Methods and compositions for rapid detection and analysis of RNA and DNA modifications

Abstract

Aspects of the present disclosure are directed to methods, compositions, and kits for detection and analysis of DNA and RNA cytosine methylation and / or RNA pseudouridylation. Certain aspects include methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids, including methylated and / or pseudouridinylated nucleic acids from low-input samples such as cell-free DNA (cfDNA) and cell-free RNA, long fragment polynucleotides, and / or cfDNA and DNA from formalin-fixed paraffin-embedded (FFPE) samples. Also disclosed herein are methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids with low nucleic acid damage, higher true positive results, and / or lower false negative results.

Description

ARCD.P0841WO / 24-T-190 METHODS AND COMPOSITIONS FOR RAPID DETECTION AND ANALYSIS OF RNA AND DNA MODIFICATIONS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 653,078, filed May 29, 2024, and U.S. Provisional Patent Application Serial No. 63 / 739,952, filed December 30, 2024, each of which is incorporated herein by reference in its entirety. STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under HG006827, and HG008935 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND I. Field of the Invention

[0003] Aspects of this invention relate to at least the fields of cell biology, nucleic acid sequencing, and epigenetics. II. Background

[0004] Nucleic acid modifications, e.g., methylation or nucleotide conversion, can control gene expression, cell functions, and may be associated with various human diseases. 5- methylcytosine (5mC) in DNA is a fundamental epigenetic mark that controls gene expression in numerous biological processes(29). In humans, the abnormal methylation patterns of 5mC reflect disease status and provide effective biomarkers for early diagnosis and monitoring of human diseases. Accurate detection of 5mC sites is critical for both basic research and clinical applications(30). Cytosine methylation in RNA (m5C) affects a variety of cell functions and plays critical roles in various human diseases. Conversion of uridine to pseudouridine (Ψ) is a prevalent RNA modification that can impact diverse biological functions of different RNA species. Ψ is also known to exist in mRNA in mammals. There are thirteen annotated pseudouridine synthetases (PUS) encoded in the human genome; most of these proteins deposit Ψ to different RNA species including mRNA, rRNA, tRNA, snRNA, etc(33). Mutations in these PUS enzymes have been associated with different human diseases(34). Thus, detection of nucleic acid modifications has useful applications in biological research and medicine. - 1 - 298000704.1ARCD.P0841WO / 24-T-190

[0005] Bisulfite sequencing (BS-seq) has been the gold standard for detecting methylation of nucleic acids due to its high resolution, robust reproducibility, and low cost, and has been widely used in basic research and clinic practices(31). While initially used for 5mC, BS-seq has been adapted for detecting m5C in RNA. However, conventional BS-seq methods suffers from several notable limitations: (1) long reaction time (the widely adopted conventional BS treatment requires ~3 hours), limiting its application in rapid clinic diagnosis; (2) in high GC DNA regions or highly structured DNA (such as mitochondrial DNA), C-to-U conversion is incomplete, resulting in high background and false positives; (3) severe RNA and / or DNA damage, limiting its application for low input samples, e.g., circulating cell-free DNA (cfDNA); (4) BS treatment leads to more severe degradation of non-methylated regions, resulting in overestimation of methylation levels; (5) conventional BS treatment has difficulties detecting RNA m5C in low abundant or highly structured RNA species, such as those with secondary structures, due to the instability of RNA; and (6) modification sites and modification levels of m5C in mRNA are much lower compared to gDNA and thus conventional methods result in high background and false positive rates.

[0006] Recent developments in bisulfite sequencing methods have improved the conversion efficiency of C-to-U conversion(32), (e.g., see PCT application publication WO 2023 / 133533 A2 published on July 13, 2023, and PCT application publication WO 2022 / 232795 A1 published on November 11, 2022, both of which are incorporated herein by reference in their entirety), however the methods can still result in substantial DNA and RNA damage, precluding their use in certain situations, such as on very low input samples and / or conditions wherein polynucleotide damage is not acceptable.

[0007] Detection of Ψ has also seen recent improvements. Several CMC (N-cyclohexyl- N’-(2-morpholino ethyl) carbodiimide methyl-p-toluenesulfonate)-based methods have been reported for Ψ detection since it can react with Ψ to form CMC-Ψ adduct, which can induce RT truncation signatures(35, 36, 37). In these methods, high modification fraction at a Ψ site is typically required to confidently read out the RT truncation signature above background noises, as many other factors may also cause RT stop. To increase sensitivity, an azide-modified CMC was later developed to enrich Ψ-containing RNA fragments for sequencing, allowing detection of more Ψ sites(38). A BID-seq based method to detect Ψ was also recently developed(39), a method based on the reaction of Ψ with BS to form the ring-opening Ψ-BS adducts which can induce deletion signatures in RT reactions—the RBS-seq method first reported by Khoddami et al In 2019(40). RBS-seq used the conventional BS conditions under acidic pH, and only allows a limited number of Ψ sites to be detected in mRNA and suffers from undesired C-to-U298000704.1- 2 -ARCD.P0841WO / 24-T-190 conversion occurring at the same time. Despite of improvements in detecting Ψ, there are still limitation for the current methods, including: (1) long reaction times, about 3 hours, to achieve a practical conversion rate; (2) high levels of RNA degradation; and (3) the inability for using low input samples.

