70. 70. The kit of any of aspects 59-69, wherein the instructions include instructions for incubating the DNA sample with the solution at a temperature of at least 95° C. for a maximum of 12 minutes.

[0207] Aspect 71. 71. The kit of any of aspects 59-70, wherein the instructions include instructions for incubating the DNA sample with the solution at a temperature of about 98° C.

[0208] Aspect 72. 72. The kit of any of aspects 59-71, wherein the instructions include instructions for incubating the DNA sample with the solution for up to 10 minutes.

[0209] Aspect 73. 73. The kit of any of aspects 59-72, wherein the instructions include instructions for incubating the DNA sample with the solution for up to 8 minutes.

[0210] Aspect 74. 74. The kit of any of Aspects 59 to 73, wherein the solution does not contain ammonium sulfite.

[0211] Aspect 75. 75. The kit of any of aspects 59-74, wherein the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0212] Aspect 76. 76. The kit of any of aspects 59 to 75, further comprising an alkaline solution.

[0213] Aspect 77. 77. The kit of any of aspects 59 to 76, further comprising one or more buffer solutions.

[0214] Aspect 78. 1. A method for RNA processing, comprising the steps of: (a) incubating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite at a temperature of at least 95° C. for up to 12 minutes, the solution not containing added sodium bisulfite; and (a) subjecting the RNA molecule to alkaline conditions.

[0215] Aspect 79. Aspect 79. The method of aspect 78, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite.

[0216] Aspect 80. Aspect 80. The method of aspect 78 or 79, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0217] Aspect 81. Aspect 71. The method of any one of Aspects 78 to 80, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0218] Aspect 82. Aspect 72. The method of any one of Aspects 78 to 81, wherein the solution has a bisulfite concentration of 6.5 M to 7.5 M.

[0219] Aspect 83. Aspect 83. The method of any of Aspects 78-82, wherein the solution is a solution with a bisulfite concentration of about 7.0 M.

[0220] Aspect 84. Aspect 84. The method of any of aspects 78 to 83, wherein the solution has a pH of 4.8 to 5.4.

[0221] Aspect 85. Aspect 85. The method of any one of aspects 78 to 84, wherein the solution comprises 5% to 15% by weight ammonium sulfite.

[0222] Aspect 86. Aspect 86. The method of any one of aspects 78 to 85, wherein the solution comprises 8% to 12% ammonium sulfite by weight.

[0223] Aspect 87. Aspect 87. The method of any of aspects 78-86, wherein the solution comprises about 10% by weight ammonium sulfite.

[0224] Aspect 88. Aspect 88. The method of any of Aspects 78-87, wherein (a) comprises incubating the solution at a temperature of about 98° C.

[0225] Aspect 89. 89. The method of any of aspects 78-88, wherein (a) comprises incubating the solution for up to 10 minutes.

[0226] Aspect 90. 90. The method of any of aspects 78-89, wherein (a) comprises incubating the solution for up to 8 minutes.

[0227] Aspect 91. 1. A method for RNA processing, comprising the steps of: (a) creating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite, the solution not containing added sodium bisulfite; (b) incubating the solution at a temperature of at least 95° C.; and (c) after up to 12 minutes of (a), removing the RNA molecules from the solution.

[0228] Aspect 92. Aspect 92. The method of aspect 91, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite.

[0229] Aspect 93. Aspect 93. The method of aspect 91 or 92, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0230] Aspect 94. Aspect 94. The method of any of aspects 91 to 93, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0231] Aspect 95. Aspect 95. The method of any one of Aspects 91 to 94, wherein the solution has a bisulfite concentration of 6.5 M to 7.5 M.

[0232] Aspect 96. Aspect 96. The method of any of aspects 91-95, wherein the solution has a bisulfite concentration of about 7.0 M.

[0233] Aspect 97. Aspect 97. The method of any of aspects 91-96, wherein the solution has a pH of 4.8 to 5.4.

[0234] Aspect 98. Aspect 98. The method of any of aspects 91-97, wherein the solution has a pH of about 5.1.

[0235] Aspect 99. Aspect 99. The method of any one of aspects 91-98, wherein the solution comprises 5% to 15% by weight ammonium sulfite.

[0236] Aspect 100. Aspect 99. The method of any one of aspects 91 to 99, wherein the solution contains 8% to 12% ammonium sulfite by weight.

[0237] Aspect 101. Aspect 91. The method of any of aspects 91-100, wherein the solution comprises about 10% by weight ammonium sulfite.

[0238] Aspect 102. Aspect 91. The method of any of Aspects 91-101, wherein (b) comprises incubating the solution at a temperature of about 98° C.

[0239] Aspect 103. Aspect 103. The method of any of Aspects 91-102, wherein (c) comprises removing the RNA molecules from the solution up to 10 minutes after (a).

[0240] Aspect 104. Aspect 96. The method of any of Aspects 91-103, wherein (c) comprises removing the RNA molecules from the solution up to 8 minutes after (a).

[0241] Aspect 105. 1. A method for processing a nucleic acid sample, comprising the steps of: Incubating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite at a temperature of at least 95°C for up to 12 minutes, wherein the solution does not contain added sodium bisulfite, each of the RNA molecules comprises one or more cytosine residues, and wherein after incubating the solution, more than 99% of the RNA molecules do not comprise a cytosine residue.

[0242] Aspect 106. Aspect 106. The method of aspect 105, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite.

[0243] Aspect 107. Aspect 107. The method of aspect 105 or 106, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0244] Aspect 108. Aspect 108. The method of any of aspects 105-107, wherein the solution has a pH of 4.8 to 5.4.

[0245] Aspect 109. Aspect 109. The method of any of aspects 105-108, wherein the solution has a pH of about 5.1.

[0246] Aspect 110. Aspect 109. The method of any one of aspects 105 to 109, wherein the solution contains 5% to 15% by weight ammonium sulfite.

[0247] Aspect 111. Aspect 111. The method of any one of aspects 105-110, wherein the solution contains 8% to 12% ammonium sulfite by weight.

[0248] Aspect 112. Aspect 112. The method of any of aspects 105-111, wherein the solution comprises about 10% by weight ammonium sulfite.

[0249] Aspect 113. Aspect 113. The method of any of Aspects 105-112, wherein (a) comprises incubating the solution at a temperature of about 98° C.

[0250] Aspect 114. 114. The method of any of aspects 105-113, wherein (a) comprises incubating the solution for up to 10 minutes.

[0251] Aspect 115. 115. The method of any of aspects 105-114, wherein (a) comprises incubating the solution for up to 8 minutes.

[0252] Aspect 116. Aspect 116. The method of any of aspects 105 to 115, wherein the solution has a bisulfite concentration of 6.5 M to 10 M.

[0253] Aspect 117. Aspect 117. The method of any of aspects 105 to 116, wherein the solution has a bisulfite concentration of 6.5 M to 7.5 M.

[0254] Aspect 118. Aspect 118. The method of any of aspects 105-117, wherein the solution has a bisulfite concentration of about 7.0 M.

[0255] Aspect 119. 119. The method of any of aspects 105 to 118, further comprising subjecting the plurality of RNA molecules to alkaline conditions.

[0256] Aspect 120. below: (a) a solution containing ammonium sulfite and ammonium bisulfite at a bisulfite concentration of 6.5M to 8M, the solution not containing added sodium bisulfite; (b) instructions for processing the RNA samples; An RNA processing kit comprising:

[0257] Aspect 121. 121. The kit of aspect 120, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite.

[0258] Aspect 122. 122. The kit of aspect 120 or 121, wherein the solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0259] Aspect 123. 123. The kit of any of Aspects 120 to 122, wherein the solution has a bisulfite concentration of about 7.0 M.

[0260] Aspect 124. 124. The kit of any of aspects 120 to 123, wherein the solution has a pH of 4.8 to 5.4.

[0261] Aspect 125. 125. The kit of any of aspects 120 to 124, wherein the solution has a pH of about 5.1.

[0262] Aspect 126. 126. The kit of any one of Aspects 120 to 125, wherein the solution contains 5% to 15% by weight ammonium sulfite.

[0263] Aspect 127. 127. The kit of any of Aspects 120 to 126, wherein the solution contains 8% to 12% ammonium sulfite by weight.

[0264] Aspect 128. 128. The kit of any of Aspects 120 to 127, wherein the solution comprises about 10% by weight ammonium sulfite.

[0265] Aspect 129. 129. The kit of any of aspects 120-128, wherein the instructions include instructions for incubating the RNA sample with the solution at a temperature of at least 95° C. for up to 12 minutes.

[0266] Aspect 130. 130. The kit of any of aspects 120-129, wherein the instructions include instructions for incubating the RNA sample with the solution at a temperature of about 98° C.

[0267] Aspect 131. 131. The kit of any of aspects 120-130, wherein the instructions include instructions for incubating the RNA sample with the solution for up to 10 minutes.

[0268] Aspect 132. 1. A method for 5-hydroxymethylcytosine analysis, comprising the steps of: (a) incubating a first solution comprising a first DNA molecule and ammonium bisulfite at a temperature of at least 95° C. for a maximum of 12 minutes; (b) incubating a second solution comprising the second DNA molecule and ammonium bisulfite at a temperature of at least 95° C. for a maximum of 12 minutes; (c) subjecting the first DNA molecule to alkaline conditions; (d) subjecting said second DNA molecule to alkaline conditions; (e) treating the second DNA molecule with an APOBEC deaminase enzyme; and (f) sequencing said first DNA molecule and said second DNA molecule.

[0269] Aspect 133. Aspect 133. The method of aspect 132, wherein the first solution does not contain added sodium bisulfite.

[0270] Aspect 134. Aspect 134. The method of aspect 132 or 133, wherein the first solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0271] Aspect 135. Aspect 135. The method of any of aspects 132-134, wherein the second solution does not contain added sodium bisulfite.

[0272] Aspect 136. Aspect 136. The method of any of aspects 132-135, wherein the second solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite.

[0273] Aspect 137. Aspect 137. The method of any of Aspects 132-136, wherein the first solution and the second solution are the same solution.

[0274] Aspect 138. Aspect 138. The method of any of Aspects 132-137, wherein the first solution and the second solution are different solutions.

[0275] Aspect 139. 139. The method of any of Aspects 132-138, wherein (a) and (b) are performed simultaneously.

[0276] Aspect 140. 140. The method of any of Aspects 132-139, wherein (c) and (d) are performed simultaneously.

[0277] Aspect 141. 141. The method of any of aspects 132-140, wherein the first DNA molecule and the second DNA molecule have the same nucleotide sequence.

[0278] Aspect 142. 142. The method of any of aspects 132-141, wherein the APOBEC deaminase enzyme is APOBEC3A. EXAMPLES

[0279] The following examples are included to demonstrate certain aspects of the invention. It should be understood by those of skill in the art that the techniques disclosed in the following examples are techniques discovered by the inventors to work well in the practice of the invention, and therefore can be considered to constitute certain aspects for practicing the invention. However, in light of this disclosure, it should be understood by those of skill in the art that many changes can be made to the specific aspects disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention.

[0280] Example 1 - m in RNA 5 Bisulfite sequencing for detection and analysis of C RNA m 5 C modification and its regulators affect various cellular functions and are important in bladder cancer 1 , hepatocellular carcinoma (HCC) 2 , glioblastoma multiforme (GBM) 3 , and leukemia4 It has been shown that RNA m 5 This suggests a regulatory role for C modifications. 5 For C mapping, m 5 C-RIP-seq 5 , 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP) 6 , and miCLIP 7 Various methods have been reported, including those described above, all of which involve an antibody enrichment step. BS sequencing remains the gold standard for 5mC sequencing, and in recent years, RNA m 5 It is increasingly being applied to the study of C. 1,8-10 Several commercially available RNA BS conversion kits are available, including Zymo Research's EZ RNA Methylation™ Kit and Epigentek's Methylamp™ RNA BS Conversion Kit. RNA BS sequencing is 5 In addition to providing a transcriptome-wide view of C attachment at single-nucleotide resolution, it is inexpensive and easy to handle. On the other hand, BS-seq can also identify mRNAs in abundant RNAs such as tRNA and rRNA. 5 Although it was effective in detecting C. 11,12 For low abundance RNAs such as mRNA, large discrepancies have been observed across studies, with m 5 Studies that detected C sites 13 Other studies found only very little methylated mRNA. 14 More recent studies have demonstrated that improved bisulfite sequencing methods and more rigorous computational approaches have revealed hundreds of mSeq sequences in the human and mouse transcriptomes. 5 It has been reported that only the C site was present. 1,15 These controversial findings suggest that the mRNA 5 Identify the C site 15 These findings have raised the need to develop more robust methods for

[0281] In a comprehensive attempt to develop quantitative methods for sequencing various RNA modifications, we focused on (i) reducing RNA degradation by improving the efficiency so that the reaction can be completed in a very short time, and (ii) using high reaction temperatures to denature the RNA and completely convert C to U. In the bisulfite (BS) sequencing mechanism (Figure 1), the reaction of cytosine with BS is fast and reversible, whereas deamination, which converts the C-BS adduct to a U-BS adduct, is rate-limiting. 16 Bisulfite is involved in both steps. 16,17 Therefore, the use of high BS concentrations can dramatically accelerate the BS conversion rate. 17 This strategy was used to 5 Its application to C sequencing has not been reported.

[0282] C-BS and U-BS adducts are the major species that generate abasic sites leading to subsequent RNA cleavage and degradation. 19 They reasoned that by converting C to U more quickly, the time that both C-BS and U-BS are present in the reaction is shorter, thus reducing RNA damage. They further reasoned that a higher temperature would not only accelerate the deamination reaction, but more importantly, aid in secondary structure denaturation in the RNA, allowing for complete bisulfite conversion in a shorter time. They hypothesized that while a higher BS concentration and higher reaction temperature could cause more RNA damage, a significantly shorter reaction time could result in less RNA damage and therefore ultimately less RNA degradation. They hypothesized that a higher concentration of BS and higher temperature could be the key to m 5 It was also important that the undesired deamination of C was not caused.

[0283] Due to the limited aqueous solubility of sodium bisulfite, current bisulfite treatments are performed at bisulfite concentrations of 3-5 M. In DNA 5mC BS-sequencing, Shiraishi et al. proposed that ammonium bisulfite has high aqueous solubility, and reported that the proposed high-concentration bisulfite reagent is more efficient than the low-concentration bisulfite reagent prepared from the sodium salt. 18,20 Shiraishi et al. proposed using ammonium bisulfite mixed with sodium bisulfite to obtain a 10 M bisulfite reagent (2.08 g NaHSO3, 0.67 g ammonium sulfite monohydrate in 5.0 mL of 50% ammonium bisulfite) for DNA 5mC sequencing. 18,20 In attempts to replicate these conditions, it was found that mixtures prepared according to this recipe needed to be heated to dissolve the solids, and that the bisulfite readily precipitated when the solution was cooled to room temperature. Furthermore, the solution was found to be very sticky and therefore difficult to work with and not a consistent recipe.