[0008] Thus, there exists a need for improved methods and compositions for detection and analysis of DNA and RNA cytosine methylation and / or pseudouridine, including improvements that allow the use of DNA and / or RNA from low-input samples. SUMMARY

[0009] To address the aforementioned problems, the inventors created new methods and compositions that allow for more widespread applications in seeking new m5C, 5mC, and / or pseudouridine disease biomarkers or in detecting known m5C, 5mC, and / or pseudouridine biomarkers starting from very low input of clinic samples. In some aspects, the problems are solved, at least in part, through a reduction in polynucleotide damage, increased reaction efficiency, increased true positive modification identification, and / or decreased false positive modification identification.

[0010] The present disclosure provides various methods, compositions, systems, and kits for nucleic acid processing, and detection and / or analysis of cytosine methylation, pseudouridine (Ψ), or simultaneously cytosine methylation and Ψ. Certain aspects of the disclosure are directed to particular bisulfite compositions useful in rapid bisulfite treatment of DNA and / or RNA for detection and analysis of 5mC, m5C, Ψ, or m5C and Ψ. Also disclosed are DNA and RNA processing methods comprising use of the disclosed compositions for cytosine deamination and preparation of DNA and / or RNA for sequencing and cytosine methylation detection and analysis. Further disclosed are methods for Ψ detection, quantification, and analysis. DNA and RNA processing kits are disclosed, including bisulfite conversion kits useful in preparation of DNA and / or RNA for cytosine methylation and / or Ψ detection and analysis.

[0011] In some aspects, methods and compositions disclosed herein, such as Ultra-Mild Bisulfite Conversion (UMBS), provide highly efficient, low-degradation chemical protocols and compositions for the conversion of unmethylated cytosine residues to uracil residues while preserving nucleic acid integrity. Unlike conventional bisulfite conversion methods, UMBS enables broader compatibility with both DNA and RNA substrates, expanding its application potential across epigenetic and transcriptomic profiling technologies. In some aspects, the improvements described herein provide means for high fidelity interrogation of any nucleic298000704.1- 3 -ARCD.P0841WO / 24-T-190 acid type, but particularly cell free DNA (cfDNA) and / or DNA from Formalin-Fixed Paraffin- Embedded (FFPE) samples.

[0012] In some aspects, compositions and methods provided herein are useful for RNA 5- methylcytosine (m5C), for example, UMBS compositions and methods allow for the selective detection of RNA m5C modifications by providing a conversion environment for unmethylated cytosines that preserves RNA structure and m5C methylation signals, thereby facilitating accurate methylation (m5C) profiling of mRNA and non-coding RNAs at single-base resolution.

[0013] In some aspects, compositions and methods provided herein are useful and compatible for investigating and / or determining nucleic acid sequences / epigenetic marks at single-cell resolution and / or in long DNA and / or RNA fragments, including but not limited to nucleic acids from cell free DNA (cfDNA) samples and / or formalin-fixed paraffin-embedded samples. In some aspects, long DNA and / or RNA fragments are greater than or about 170 bases (e.g., cfDNA), greater than or about 200 bases, greater than or about 300 bases, greater than or about 400 bases, greater than or about 500 bases, greater than or about 600 bases, greater than or about 700 bases, greater than or about 800 bases, greater than or about 900 bases, greater than or over 1 kb, or any range derivable therein, or longer. In some aspects, UMBS methods and compositions are specifically suitable and fully compatible and with single-cell workflows, enabling the detection of DNA 5-methylcytosine (5mC) and RNA m5C at single-cell resolution using minimal input material. In some aspects, compositions and methods provided herein comprise ultra-mild conditions that effectively preserve both DNA and RNA integrity, making them particularly well-suited for sensitive applications. Furthermore, in some aspects, the high preservation DNA and / or RNA fragments and structural integrity renders the UMBS method (e.g., when combined with PCR amplification) ideal for detecting DNA 5-methylcytosine (5mC) and / or RNA methylcytosine (m5C) on long DNA and / or RNA fragments using long- read sequencing platforms, including for example, third-generation sequencing technologies such as those designed by Oxford Nanopore and PacBio. In some aspects, this compatibility facilitates high-resolution methylation profiling across extensive genomic regions, including repetitive elements, imprinted loci, and / or full-length RNA transcripts.

[0014] In some aspects, compositions and methods provided herein are suitable for seamless integration with downstream molecular biology tools, including but not limited to: methylation Polymerase Chain Reaction (PCR) and quantitative PCR (qPCR) for targeted methylation analysis, DNA methylation microarrays for high-throughput epigenetic screening,298000704.1- 4 -ARCD.P0841WO / 24-T-190 and / or library preparation workflows compatible with both short- and long-read sequencing platforms.

[0015] In some aspects, compositions and methods provided herein comprise distinct technological advantages relative to standard bisulfite sequencing methodologies and compositions. For example, in some aspects, by minimizing nucleic acid degradation while maintaining efficient deamination of unmethylated cytosines, compositions and methods provided herein overcome limitations of traditional bisulfite treatments, offering a platform technology adaptable to both high-resolution research and clinical diagnostic applications and methods comprising epigenetic and / or epitranscriptomic interrogation.