[0284] Next, we created a bisulfite recipe consisting of ammonium bisulfite and ammonium sulfite alone. We screened a series of BS conditions, including BS salt, concentration, pH, temperature, and reaction time.

[0285] A clear solution, a mixture containing 50% ammonium bisulfite (1 mL) and ammonium sulfite (100 mg) (referred to as "R-1G"; bisulfite concentration of about 7.0 M, pH about 5.1), was mixed (9 μL) with 5-mer RNA oligo AGCGA (SEQ ID NO:1) (100 ng) in water (1 μL) and incubated at 98 °C. The reaction was monitored using MALDI TOF mass spectrometry (MS). Since neither A nor G reacts with BS, the mass change before and after BS treatment should only reflect the reaction of cytosine residues with BS. As shown in Figure 2A, most of the starting material was consumed within 1 min, resulting in an intermediate with MS +83. This suggests that cytosine was directly converted to the corresponding U-BS adduct. Cytosine was almost completely consumed within 2 min and completely converted to the U-BS adduct within 3 min.

[0286] Interestingly, no C-BS adducts (MS+82 compared to the control) were observed at any time point in the reaction. This suggests that the use of the new BS recipe dramatically accelerated the C-BS deamination to form the U-BS adduct such that it was no longer the rate-limiting step. This observation likely explains why the new BS conditions can dramatically accelerate the entire reaction from C to U-BS. Subsequent treatment of the U-BS adduct with base resulted in quantitative conversion of U-BS to U (MS+1 compared to the control, Figure 2A). In addition to the significantly greater C to U conversion rate observed under the new BS conditions, secondary structures in the RNA fragments should also be fully denatured at 98 °C (e.g., if the incubation time is appropriately long). The combination of these changes is what allows the conventional RNA m 5 We hypothesized that this would dramatically reduce the false positives that are frequently encountered in C bisulfite sequencing.

[0287] New BS conditions are undesirable 5 To determine whether the corresponding m 5 C RNA oligonucleotide (SEQ ID NO:2) was treated under the same conditions for various times. Even after 30 minutes of treatment, 5No reaction between C and BS was observed, suggesting that the new BS reaction would not produce false negatives within at least 30 min (Figure 2B). To test whether the new BS conditions degrade RNA into too small fragments, HeLa cell total RNA was treated with the R-1G BS recipe at 95°C or 98°C for various times, and then PAGE gels were run to assess RNA fragment size. The majority of RNA fragments showed a size distribution of 150-300 bp within 10 min of treatment (Figure 3). Fragmented RNA with this size range could be used directly to assemble libraries by random priming methods, or could be further fragmented to smaller sizes (50-100 bp) to assemble libraries using ligation-based methods.

[0288] Using the new BS method, m 5 To verify that libraries with good read lengths without C deamination, efficient C to U conversion, and low false positives caused by secondary structures can be assembled, the method was applied to total RNA isolated from a range of different biological samples, including A549 cells. Individual biological sample total RNA was treated with recipe R1-G at a range of different temperatures and times. Oligonucleotide libraries were constructed using the NEB small RNA kit and then subjected to next generation sequencing. Human 28S RNA contains two confirmed mRNAs. 5 C sites, whereas other cytosine sites remain unmethylated. These studies have demonstrated that these two 5 Whether the C site can be detected using the new BS method 5 The C site is free of any undesirable m 5 In addition, human 28S rRNA contains abundant secondary and tertiary structures, and therefore conventional BS sequencing usually produces many false positives (e.g., incomplete conversion of unmethylated cytosine to uracil). As shown in Figure 4, as the reaction time increases, the number of known m 5Undesirable m at C site 5 The background also decreased with increasing time, while C deamination increased. The incubation conditions characteristic of one particular improvement described herein were identified as incubation at 98°C for 9 minutes. Under this incubation, two known m 5 The average non-conversion rate of C sites was over 95%, whereas the non-conversion rate of all C sites was less than 5% (Fig. 5). 5 We constructed libraries using the new BS method in parallel with the EZ RNA Methylation Kit® (Zymo Research), the most widely used kit for the detection of C, and compared the false positive rates. Indeed, as shown in Figure 6A, the libraries prepared using the Zymo kit showed high levels of both known m 5 C sites were detected (green dots with vertical lines indicate these locations). However, a large number of false positives (red dots) also appeared. We downloaded the literature dataset obtained using the "optimized" bisulfite conditions (75°C, 4 hours). 10 As shown in Figure 6, false positives were indeed reduced compared to the standard Zymo kit protocol (false positive percent (FP%) reduced from 17.60% to 0.67%), but some false positives remained. 5 The modification rate of the C site was also significantly and observably reduced (from 97.52% efficiency to 87.64%) (Figure 6B). 15,21 We downloaded two additional data sets from. In these papers, the researchers used Zymo BS reagent but performed three cycles of BS treatment at a higher temperature. The false positives were further reduced, but a significant number of false positives were still detected (Figures 6C and 6D). In addition, two known m 5 Significant undesired deamination at the C site was also observed, indicating that all of the BS conditions in these publications were suboptimal.

[0289] A statistical comparison of the BS treatment disclosed herein with standard BS treatment (e.g., Zymo-BS) and additional literature conditions is summarized in Figure 7. The false positive rate was lowest using the BS treatment conditions disclosed herein (Figure 7E; "optimal conditions"), whereas m was detected at two known sites using the BS treatment conditions disclosed herein. 5 The C signal was the highest (Figure 7F; "optimal condition"). Furthermore, we compared the read distribution patterns between the BS treatment conditions disclosed herein and other methods. Various lengths of incubation time and temperature were screened using the R-1G recipe. It was found that in the majority of conditions, no false positive sites were detected when using a 5% non-conversion ratio cutoff (Figure 7A). Using longer reaction times and higher temperatures reduced the undesirable m 5 Based on these observations, it was confirmed that the C deamination rate increased with the R-1G recipe (Figure 7B). 5 C ratio of two known m 5 C sites were detected, whereas the false positive rate was zero when using a 5% non-converting ratio cutoff (Figure 7C). From these studies, the new BS conditions were 5 It was suggested that the method solves the major problem in BS sequencing of C. Furthermore, as shown in Figures 7D and 8, the blue (Figure 7D) and black (Figure 8) curves represent the read distribution of our method, while the red (Figure 7D) and black (Figure 8) curves represent some other literature methods. These cytosine-rich regions (e.g., 28S rRNA gene) showed sequencing coverage in all published data. The low-depth regions contained many cytosines and therefore caused many fragmentations. This was consistent with all methods. This indicates that reactive cytosines in RNA cause RNA fragmentation, which is consistent with the proposed RNA fragmentation mechanism during BS treatment. However, the variation in read depth using the disclosed method was much smaller compared to the literature methods. This indicates that the new BS conditions produced very little RNA fragmentation, and therefore, m 5This suggests that there was very little bias in the estimation of the C fraction.

[0290] To further validate the disclosed method, we sequenced small RNA fractions derived from the wild-type A549 cell line and its NSUN2 KO line. 5 C is present at positions 48, 49, or 50 in some tRNA species, which are known to be substrates for NSUN2 methyltransferase (Fig. 9A). 5 We expected that the C region should be sensitive to NSUN2 knockout. In contrast, the m 5 The C site is a substrate for DNMT2 (Figure 9A), so that site should be less sensitive to NSUN2 knockout. Indeed, as shown in Figures 9B-9D, no mC-related mutations were detected at sites 48, 49, or 50. 5 The C proportion was significantly reduced, whereas the m 5 The C ratio did not change upon NSUN2 knockout, further supporting the validity of the disclosed method. Further analysis of the small RNA library showed that m 5 The majority of C sites were found to have high modification rates (Figure 10A). CCC All C and m in 5 The non-conversion rates at the C site are shown in Figure 10B. All C sites showed very low backgrounds, whereas the two m sites at 49 and 50 showed very low backgrounds. 5 The C site showed a very high modification rate (>90%), whereas site 48 showed a very low modification rate (<25%). 5 Accurate and quantitative detection of C-sites can facilitate the study of associated biological functions.