[0016] Aspects of the disclosure can include or exclude at least bisulfite solutions, ammonium sulfite solutions, ammonium bisulfite solutions, sodium bisulfite solutions, sodium metabisulfite solutions, sodium sulfite solutions, potassium sulfite solutions, potassium bisulfite solutions, potassium metabisulfite solutions, guanidinium bisulfite solutions, guanidinium hydrogen sulfite solutions, guanidinium ammonium bisulfite solutions, nucleic acid processing methods, DNA processing methods, RNA processing methods, methods for 5mC detection and analysis, methods for m5C detection and analysis, methods for Ψ detection and analysis, bisulfite sequencing methods, methylation detection and analysis methods, bisulfite treatment methods, nucleic acid processing kits, DNA processing kits, and RNA processing kits.

[0017] Methods of the disclosure can include or exclude at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the following steps: generating a bisulfite solution, mixing two or more solutions, mixing a first bisulfite solution with a second sulfite solution, mixing a bisulfite solution with a base, mixing a sulfite solution with a base, denaturing a nucleic acid, incubating a DNA molecule in a bisulfite solution, incubating an RNA molecule in a bisulfite solution, removing a DNA molecule from a bisulfite solution, removing an RNA molecule from a bisulfite solution, subjecting a DNA molecule to acidic or neutral conditions, subjecting an RNA molecule to acidic or neutral conditions, performing an additional desulphonation step, detecting a nucleotide methylation, quantifying nucleotide methylation, obtaining a sample from a subject, isolating nucleic acid molecules from a sample, sequencing a DNA molecule, and / or sequencing an RNA molecule. Any one or more of the preceding steps may be excluded from certain aspects. Compositions (e.g., solutions) of the disclosure can include, include at least, or include at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the following components: ammonium bisulfite, ammonium sulfite, ammonium sulfite monohydrate, sodium bisulfite, sodium metabisulfite, sodium sulfite, potassium sulfite, potassium bisulfite, potassium298000704.1- 5 -ARCD.P0841WO / 24-T-190 metabisulfite, guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, potassium hydroxide, and / or one or more organic solvents. Any one or more of the preceding components may be excluded from certain aspects. Kits of the disclosure can include, include at least, or include at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the following components: a bisulfite solution, a sodium bisulfite solution, an ammonium bisulfite solution, a sodium metabisulfite solution, a sodium sulfite solution, a potassium sulfite solution, a potassium bisulfite solution, a potassium metabisulfite solution, a guanidinium bisulfite solution, a guanidinium hydrogen sulfite solution, a guanidinium ammonium bisulfite solution, a basic solution, a buffer, a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, an antioxidant solution, instructions for DNA processing, instructions for RNA processing, instructions for bisulfite treatment of DNA, and instructions for bisulfite treatment of RNA. Any one or more of the preceding components may be excluded from certain aspects.

[0018] In some aspects, a method further comprises or does not further comprise subjecting the plurality of DNA molecules to acidic conditions. In some aspects, a solution does not comprise ammonium sulfite or added ammonium sulfite. In some aspects, a solution comprises, consists essentially of, or consists of ammonium bisulfite and potassium hydroxide. In some aspects, a solution comprises, consists essentially of, or consists of sodium metabisulfite and potassium hydroxide. In some aspects, the solution comprises, consists essentially of, or consists of potassium metabisulfite and potassium hydroxide.

[0019] In some aspects, a solution comprises between 30% and 80%, 40% and 80%, 50% and 80%, 50% and 70%, or 50% and 72% ammonium bisulfite by weight. In some aspects, a solution comprises, comprises at least, or comprises at most 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%, 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%, 55.1%, 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%, 56.7%, 56.8%, 56.9%, 57%, 57.1%, 57.2%, 57.3%, 57.4%, 57.5%, 57.6%, 57.7%, 57.8%, 57.9%, 58%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.6%, 58.7%, 58.8%, 58.9%, 59%, 59.1%, 59.2%, 59.3%, 59.4%, 59.5%, 59.6%, 59.7%, 59.8%, 59.9%, 60%, 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%, 61.6%, 61.7%, 61.8%, 61.9%, 62%, 62.1%, 62.2%, 62.3%, 62.4%, 62.5%, 62.6%, 62.7%, 62.8%, 62.9%, 63%, 63.1%, 63.2%, 63.3%, 63.4%, 63.5%, 63.6%, 63.7%,298000704.1- 6 -ARCD.P0841WO / 24-T-190 63.8%, 63.9%, 64%, 64.1%, 64.2%, 64.3%, 64.4%, 64.5%, 64.6%, 64.7%, 64.8%, 64.9%, 65%, 65.1%, 65.2%, 65.3%, 65.4%, 65.5%, 65.6%, 65.7%, 65.8%, 65.9%, 66%, 66.1%, 66.2%, 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. In some aspects, a solution comprises between 65% and 70% ammonium bisulfite by weight. In some aspects, a solution comprises or comprises about 70% ammonium bisulfite by weight. In some aspects, a solution comprises or comprises about 71% ammonium bisulfite by weight. In some aspects, a solution comprises or comprises about 72% ammonium bisulfite by weight.

[0020] In some aspects, a solution is at a bisulfite concentration between 4 M and 12 M, or any range or value derivable therein. In some aspects, a solution is at a bisulfite concentration greater than or equal to 6 M. In some aspects, a solution is at a bisulfite concentration between 8 M and 10 M. In some aspects, a solution is at a bisulfite concentration between 9 M and 10 M. In some aspects, a solution is at a bisulfite concentration between 9.5 M and 10 M. In some aspects, a solution is at a bisulfite concentration of, of at least, or of at most 4.0 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5.0 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10 M, 10.1 M, 10.2 M, 10.3 M, 10.4 M, 10.5 M, 10.6 M, 10.7 M, 10.8 M, 10.9 M, 11.0 M, 11.1 M, 11.2 M, 11.3 M, 11.4 M, 11.5 M, 11.6 M, 11.7 M, 11.8 M, 11.9 M, 12.0 M, or any range or value derivable therein. In some aspects, a solution is at a bisulfite concentration of or of about 7 to 7.5 M, or of or about 7.37 M. In some aspects, a solution is at a bisulfite concentration of or of about 9.5 to 10 M, or of or of about 9.8 M. In some aspects, a solution is at a bisulfite concentration of at least 9.8 M.

[0021] In some aspects, a solution has a pH between 4.8 and 8. In some aspects, the solution has a pH of, of at least, or of 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, 5.9, 6.0, 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, 8.0 or any range or value derivable therein. In some aspects, a solution has a pH between 5 and 7. In some aspects, a solution has a pH between 4.5 and 6. In some aspects, a solution has a pH between 5.2 and 6. In some aspects, a solution has a pH of or of about 5.7. In some aspects, a solution has a pH of or of about 5.3. In some aspects, a solution has a pH of or of about 5.1. In some aspects, a solution has a pH of or of about 4.9.298000704.1- 7 -ARCD.P0841WO / 24-T-190

[0022] In some aspects, a solution further comprises or does not comprise a bisulfite stabilizer, an antioxidant, and / or a stabilizer of single stranded (or denatured) nucleic acids). In some aspects, a composition further comprises or does not comprise an organic solvent. In some aspects, a composition comprises about 0-30% of an organic solvent. In some aspects, an organic solvent comprises or does not comprise methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof. In some aspects, a composition comprises or does not comprise hydroquinone, vitamin C, glutathione, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), allium sulfur compounds, beta-carotene, or a combination thereof. In some aspects, a composition comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA). In some aspects, a composition comprises about 0-25%, or 0.1-20%, or 5 to 15%, of Trolox dissolved in THFA. In some aspects, a composition comprises 7.5% of Trolox dissolved in THFA. In some aspects, a composition comprises about 0-25% of THFA. In some aspects, a composition comprises about 15% of THFA.

[0023] In some aspects, a method comprises incubating the solution at a temperature of, of at least, or of at most 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 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, a method comprises incubating the solution at a temperature of or of less than 95 °C, 90 °C, 85 °C, 70 °C, 65 °C, 60 °C, 55 °C, 50 °C, or any range derivable therein. In some aspects, a method comprises incubating the solution at a temperature of or of greater than or equal to 70 °C but less than or equal to 90 °C. In some aspects, a method comprises incubating the solution at a temperature of or of at least 98 °C. In some aspects, a method comprises incubating the solution for, for at least, or for at most 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, 60, 90, 120 minutes, or any range or value derivable therein. In some aspects, a method comprises incubating the solution for at most 90 minutes. In some aspects, a method comprises incubating the solution for at most 20 minutes. In some aspects, a method comprises incubating the solution for at most 10 minutes. In some aspects, a method comprises incubating the solution for at most 8 minutes.

[0024] In some aspects, a method further comprises a denaturation step that occurs prior to the step of incubating a solution comprising a polynucleotide with bisulfite. In some aspects, a denaturation step comprises incubating a solution comprising a polynucleotide with a KOH298000704.1- 8 -ARCD.P0841WO / 24-T-190 solution. In some aspects, the KOH solution comprises a concentration of about 1.0-3.0 M. In some aspects, the composition comprising a polynucleotide and KOH comprises a final KOH concentration between about 0.1-1.0 M. In some aspects, the composition comprising a polynucleotide and KOH comprises a final KOH concentration of about 0.3 M. In some aspects, the denaturation step comprises incubating the solution comprising a polynucleotide with KOH at about 35-55 °C. In some aspects, the denaturation step comprises incubating the solution comprising a polynucleotide with KOH at about 42 °C. In some aspects, the denaturation step comprises incubating the solution comprising a polynucleotide with KOH for 1-40 minutes. In some aspects, the denaturation step comprises incubating the solution comprising a polynucleotide with KOH for 20 minutes. In some aspects, the denaturation step comprises incubating the solution comprising a polynucleotide with a KOH solution at a final concentration of KOH of about 0.3 M at about 42 °C for 20 minutes.

[0025] In some aspects, a method further comprises or does not comprise a desulphonation step that occurs after the step of incubating a solution comprising a polynucleotide with bisulfite. In some aspects, the desulphonation step comprises in-column or on-bead desulphonation. In some aspects, the desulphonation step comprises on-bead desulphonation.