[0291] The BS-seq protocol disclosed herein was then applied to HeLa mRNA. 5The majority of C sites were found to be located in protein-coding RNA (Figure 11A), and of these, half of the sites were located in coding sequence (CDS) regions (Figure 11B). Using the protocol described herein, we found even more m 5 We were able to identify the C site (Figure 12). Huang, et al., 15 and Zhang et al. 21 m specified in 5 The majority of C were also found herein (e.g., 376 / 565 and 222 / 343, respectively), suggesting that the BS processing method described herein was more accurate and sensitive than previously described methods. 5 The C sites had various modification rates ranging from 10 to 100%, with many sites being highly modified (Figure 13). 5 Most of the genes containing C modifications 5 There was only one C site, whereas there were 2–5 m 5 C sites were detected in 159 genes (Figure 14A). Further gene ontology (GO) analysis revealed that m 5 We found that genes modified by C are involved in various gene functions, including glycoprotein metabolism, cytoskeleton organization, and cellular localization (Figure 14B). 5 It is suggested that the C modification may have important biological functions.

[0292] In addition to HeLa mRNA, we also sequenced polyA+ RNA extracted from HEK293T cells. 5 The overall modification level of C sites was consistent between the HeLa and HEK293T cell lines. However, there were some differentially modified sites. 5 C sites showed more G-rich motifs, whereas m 5The C site showed more CUCCA motifs (Figure 15B). NSUN2 and NSUN6 5 It has been reported that NSUN2 is a methyltransferase that places mC on mRNA. Therefore, we applied the currently disclosed BS-seq protocol to NSUN2 or NSUN6 knockdown HeLa cell mRNA extracts and the corresponding shRNA controls (Figure 16). Sequencing results showed that the NSUN2 knockdown cell mRNA extracts had a reduction of more than 90% of the modified sites, mainly in G-rich motifs. This suggests that NSUN2 is a methyltransferase that places mC on mRNA in HeLa cells. 5 These results suggest that NSUN6 may play a major role in C modification. Furthermore, we also found that m 5 A portion of the C-sites was also detected, mainly in the CUCCA motif. These results also suggest that the differences in modification profiles between cell lines may be related to differences in methyltransferase expression levels.

[0293] Interestingly, both HeLa and HEK293T cells expressed m 5 In conjunction with the ribosome profiling data, we found that the mC site at the 5' end of the transcript showed a similar enrichment pattern (Figure 17). 5 We found evidence that C modifications can modulate translation efficiency. Compared to unmethylated genes, 5 Genes containing C sites were enriched for ribosome density signals in the 5′-UTR of their transcripts ( p -value = 1.05 × 10 -6 In contrast, 5 Genes containing C sites did not show significant enrichment of ribosome density signals (p=0.37, FIG. 18A). Furthermore, both 5'- and 3'-end methylated genes did not show ribosome density enrichment within the CDS regions (FIG. 18B).

[0294] Experimental procedure (1) Identification of BS conditions using RNA model oligos by Maldi-TOF MS 50% ammonium bisulfite and ammonium sulfite were mixed in various ratios to prepare various BS reagents. Then, 9 μL of BS reagent was mixed with model RNA (AGCGA, 100 ng) (SEQ ID NO:1) dissolved in water (1 μL). The mixtures were incubated at various temperatures from 70 to 98 °C for various times, and the reactions were monitored by Maldi-TOF MALDI.

[0295] [Table 1]

[0296] The most efficient conditions were found to be the use of BS reagent R-1G (a solution of 1 mL of 50% ammonium bisulfite and 100 mg of ammonium sulfite) and incubation of the reaction mixture at 98°C. This resulted in quantitative conversion of C to the U-BS adduct within 3 min. Further alkaline treatment converted U-BS to U and C to U completely. Using a similar model RNA (AGm 5 Under the same conditions using 1,2-dichlorophenyl ... 5 It was found that no C-BS adducts were formed, suggesting that the new BS conditions were highly selective and did not produce false negatives.

[0297] (2) Analysis of BS conditions by sequencing 28S rRNA derived from HeLa cells using R-1G Incubation temperatures from 70 to 90 °C were tested, along with incubation times of 20 to 40 min, with or without the addition of urea. The average cytosine conversions were all above 98%, with both m 5 All C sites showed rates above 90%. There was no clear benefit from the addition of urea.

[0298] [Table 2]

[0299] Additional conditions were tested with R-1G, and treatment at 98° C. was found to result in the greatest efficiency of cytosine conversion.

[0300] [Table 3]

[0301] After identifying 98°C as the temperature that yielded the highest cytosine conversion efficiency, various reaction times were tested. The highest cytosine conversion efficiency (99.7%) was achieved at 9 min, with high m 5 It was found that a high C percentage (94.5%) was detected (see Table 4 below).

[0302] [Table 4]

[0303] (3) Study of treatment time and temperature using BS Reagent R-1G and A549 total RNA by next-generation sequencing A mixture of 9 μL of BS Reagent R-1G and 1 μL of A549 total RNA (200 ng) was incubated at 70-98°C for various times, and then 140 μL of water was added. In-column desulphonation was performed by following the instructions of standard BS treatment (e.g., Zymo EZ RNA Methylation™ Kit instructions). The RNA was further treated with 0.1 M NaHCO3 at 95°C for 3 min to fragment to sizes of 50-80 nt. After OCC purification and 3'-repair and 5'-phosphorylation with T4 PNK, the RNA fragments were further purified by OCC and eluted with 7 μL of water. 6 μL was used to assemble libraries using the NEB small RNA libraries kit, and the libraries were sequenced by Nova-seq. After data analysis, incubation at 98°C for 9 min was found to be the condition that eliminated all false positives and identified two known mRNAs in 28S rRNA. 5A parallel library was also constructed starting from the same amount of A549 total RNA (200 ng) using standard RNA methylation procedures (e.g., Zymo Research's EZ RNA Methylation™ Kit). Sequencing results showed that the two known m 5 C part is high m 5 Although the C percentage was shown, it was found that many false positive sites, suggesting many unconverted cytosine sites, also appeared.

[0304] Example 2 - m in RNA from low input samples 5 Detection and analysis of C For patient samples such as blood or embryo samples, the amount of sample available is usually limited (e.g., 10-100 ng of total RNA). With this small amount of total RNA, it is not practical to enrich mRNA using poly-T beads or to deplete rRNA using ribo-minus to obtain enough RNA sample to assemble a good quality library using ligation-based methods. Therefore, a short DNA probe is added to the RNA obtained from a low-input sample (e.g., blood sample, single-cell RNA) to anneal with the rRNA, and then RNase H is added to digest the rRNA into small fragments. After purifying other undigested RNA using paramagnetic beads, the RNA is subjected to BS treatment using R-1G bisulfite reagent at 98 °C for 9 min, followed by random priming to synthesize cDNA, and then the library is assembled using a ssDNA library construction kit. m in the non-rRNA of the low-input total RNA sample 5 The C site is detected.

[0305] Example 3 - Bisulfite sequencing for detection and analysis of 5mC in DNA First, bisulfite conversion on DNA was tested using the R-1G BS recipe applied to the DNA oligonucleotide AGCGA (SEQ ID NO:3). The reaction was observed to be slower than that observed for the RNA oligonucleotide, requiring 5 min at 98° C. to complete (FIG. 19A). Further recipes were screened and a new recipe was identified, named A7 (1 mL 70% ammonium bisulfite + 100 μL 50% ammonium bisulfite; bisulfite concentration of about 9.5 M, pH about 5.1). This recipe quantitatively converted dC to dU within 3 min (FIG. 19B). No obvious 5mC deamination was observed within 10 min of treatment (FIG. 20).

[0306] When an 82-mer synthetic DNA oligo (SEQ ID NO:8) containing both C and 5mC was treated with BS recipe A7 at 98°C for 4-10 min, Sanger sequencing results showed that 5mC was read as C in all cases, whereas C was quantitatively read as T after 8-12 min (Figure 21), confirming that this recipe is not only highly efficient in inducing C to U conversion in a very short time, but also highly selective, avoiding undesired 5mC deamination. SEQ ID NO:8(DO-16-20) TIFF2025502057000006.tif12158

[0307] For DNA BS sequencing, it may also be important to distinguish 5mC from 4mC, which is known to exist in bacteria and has recently been detected in eukaryotic genomic DNA. 27 Previously, we reported that standard BS treatment (e.g., Zymo BS conditions) could only deaminate 4mC with approximately 50% efficiency. 28, and therefore discovered that 4mC sites may be a source of false-positive 5mC detection sites when using BS sequencing. We reasoned that the reaction conditions disclosed herein, including high temperature and BS recipe concentration, may facilitate deamination of 4mC. To test this, a short DNA oligo containing 4mC modification (TA4mCTT; SEQ ID NO:9) was treated with the BS conditions disclosed herein in parallel with standard BS treatment (e.g., Zymo BS conditions). Maldi TOF MS data showed that when standard BS treatment was used, 4mC was partially deaminated to generate the corresponding dU-containing oligo with approximately 50% efficiency, whereas when the new BS conditions disclosed herein were used, 4mC was quantitatively converted to dU (Figure 22A). To test the deamination efficiency of C, 5mC, and 4mC using the BS conditions disclosed herein compared to the standard BS treatment previously disclosed (e.g., Zymo kit BS treatment), a synthetic 100 bp DNA oligo (SEQ ID NO: 12) containing both C and 5mC and 4mC modifications was synthesized and treated with the BS conditions disclosed herein or standard BS treatment (e.g., Zymo BS conditions). After treatment, Sanger sequencing was performed to evaluate the deamination efficiency of C, 5mC, and 4mC. The results showed that after incubation with the BS reagent of the present disclosure at 98°C for 10 minutes, the 4mC and C sites were all quantitatively read as T, while the 5mC site still read as C (Figure 22B). In contrast, when standard BS treatment (e.g., Zymo kit BS treatment) was utilized, both of the two 4mC sites were read as C and T in a 1:1 ratio. These results suggest that, in stark contrast to previously disclosed standard BS procedures, the presently disclosed BS conditions can avoid, and in certain cases completely avoid, false positives caused by the presence of 4mC in the genome. SEQ ID NO:12 TIFF2025502057000007.tif11158

[0308] DNA degradation is a known problem in BS sequencing. It can not only cause DNA material loss, which is a significant problem in low-input DNA samples, but also cause biased DNA breaks that may overestimate the detected 5mC fraction. 27 Based on the suggested mechanism of DNA degradation in conventional BS treatment, 19 In BS treatment, the C-BS adducts formed are the major species that cause deglycosylation to form AP sites, which lead to further DNA backbone cleavage via β-elimination. 5mC does not react with BS, whereas C sites are much more susceptible to cleavage than 5mC. Thus, BS treatment causes greater DNA damage in DNA sequences where C is abundant, and therefore DNA fragments containing abundant C are under-represented in the library, which leads to an overestimation of 5mC levels.

[0309] Some recipes disclosed herein, including A7, were found to be highly efficient in converting C-BS to U-BS adducts, so that using these recipes and the disclosed conditions, the deamination step was no longer the rate-limiting step, and therefore the C-BS adducts were present in the reaction for only a very short time and at very low concentrations. Therefore, it was expected that using the disclosed recipes (including A7) and treatment conditions would result in significantly less DNA damage compared to other BS conditions. Although the very high BS concentrations and very high temperatures used to accelerate the BS reaction in the disclosed methods could hypothetically accelerate DNA degradation, it was hypothesized that the very short reaction time would be superior to the accelerated DNA degradation caused by high temperatures and high BS concentrations. To test this hypothesis, fish gDNA and synthetic 164-mer dsDNA (mimicking the size of cfDNA; (SEQ ID NO: 13, and antisense SEQ ID NO: 14) were treated with BS recipe A7 for various periods of time and compared in parallel with standard BS treatment (e.g., Zymo EZ DNA Methylation-Gold® Kit). As shown in Figures 23A-23B, in all cases, within 4-10 min, less DNA damage was observed with recipe A7 (1 mL of 70% ammonium bisulfite + 100 μL of 50% ammonium bisulfite) compared to standard BS treatment (e.g., Zymo kit, 98 °C for 10 min followed by 64 °C for 2.5 h). This suggests that recipe A7 may be applicable to low-input DNA and may overcome the problem of overestimation of 5mC percentage. SEQ ID NO:13 TIFF2025502057000008.tif19158SEQ ID NO:14 TIFF2025502057000009.tif19158

[0310] With the principles established above, we moved on to evaluate the new method using biological DNA containing synthetic spike-ins. Given that standard BS treatment options such as Zymo EZ DNA Methylation-Gold® Kit are the gold standard for BS sequencing, libraries were assembled in parallel for direct comparison. Arabidopsis thaliana gDNA has a small genome (approximately 135 megabases) that was previously reported to have 5mC sites, and was therefore selected as an exemplary gDNA for these studies. In order to compare the conversion efficiency of all C sites, spike-in λ DNA containing no 5mC sites was added to assess background. To assess the rate of undesired 5mC demethylation, a spike-in synthetic 164mer dsDNA (SEQ ID NO:13, and antisense SEQ ID NO:14) containing four 5mC sites was also added. After BS treatment, library construction (single-stranded DNA library construction) using the Swift Accel-NGS Methyl-Seq DNA Library Kit, and NGS sequencing, the sequencing data showed that the background was lowest after the incubation time reached 10 min, and the average C → U conversion rate of recipe A7 after 10 min reaction was greater than or equal to about 99.2% for all C sites in λDNA (the average non-conversion rate was 0.82% as shown in Figure 24C. Additional assays yielded a conversion rate of 99.6% and a non-conversion rate of 0.4% as shown in Figure 24A). In comparison, for standard BS treatment (e.g., Zymo kit), the average conversion rate was 98.2% as shown in Figure 24C, and the average non-conversion rate was 1.81%. Additional assays yielded an average conversion rate of 97.8% and a non-conversion rate of 2.2% as shown in Figure 24A (Figures 24C and 24A, respectively). This shows that high conversion efficiency was obtained from the disclosed BS recipe and conditions ("New BS") (FIGS. 24A to 24E).Importantly, using standard BS treatments (e.g., Zymo kit), the non-conversion rates at each C site showed considerable variability, necessitating a large cutoff (10%) to avoid false positives, whereas with the new BS recipe and conditions, the non-conversion rates at each site were more uniform than with the standard BS treatment, with almost all sites showing non-conversion rates less than 2% (Figures 24B and 24E).