[0026] In some aspects, disclosed herein are compositions for facilitating detection of 5mC modifications in DNA and / or m5C modifications in RNA. In some aspects, disclosed herein are compositions comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 M and 12 M and the pH of the composition is between or between about 4.0 and 6.5. In some aspects, a concentration of bisulfite in the composition is between 9 M and 10 M and the pH of the composition is between 5 and 6. In some aspects, a bisulfite solution comprises ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), sodium bisulfite (NaHSO3), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a bisulfite solution consists essentially of, or consists of ammonium bisulfite and water. In some aspects, a composition does not comprise added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium bisulfite (NaHSO3), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3). In some aspects, an298000704.1- 9 -ARCD.P0841WO / 24-T-190 ammonium bisulfite solution comprises or comprises about 30-80%, 40-80%, 50-80%, or 60- 80%, or 65-80%, or 72% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite solution comprises or comprises about 68-70% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite solution comprises or comprises about 70% by weight ammonium bisulfite. In some aspects, a composition comprises ammonium bisulfite at or at about 68-70% by weight. In some aspects, a composition comprises ammonium bisulfite at or at about 69-69.5% by weight, or at or about 69.3% by weight. In some aspects, a composition comprises ammonium bisulfite at or at about 68-68.5% by weight or at or at about 68.3% by weight. In some aspects, a composition further comprises, or does not comprise, a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

[0027] In some aspects, a base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 15-30 M, 15-25 M, 15-20 M, or 20 M.

[0028] In some aspects, a composition comprises a concentration of bisulfite of, of greater than, or of about 9.65 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 9.8 M. In some aspects, a pH of the composition is between or between about 5 and 7. In some aspects, a pH of the composition is between or between about 4.9 and 5.3. In some aspects, a pH of the composition is between or between about 5.2 and 6. In some aspects, a pH of the composition is or is about 4.9. In some aspects, a pH of the composition is or is about 5.1. In some aspects, a pH of the composition is or is about 5.3. In some aspects, a composition is clear and has little-to-no precipitation at room temperature. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C.

[0029] In some aspects, a composition further comprises nucleic acids. In some aspects, a nucleic acid comprises deoxyribonucleic acid (DNA). In some aspects, DNA comprises 5- methylcytosine (5mC) modifications. In some aspects, DNA comprises 5mC modifications and cytosine to uracil (C-to-U) conversions. In some aspects, greater than 90%, 95%, 96%, 97%, 98%, or 99%, or any range derivable therein, of the DNA cytosines are converted to uracil. In some aspects, DNA comprises a low level of DNA damage. In some aspects, DNA damage is assessed as DNA fragmentation. In some aspects, DNA damage is measured using gel298000704.1- 10 -ARCD.P0841WO / 24-T-190 electrophoresis and / or a fragment analyzer. In some aspects, a composition comprises nucleic acids wherein the majority (e.g., greater than 50%) of the nucleic acid molecules exist in a single stranded or denatured state.

[0030] Also disclosed herein, in some aspects, are methods of detecting 5mC modifications in DNA, the method comprising use of any one or more compositions disclosed herein. In some aspects, a method of detecting 5mC further comprises a denaturation step and / or a desulphonation step. In some aspects, a method of detecting 5mC modifications in DNA comprises sequencing the DNA that was comprised within a bisulfite containing composition. In some aspects, a nucleic acid comprises ribonucleic acid (RNA). In some aspects, RNA comprises 5-methylcytosine (m5C) modifications. In some aspects, RNA comprises m5C modifications and cytosine to uracil (C-to-U) conversions. In some aspects, greater than 90%, 95%, 96%, 97%, 98%, or 99% of the RNA cytosines are converted to uracil. In some aspects, RNA comprises a low level of RNA damage. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer. Also disclosed herein are methods of detecting m5C modifications in RNA, the methods comprising use of any one or more compositions disclosed herein. In some aspects, a method of detecting m5C modifications in RNA comprises sequencing the RNA of that was comprised within a bisulfite containing composition.

[0031] Also disclosed herein, in some aspects, are methods for converting cytosine to uracil (C-to-U conversion) in a polynucleotide, the method comprising incubating a solution comprising a polynucleotide and ammonium bisulfite, wherein the solution is a) at a bisulfite concentration of or of about 4 M to 12 M, b) at a pH between or between about 4.5-6.5, c) at an incubation temperature between or between about 37 to 98 °C, or 70 to 98 °C, and d) incubated between 0.5-180 minutes. In some aspects, a bisulfite concentration is or is about 9 M to 10 M, and the pH is between or between about 4.9 to 5.3. In some aspects, a bisulfite concentration is or is greater than 9.65 M. In some aspects, a bisulfite concentration is, or is greater than, or is equal to, or is about, 9.8 M. In some aspects, ammonium bisulfite is provided as a solution at or at about 70%, 71%, or 72% ammonium bisulfite by weight. In some aspects, pH is or is about 4.9, 5.1, or 5.3. In some aspects, pH is adjusted using potassium hydroxide (KOH). In some aspects, potassium hydroxide is provided as a solution at a concentration of or of about 10 M to 25 M, or of or of about 20 M. In some aspects a temperature is or is about 37, 55, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable therein. In some aspects, an298000704.1- 11 -ARCD.P0841WO / 24-T-190 incubation temperature is or is about 70-98 °C. In some aspects, an incubation temperature is or is about 85 °C. In some aspects, an incubation temperature is or is about 55 °C.