[0311] (1) Identification of BS conditions using DNA model oligos by Maldi-TOF MS To prepare various BS reagents, 70% ammonium bisulfite and 50% ammonium bisulfite were mixed in various ratios. Then, 9 μL of BS reagent was taken and mixed with model DNA (AGCGA, 100 ng) dissolved in water (1 μL). The mixtures were incubated at various temperatures at 98° C. for various times, and the reactions were monitored by Maldi-TOF MALDI. See Table 5 and Table 6 below.

[0312] [Table 5]

[0313] [Table 6]

[0314] The best results were obtained using BS reagent A7 (a mixture of 1 mL 70% ammonium bisulfite and 100 μL 50% ammonium bisulfite), where C was found to be quantitatively converted to U-BS adduct within 3 min upon incubation of the reaction mixture at 98 °C. Further conversion of U-BS to U by alkaline treatment resulted in complete conversion of C to U. Under the same conditions using a similar model DNA (AG5mCGA; SEQ ID NO:4) as substrate, Maldi-TOF-MS showed that almost no T-BS adduct was formed within 20 min, indicating that the new BS conditions are highly selective.

[0315] (2) Process plant gDNA containing λDNA and a synthetic 164 nt DNA oligo containing two 5mC sites by next-generation sequencing using BS Reagent A7. A mixture of 9 μL of BS reagent A7, 1 μL of plant gDNA (50 ng) containing 0.5 ng of λDNA without 5mC modification, and 0.1 ng of synthetic 164-mer dsDNA containing four 5mC sites (SEQ ID NO: 13, and antisense SEQ ID NO: 14) was incubated at 98°C for various times, and then 140 μL of water was added. In-column desulfonation was performed by following standard BS treatment (Zymo EZ DNA Methylation-Gold® Kit) and eluted with 7 μL of water. 6 μL was used to assemble libraries using Swift Accel-NGS Methyl-Seq DNA Library Kit, and the libraries were sequenced by Nova-seq. After data analysis, it was confirmed that incubation at 98°C for 10 minutes was the best condition, and under this condition the average conversion efficiency of all cytosine sites in λDNA was 99.18% (see also Table 7 below). For the two known 5mC sites in the 164bp spike-in DNA, the detection rate was greater than 96%. A side-by-side library was also assembled starting from the same amount of DNA using standard BS treatment (e.g., Zymo EZ DNA Methylation-Gold® Kit). Sequencing results showed that the average conversion efficiency of all cytosine sites in lambda DNA using standard BS treatment was 98.2%. For the two known 5mC sites in the 164nt spike-in DNA, the detection rate was 98% using standard BS treatment.

[0316] [Table 7]

[0317] Example 4 - Detection and analysis of 5mC in DNA from low input samples Using the A7 recipe, libraries were assembled starting from 0.1, 1.0, or 10 ng of mouse embryonic stem cell (mES) genomic DNA (gDNA) or 0.1, 1.0, or 10 ng of human cell-free DNA (cfDNA) using the Swift kit in combination with the new BS treatment conditions. Sequencing results were analyzed to identify methylation sites in DNA.

[0318] We applied the BS protocol disclosed herein (e.g., recipe A7 and incubation at 98°C for 10 minutes) to mouse embryonic stem cell (mESC) gDNA. Because the recipes and conditions disclosed herein caused less DNA damage than traditional BS conditions, we reasoned that these protocols could be used to assay with low-input gDNA. To evaluate conversion efficiency, we generated gDNA sequencing libraries treated with the BS protocol disclosed herein, using starting concentrations of 10ng or 3.3ng mESC gDNA and lambda DNA without spike-in 5mC sites. In addition, synthetic dsDNA containing 5mC was also spiked in to evaluate the rate of undesired 5mC conversion. To facilitate direct comparison of the disclosed protocol with current traditional BS protocols, we also generated parallel libraries using standard BS treatment (e.g., Zymo EZ DNA Methylation-Gold® Kit). After sequencing, we analyzed the conversion rates of all C sites and two known 5mC sites in the synthetic dsDNA. As shown in Figure 25, the two libraries starting from 3.3ng mESC gDNA gave slightly higher background than the one starting from 10ng (Figure 25B). When the two libraries made using the BS protocol disclosed herein were compared with the standard BS treatment (compare Figure 25A with Figure 25C), the new BS protocol gave a much smaller background. On the other hand, the undesired 5mC conversion rate was low in all four libraries (Figure 25D).

[0319] Moreover, the methylation levels detected from the sequencing libraries systematically generated by standard BS treatment showed a higher ratio than the BS treatment protocol disclosed herein (Figure 26). This may be due to the large background noise level of standard BS treatment compared to the BS protocol disclosed herein. Studies using standard BS treatment may overestimate the methylation level in the genome. Meanwhile, standard BS treatment data reported many methylation sites in non-CpG motifs (Figure 27). This may also be the result of the relatively large background noise level compared to the protocol disclosed herein. Background noise is a random signal and is more likely to be found in non-CpG sites. The increased background when studying non-CpG methylation may misinform researchers about biological significance and potentially lead to erroneous conclusions. Samples treated with the BS protocol disclosed herein showed similar genome coverage in different GC% regions when compared to samples treated with standard BS (Figure 28A). However, the sample treated with standard BS showed a higher percentage of unconverted C, especially in the high GC% region (Figure 28B). Furthermore, the two libraries produced using the BS protocol disclosed herein also showed more evenly distributed genome coverage than those produced using standard BS treatment (Figures 29A and 29B). This demonstrates further advantages of the methods and compositions disclosed herein when compared to standard BS treatment.

[0320] The BS protocol described herein was utilized to generate ultra-low or low gDNA input libraries generated using mES cells (1, 10, and 100 cells, respectively) and spike-in λ DNA. BS conversion efficiency from λ DNA and mitochondrial DNA (mtDNA) was evaluated because all cytosine sites were free of 5mC modifications. As shown in Figures 30 and 31, the non-converted C background noise decreased with increasing input amount. For example, the 1-cell sample showed a larger background than the 10-cell sample, whereas the 10-cell sample showed a larger background than the 100-cell sample. The BS protocol described herein resulted in a significantly smaller background level when compared to standard BS treatment. In the case of λDNA, when 1% was set as the background cutoff, the standard BS treatment showed more than 10-fold higher false positive levels (e.g., % unconverted C) than the BS protocol disclosed herein (e.g., average of about 4.9% vs. about 0.36% for three 10-cell sample tests, FIG. 30). In the case of highly structured mitochondrial DNA (which generally has a higher background level than λDNA, potentially due to the challenges associated with achieving high conversion levels due to the highly structured nature of mtDNA and incomplete denaturation), when 10% was set as the background cutoff, the false positive ratio using the standard BS treatment was more than 80-fold higher than the BS protocol disclosed herein (e.g., average of about 86.3% vs. about 1.3% for three 10-cell sample tests, FIG. 31). These results showed that the BS treatment protocol disclosed herein was superior to the standard BS treatment protocol when using ultra-low input gDNA.

[0321] Example 5 - Detection and analysis of 5hmC in DNA DNA oligonucleotides AGXGA (X = 5hmC, 5fC, or 5caC) (SEQ ID NO: 5, 6, 7) were incubated with BS recipe A7 at 98 °C for various times, and the reaction was monitored using MALDI-TOF MS. The reaction of 5hmC with BS was found to be the most efficient, with 5hmC being converted to the corresponding cytosine methylene sulfonate (CMS) within only 1 min (Figure 32A). In contrast, the reaction with 5fC was the slowest, with 5fC being converted to the U-BS adduct within 30 min. After desulfonation under basic conditions, U-BS was quantitatively converted to U. Thus, 5fC would be converted to U in BS sequencing (Figure 33A). 5caC also reacted very quickly with the BS reagent. This reaction was completed within 3 min, quantitatively converting 5caC to the U-BS adduct (Figure 34A). Thus, 5fC and 5caC would both be read as U in BS-seq, whereas 5mC and 5hmC would still be read as C.