[0032] In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than, of equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 90 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.1, and c) temperature of or of about 70 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 95 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, or of greater than or equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 80 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.1, and c) temperature of or of about 70 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 85 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.1, and c) temperature of or of about 85 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.3, and c) temperature of or of about 50 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 98 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.1, and c) temperature of or of about 98 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 4.9, and c) temperature of or of about 55 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.1, and c) temperature of or of about 55 °C. In some aspects, a method for converting C-to-U comprises: a) bisulfite concentration of, of greater than or equal to, or of about 9.8 M, b) pH of or of about 5.3, and c) temperature of or of about 55 °C.

[0033] In some aspects, an incubating time is 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, an298000704.1- 12 -ARCD.P0841WO / 24-T-190 incubation time is 5 to 50 minutes. In some aspects, an incubation time is 5 to 10 minutes. In some aspects, an incubation time is 5 to 8 minutes. In some aspects, an incubation time is 10 minutes. In some aspects, an incubation time is 20 to 50 minutes. In some aspects, a polynucleotide is DNA. In some aspects, a polynucleotide is RNA. In some aspects, a polynucleotide comprises a low level of damage. In some aspects, polynucleotide damage is assessed as RNA and / or DNA fragmentation. In some aspects, polynucleotide damage is measured using gel electrophoresis and / or a fragment analyzer.

[0034] In some aspects, methods disclosed herein comprise detecting 5-methylcytosine in a polynucleotide, the method comprising sequencing the polynucleotide after incubation in a composition disclosed herein. In some aspects, a polynucleotide comprises a total mass less than about 50 nanograms. In some aspects, a polynucleotide comprises a total mass between about 0.01 – 10 nanograms. In some aspects, a polynucleotide comprises a total mass between or between about 0.01, 0.05, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nanograms, or any range derivable therein. In some aspects, a C-to-U unconverted rate is or is less than about 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%, or any range derivable therein. In some aspects, a C-to-U unconverted rate is below or is below about 0.2%. In some aspects, a C-to-U unconverted rate is less than or less than about 0.1%. In some aspects, a C-to-U unconverted rate is, is less than, or is about 0.093%, 0.058%, or 0.048%.