[0322] During BS treatment, 5mC does not react with BS at all, whereas 5hmC is converted to CMS, so their chemical properties are different. However, after BS treatment, both 5mC and 5hmC are read as C, so a known difficulty with BS sequencing is that it cannot distinguish between 5mC and 5hmC. Recently, ACE-seq 28 reported sequencing 5hmC by taking advantage of the high deamination reactivity of APOBEC3A toward C and 5mC, but 5hmC may also be partially deaminated.

[0323] Since the disclosed BS conditions convert 5hmC to CMS spontaneously and quantitatively, we hypothesized that CMS is not deaminated by APOBEC3A treatment. To test this hypothesis, DNA 5mer oligos containing 5hmC were treated with BS recipe A7 to convert 5hmC to CMS, and then 5mC- or CMS-containing probes were treated in parallel with APOBEC3A. Maldi-TOF MS showed that 5mC was efficiently converted to T, which is consistent with the literature. However, no reaction to CMS was observed (Figure 35). This was further tested with 82mer DNA oligos containing 5mC, 5hmC, or CMS. Without APOBEC3A treatment, all of these were read as C in Sanger sequencing. However, after APOBEC3A treatment, 5mC was quantitatively read as T, 5hmC was partially read as T, while CMS was quantitatively read as C (Figure 36). This provides further support that this novel property of CMS can be used to distinguish between 5mC and 5hmC.

[0324] A new approach to sequence 5mC and 5hmC and a method to distinguish between them are provided herein.As shown in Figure 37, biological DNA can be treated with new BS conditions, and then the sample is divided into two parts.One part that is not further treated with APOBEC3A provides 5mC+5hmC sites, while the other part that is further treated with APOBEC3A converts 5mC to T but does not change CMS, so only the original 5hmC sites are read as C.Then, the two sets of data are subtracted to obtain only 5mC sites.

[0325] All of the methods disclosed and claimed herein can be made and carried out without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of certain specific aspects, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0326] References The following references, to the extent that they provide exemplary procedural details or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025502057000013.tif179160TIFF2025502057000014.tif245160TIFF2025502057000015.tif55160

Claims

1. 1. A method for processing DNA, comprising: (A) A method comprising the steps of: (a) incubating a solution comprising DNA molecules and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes, wherein the solution does not contain added sodium bisulfite; and (b) subjecting the DNA molecule to alkaline conditions; or (B) A method comprising the steps of: (a) preparing a solution comprising DNA molecules and ammonium bisulfite, wherein the solution does not contain added sodium bisulfite; (b) incubating the solution at a temperature of at least 95°C; and (c) removing the DNA molecules from the solution after a maximum of 12 minutes of step (a).

2. The method according to claim 1, which is the method (A), (ia) The solution does not contain added ammonium sulfite; or (ib) The solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (iii-a) The solution has a bisulfite concentration of 6.5 M to 10 M; (iii-b) The solution has a bisulfite concentration of 8M to 10M; (iii-c) The solution has a bisulfite concentration of 9M to 10M; or (iii-d) The solution has a bisulfite concentration of about 9.5 M; and / or (iv-a) the solution contains 50% to 70% by weight of ammonium bisulfite; (iv-b) the solution contains 60% to 70% by weight of ammonium bisulfite; (iv-c) the solution contains 65% to 68% by weight of ammonium bisulfite; or (iv-d) the solution contains about 66.7% by weight of ammonium bisulfite; and / or (va) The solution has a pH of 4.8 to 5.4; or (vb) the solution has a pH of about 5.1; and / or (vi) step (a) comprises incubating the solution at a temperature of about 98°C; and / or (vii-a) step (a) comprises incubating the solution for a maximum of 10 minutes; or (vii-b) step (a) comprises incubating the solution for up to 8 minutes; and / or (viii-a) the DNA molecule contains N4-methylcytosine (4mC), and after incubation, more than 50% of the 4mC is deaminated; (viii-b) the DNA molecule contains 4mC and after incubation, more than 75% of the 4mC is deaminated; or (viii-c) the DNA molecule contains 4mC, and after incubation, substantially all of the 4mC is deaminated; 10. The method of claim 1.

3. The method according to claim 1, which is the method (B), (ia) The solution does not contain added ammonium sulfite; or (ib) The solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (iii-a) The solution has a bisulfite concentration of 6.5 M to 10 M; (iii-b) The solution has a bisulfite concentration of 8M to 10M; (iii-c) The solution has a bisulfite concentration of 9M to 10M; or (iii-d) The solution has a bisulfite concentration of about 9.5 M; and / or (iv-a) the solution contains 50% to 70% by weight of ammonium bisulfite; (iv-b) the solution contains 60% to 70% by weight of ammonium bisulfite; (iv-c) the solution contains 65% to 68% by weight of ammonium bisulfite; or (iv-d) the solution contains about 66.7% by weight of ammonium bisulfite; and / or (va) The solution has a pH of 4.8 to 5.4; or (vb) the solution has a pH of about 5.1; and / or (vi) step (b) comprises incubating the solution at a temperature of about 98°C; and / or (vii-a) step (c) comprises removing the DNA molecules from the solution at most 10 minutes after step (a); or (vii-b) step (c) comprises removing the DNA molecules from the solution at most 8 minutes after step (a); and / or (viii) step (a) comprises mixing a 70% ammonium bisulfite solution with a 50% bisulfite solution; and / or (ix-a) The DNA molecule contains 4mC, and after incubation, more than 50% of the 4mC is deaminated; (ix-b) the DNA molecule contains 4mC and after incubation, more than 75% of the 4mC is deaminated; or (ix-c) The DNA molecule contains 4mC, and after incubation, substantially all of the 4mC is deaminated.

10. The method of claim 1.

4. 1. A method for processing a nucleic acid sample, comprising: (A) A method comprising the steps of: incubating a solution comprising DNA molecules and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes, wherein the solution does not contain added sodium bisulfite, each of the DNA molecules contains one or more cytosine residues, and after incubating the solution, more than 99% of the DNA molecules do not contain cytosine residues; or (B) A method comprising the steps of: Incubating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes, wherein the solution does not contain added sodium bisulfite, each of the RNA molecules contains one or more cytosine residues, and wherein after incubating the solution, more than 99% of the RNA molecules do not contain cytosine residues.

5. The method according to claim 4, which is the method (A), (i) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii) further comprising the step of subjecting the plurality of DNA molecules to alkaline conditions; and / or (iii-a) the solution comprises 50% to 70% by weight of ammonium bisulfite; (iii-b) the solution contains 60% to 70% by weight of ammonium bisulfite; (iii-c) the solution contains 65% to 68% by weight of ammonium bisulfite; or (iii-d) the solution contains about 66.7% by weight of ammonium bisulfite; and / or (iv-a) The solution does not contain added ammonium sulfite; or (iv-b) the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (va) The solution has a bisulfite concentration of 6.5M to 10M; (vb) The solution has a bisulfite concentration of 8M to 10M; (vc) the solution has a bisulfite concentration of 9M to 10M; or (vd) The solution has a bisulfite concentration of about 9.5 M; and / or (vi) the solution has a pH of 4.8 to 5.4; and / or (vii-a) the DNA molecule contains 4mC, and after incubation, more than 50% of the 4mC is deaminated; (vii-b) greater than 75% of the 4mC is deaminated after incubation; or (vii-c) Substantially all of the 4mC is deaminated after incubation; 5. The method of claim 4.