[0035] Also disclosed herein, in some aspects, are polynucleotide processing kits comprising: a) a first component comprising a first solution comprising ammonium bisulfite and water having a bisulfite concentration between about 60-70% by weight; b) a second component comprising a potassium hydroxide salt or a second solution comprising potassium hydroxide and water having a potassium hydroxide concentration of or of about 10 M, 15 M, or 20 M; and c) optionally instructions for processing a polynucleotide sample. In some aspects, a kit further comprises a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, and / or an antioxidant solution. In some aspects, a kit comprises an ammonium bisulfite solution that has an ammonium bisulfite concentration of or of about 70% by weight. In some aspects, a kit further comprises a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M. In some aspects, the third component comprises a KOH solution comprising a KOH concentration of about 3.0 M. In some aspects, a kit further comprises a fourth component comprising a bisulfite stabilizer. In some aspects, the bisulfite stabilizer comprises or does not comprise an organic solvent. In some aspects, the bisulfite stabilizer comprises or does not comprise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone,298000704.1- 13 -ARCD.P0841WO / 24-T-190 acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2- Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, the bisulfite stabilizer comprises 1-20% methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, a kit further comprises or does not comprise a fifth component comprising a stabilizer of single stranded nucleic acids. In some aspects, the stabilizer of single stranded nucleic acids comprises or does not comprise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof. In some aspects, the stabilizer of single stranded nucleic acids comprises THFA. In some aspects, a kit further comprises or does not comprise a sixth component comprising a desulphonation solution. In some aspects, a kit further comprises or does not comprise a seventh component comprising an antioxidant. In some aspects, the antioxidant comprises or does not comprise hydroquinone, vitamin C, glutathione, Trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), allium sulfur compounds, beta- carotene, or a combination thereof. In some aspects, the antioxidant comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA). In some aspects, the antioxidant comprises 0-25% of Trolox dissolved in THFA. In some aspects, the antioxidant comprises 7.5% of Trolox dissolved in THFA. In some aspects, a kit further comprises desulphonation columns or beads. In some aspects, the first, second, third, fourth, fifth, sixth, and / or seventh components are provided separately. In some aspects, a first and second components are provided as a premixed solution. In some aspects, the first, second, fourth, fifth, and / or seventh components are provided as a premixed solution. In some aspects, the first, second, fourth, fifth, and / or seventh components are provided in suitable containers. In some aspects, a premixed solution is stable at or below room temperature and / or forms little to no precipitate. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, a premixed solution comprises a bisulfite concentration between or between about 6 M to 12 M, or 9 M to 10 M. In some aspects, a premixed solution comprises a bisulfite concentration greater than 9.65 M. In some aspects, a bisulfite concentration is or is about 9.8298000704.1- 14 -ARCD.P0841WO / 24-T-190 M. In some aspects, a premixed solution has a pH between or between about 4.9-5.3. In some aspects, a premixed solution has a pH of or of about 4.9, 5.1, or 5.3. In some aspects, instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at an incubation temperature between or between about 50-98 °C for an incubation time between or between about 0.5-180 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the first, second, third, fourth, fifth, sixth, and / or seventh components or a premixed solution of the first, second, fourth, fifth, and / or seventh components at a temperature between about 35- 98 °C for a time between 0.5-180 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the third component at about 42 °C for 20 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the sixth component. In some aspects, an incubation temperature is or is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 °C. In some aspects, an incubation temperature is or is about 80, or 85 °C. In some aspects, an incubation time is 5, 8, 10, 15, 20, 25, 30, 35, 80, 85, 90, 95, or 100 minutes, or any range derivable therein. In some aspects, a kit further comprises one or more buffer solutions and / or enzymes. In some aspects, one or more buffers comprise polynucleotide sequencing reagents. In some aspects, sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0036] Also disclosed herein, in some aspects, are compositions for facilitating detection of m5C modifications in RNA. In some aspects, disclosed herein are compositions comprising a mixture of two or more sulfites, wherein the final concentration of bisulfite in the composition is between 4 M and 12 M, and the pH value is between 4.0 to 6.5. In some aspects, a final concentration of bisulfite in a composition is between or between about 7 M and 8 M, and a pH value is between 4.0 to 6.5. In some aspects, a first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5; also known as sodium pyrosulfite or disodium metabisulfite), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) or ammonium sulfite monohydrate ((NH4)2SO3•H2O). In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of or of about 760 to 950 mg per 1000 μl water, and / or of or of about 4 M to 5 M. In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of or of about 840 mg per 1000 μl water, and / or of or of about 4.42 M. In some aspects, an ammonium sulfite is hydrated as ammonium sulfite monohydrate ((NH4)2SO3•H2O). In some aspects, an ammonium sulfite monohydrate is at a concentration of298000704.1- 15 -ARCD.P0841WO / 24-T-190 or of about 60 to 100 mg per 1000 μl water, and / or of or of about 447 mM to 746.3 mM. In some aspects, an ammonium sulfite monohydrate is at a concentration of or of about 80 mg per 1000 μl water, and / or of or of about 597 mM. In some aspects, a bisulfite concentration is of or of about 7.2 M to 7.5 M, or 7.37 M. In some aspects, a composition further comprises or does not comprise a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant. In some aspects, a composition further comprises nucleic acids. In some aspects, a nucleic acid comprises ribonucleic acid (RNA). In some aspects, an RNA comprises m5C modifications. In some aspects, an RNA comprises m5C modifications and C-to-U (cytosine to uracil) conversions. In some aspects, greater than 90%, 95%, 96%, 97%, 98%, or 99%, or any range derivable therein, of the RNA cytosines are converted to uracil. In some aspects, an RNA comprises a low level of RNA damage. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer.

[0037] Also disclosed herein, in some aspects, are methods of detecting m5C modifications in RNA, the method comprising use of any one or more of the compositions disclosed herein. In some aspects, methods of detecting m5C modifications in RNA comprise sequencing the RNA incubated in any one or more of the solutions disclosed herein.

[0038] Also disclosed herein, in some aspects, are methods of converting cytosine to uracil in a polynucleotide, the methods comprising incubating a composition comprising a polynucleotide and a mixture of two or more sulfites, wherein the composition is a) at a bisulfite concentration of or of about 4-12 M, b) at an incubation temperature between or between about 50 to 98 °C, and d) incubated between or between about 1-180 minutes. In some aspects, a bisulfite concentration is between or between about 7 to 8 M, and the temperature is between or between about 50 to 98 °C or 70 to 98 °C. In some aspects, a first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5; also known as sodium pyrosulfite or disodium metabisulfite), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) and / or ammonium sulfite monohydrate ((NH4)2SO3•H2O). In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of or of about 760 to 950 mg per 1000 μl water, and / or of or of about 4 M to 5 M. In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of or of about 840 mg per 1000 μl water, and / or of or of about 4.42 M. In some aspects, an ammonium sulfite monohydrate is at a concentration of or of about 60 to 100 mg per 1000 μl water, and / or of or about 447 mM to 746.3 mM. In some aspects, an ammonium sulfite monohydrate is at a concentration of or of about 80 mg per298000704.1- 16 -ARCD.P0841WO / 24-T-190 1000 μl water, and / or of or of about 597 mM. In some aspects, a bisulfite concentration is of or of about 7.2 M to 7.5 M, or 7.37 M. In some aspects, an incubation temperature is or is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable therein. In some aspects, an incubation time is 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, a polynucleotide is RNA. In some aspects, a polynucleotide comprises a low level of damage. In some aspects, polynucleotide damage is assessed as DNA and / or RNA fragmentation. In some aspects, polynucleotide damage is measured using gel electrophoresis and / or a fragment analyzer. In some aspects, a method further comprises a denaturation step and / or a desulphonation step. In some aspects, a method of detecting 5-methylcytosine in a polynucleotide comprises sequencing the polynucleotide, or a reverse transcribed copy thereof, after incubation. In some aspects, a polynucleotide comprises a total mass less than about 250, 200, 150, 100, or 50 nanograms. In some aspects, a polynucleotide comprises a total mass between about 50-200 nanograms. In some aspects, a polynucleotide comprises a total mass of or of about 50, 100, or 200 nanograms. In some aspects, a C-to-U unconverted ratio is, is less than, or is about 0.4%. In some aspects, a C-to- U unconverted ratio is, is less than, or is about 0.4%, or 0.3%.