6. below: (a) a solution containing ammonium bisulfite having a bisulfite concentration of 6.5 M to 10 M, the solution not containing added sodium bisulfite; (b) instructions for processing the DNA sample; A DNA processing kit comprising:

7. (i) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii-a) The solution has a bisulfite concentration of 8M to 10M; (ii-b) The solution has a bisulfite concentration of 9M to 10M; or (ii-c) The solution has a bisulfite concentration of about 9.5 M; and / or (iii-a) the solution contains 50% to 70% by weight of ammonium bisulfite; (iii-b) the solution contains 60% to 70% by weight of ammonium bisulfite; (iii-c) the solution contains 65% to 68% by weight of ammonium bisulfite; or (iii-d) the solution contains about 66.7% by weight of ammonium bisulfite; and / or (iv-a) the solution has a pH of 4.8 to 5.4; or (iv-b) the solution has a pH of about 5.1; and / or (v) the instructions include instructions to incubate the DNA sample with the solution at a temperature of at least 95°C for a maximum of 12 minutes; and / or (vi) the instructions include instructions for incubating the DNA sample with the solution at a temperature of about 98°C; and / or (vii-a) The instructions include instructions to incubate the DNA sample with the solution for a maximum of 10 minutes; or (vii-b) the instructions include instructions to incubate the DNA sample with the solution for up to 8 minutes; and / or (viii-a) The solution does not contain added ammonium sulfite; or (viii-b) the solution does not contain ammonium sulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ix) further comprising an alkaline solution; and / or (x) further comprising one or more buffer solutions; The kit of claim 6.

8. 1. A method for RNA processing, comprising: (A) A method comprising the steps of: (a) incubating a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite at a temperature of at least 95° C. for a maximum of 12 minutes, wherein the solution does not contain added sodium bisulfite; and (b) subjecting the RNA molecule to alkaline conditions; or (B) A method comprising the steps of: (a) preparing a solution comprising RNA molecules, ammonium sulfite, and ammonium bisulfite, wherein the solution does not contain added sodium bisulfite; (b) incubating the solution at a temperature of at least 95°C; and (c) removing the RNA molecules from the solution after step (a) for up to 12 minutes.

9. The method according to claim 8, which is the method (A), (ia) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite; or (ib) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii-a) The solution has a bisulfite concentration of 6.5 M to 10 M; (ii-b) The solution has a bisulfite concentration of 6.5M to 7.5M; or (ii-c) The solution has a bisulfite concentration of about 7.0 M. and / or (iii) the solution has a pH of 4.8 to 5.4; and / or (iv-a) the solution contains 5% to 15% by weight of ammonium sulfite; (iv-b) the solution contains 8% to 12% by weight of ammonium sulfite; or (iv-c) the solution contains about 10% by weight of ammonium sulfite; and / or (v) step (a) comprises incubating the solution at a temperature of about 98°C; and / or (vi-a) step (a) comprises incubating the solution for a maximum of 10 minutes; or (vi-b) step (a) comprises incubating the solution for up to 8 minutes; 9. The method of claim 8.

10. The method according to claim 8, which is the method (B), (ia) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite; or (ib) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii-a) the solution has a bisulfite concentration of 6.5 M to 10 M; (ii-b) The solution has a bisulfite concentration of 6.5M to 7.5M; or (ii-c) the solution has a bisulfite concentration of about 7.0 M; and / or (iii-a) the solution has a pH of 4.8 to 5.4; or (iii-b) the solution has a pH of about 5.1; and / or (iv-a) the solution contains 5% to 15% by weight of ammonium sulfite; (iv-b) the solution contains 8% to 12% by weight of ammonium sulfite; or (iv-c) the solution contains about 10% by weight of ammonium sulfite; and / or (v) step (b) comprises incubating the solution at a temperature of about 98°C; and / or (vi-a) step (c) comprises removing the RNA molecules from the solution at most 10 minutes after step (a); or (vi-a) step (c) comprises removing the RNA molecules from the solution at most 8 minutes after step (a); 9. The method of claim 8.

11. The method according to claim 4, which is the method (B), (ia) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite; or (ib) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii-a) the solution has a pH of 4.8 to 5.4; or (ii-b) the solution has a pH of about 5.1; and / or (iii-a) the solution contains 5% to 15% by weight of ammonium sulfite; (iii-b) the solution contains 8% to 12% by weight of ammonium sulfite; or (iii-c) the solution contains about 10% by weight of ammonium sulfite; and / or (iv) step (a) comprises incubating the solution at a temperature of about 98°C; and / or (va) step (a) comprises incubating the solution for a maximum of 10 minutes; or (vb) step (a) comprises incubating the solution for up to 8 minutes; and / or (vi-a) the solution has a bisulfite concentration of 6.5 M to 10 M; (vi-b) the solution has a bisulfite concentration of 6.5M to 7.5M; or (vi-c) the solution has a bisulfite concentration of about 7.0 M; and / or (vii) further comprising the step of subjecting the plurality of RNA molecules to alkaline conditions; 5. The method of claim 4.

12. below: (a) a solution containing ammonium sulfite and ammonium bisulfite at a bisulfite concentration of 6.5M to 8M, the solution being free of sodium bisulfite; (b) instructions for processing RNA samples; An RNA processing kit comprising:

13. (ia) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium sulfite; or (ib) The solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii) the solution has a bisulfite concentration of about 7.0 M; and / or (iii-a) the solution has a pH of 4.8 to 5.4; or (iii-b) the solution has a pH of about 5.1; and / or (iv-a) the solution contains 5% to 15% by weight of ammonium sulfite; (iv-b) the solution contains 8% to 12% by weight of ammonium sulfite; or (iv-c) the solution contains about 10% by weight of ammonium sulfite; and / or (v) the instructions include instructions to incubate the RNA sample with the solution at a temperature of at least 95°C for a maximum of 12 minutes; and / or (vi) the instructions include instructions for incubating the RNA sample with the solution at a temperature of about 98°C; and / or (vii) the instructions include instructions to incubate the RNA sample with the solution for up to 10 minutes; 13. The kit of claim 12.

14. 1. A method for 5-hydroxymethylcytosine analysis, comprising the steps of: (a) incubating a first solution comprising a first DNA molecule and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes; (b) incubating a second solution comprising the second DNA molecule and ammonium bisulfite at a temperature of at least 95°C for a maximum of 12 minutes; (c) subjecting the first DNA molecule to alkaline conditions; (d) subjecting the second DNA molecule to alkaline conditions; (e) treating the second DNA molecule with an APOBEC deaminase enzyme; and (f) sequencing the first DNA molecule and the second DNA molecule.

15. (ia) the first solution does not contain added sodium bisulfite; or (ib) the first solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (ii-a) the second solution does not contain added sodium bisulfite; or (ii-b) the second solution does not contain sodium bisulfite at a level greater than about 1 / 10 the level of ammonium bisulfite; and / or (iii-a) the first solution and the second solution are the same solution; or (iii-b) the first solution and the second solution are different solutions; and / or (iv) steps (a) and (b) are carried out simultaneously; and / or (v) steps (c) and (d) are carried out simultaneously; and / or (vi) the first DNA molecule and the second DNA molecule have the same nucleotide sequence; and / or (vii) the APOBEC deaminase enzyme is APOBEC3A; 15. The method of claim 14.

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