[0039] Also disclosed herein, in some aspects, are kits for facilitating detection of m5C modifications in RNA. In some aspects, a polynucleotide processing kit comprises: a) two or more sulfites; and b) optionally instructions for processing a polynucleotide sample. In some aspects, a first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5; also known as sodium pyrosulfite or disodium metabisulfite), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) and / or ammonium sulfite monohydrate ((NH4)2SO3•H2O). In some aspects, a first and second sulfites are provided separately. In some aspects, a sodium metabisulfite (Na2S2O5) is provided at a concentration of or of about 760 to 950 mg per 1000 μl water, and / or of or of about 4 M to 5 M. In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of or of about 840 mg per 1000 μl water, and / or of or of about 4.42 M. In some aspects, an ammonium sulfite monohydrate is at a concentration of or of about 60 to 100 mg per 1000 μl water, and / or of or of about 447 mM to 746.3 mM. In some aspects, an ammonium sulfite monohydrate is at a concentration of or of about 80 mg per 1000 μl water, and / or of or of about 597 mM. In some aspects, a first and second sulfites are provided as a premixed solution. In some aspects, a premixed solution comprises a bisulfite concentration between about 7M to 8 M. In some aspects, a premixed solution comprises a bisulfite concentration of or of about 7.2 M to 7.5 M,298000704.1- 17 -ARCD.P0841WO / 24-T-190 or 7.37 M. In some aspects, a kit further comprises a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M. In some aspects, the third component comprises a KOH solution comprising a KOH concentration of about 3.0 M. In some aspects, a kit further comprises a fourth component comprising a bisulfite stabilizer. In some aspects, the bisulfite stabilizer comprises or does not comprise an organic solvent. In some aspects, the bisulfite stabilizer comprises or does not comprise methanol, ethanol, 1- propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, the bisulfite stabilizer comprises 1-20% methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, a kit further comprises a fifth component comprising a stabilizer of single stranded nucleic acids. In some aspects, the stabilizer of single stranded nucleic acids comprises or does not comprise methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, the stabilizer of single stranded nucleic acids comprises THFA. In some aspects, a kit further comprises a sixth component comprising a desulphonation solution. In some aspects, a kit further comprises a seventh component comprising an antioxidant. In some aspects, the antioxidant comprises or does not comprise hydroquinone, vitamin C, glutathione, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), allium sulfur compounds, beta-carotene, or a combination thereof. In some aspects, the antioxidant comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA). In some aspects, the antioxidant comprises 0-25% of Trolox dissolved in THFA. In some aspects, the antioxidant comprises 7.5% of Trolox dissolved in THFA. In some aspects, a kit further comprises desulphonation columns or beads. In some aspects, the first, second, third, fourth, fifth, sixth, and / or seventh components are provided separately. In some aspects, a first and second components are provided as a premixed solution. In some aspects, the first, second, fourth, fifth, and / or seventh components are provided as a premixed solution. In some aspects, a premixed solution is stable at or below room temperature and / or forms little to no precipitate. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about298000704.1- 18 -ARCD.P0841WO / 24-T-190 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, instructions comprise instructions for incubating the polynucleotide sample with the first and second components or the premixed solution at an incubation temperature between or between about 35-98 °C for an incubation time between 1- 180 minutes. In some aspects, an incubation temperature is or is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable there. In some aspects, an incubation temperature is or is about 80, or 85 °C. In some aspects, an incubation time is about 5, 8, 10, 15, 20, 25, 30, 35, 80, 85, 90, 95, or 100 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the first, second, third, fourth, fifth, sixth, and / or seventh components or a premixed solution of the first, second, fourth, fifth, and / or seventh components at a temperature between about 35-98 °C for a time between 0.5- 180 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the third component at about 42 °C for 20 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the sixth component. In some aspects, a kit further comprises one or more buffer solutions and / or enzymes. In some aspects, one or more buffers comprise sequencing reagents. In some aspects, sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0040] Also disclosed herein, in some aspects, are compositions for facilitating detection of pseudouridine modifications in RNA. In some aspects, disclosed herein are compositions comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 and 10 M, and the pH of the composition is between 6.0 and 8.5. In some aspects, a concentration of bisulfite in the composition is between 4 and 6 M. In some aspects, a bisulfite solution comprises or does not comprise potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1.66 M to 2.46 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 2.06 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or298000704.1- 19 -ARCD.P0841WO / 24-T-190 of about 1.76 M to 2.71 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 2.31 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 2.16 M. In some aspects, a composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfate (KHSO3), and / or added potassium sulfite (K2SO3). In some aspects, a composition further comprises a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

[0041] In some aspects, a base or acid solution comprises or does not comprise potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10- 30 M, 15-25 M, 15-20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 20-30% or 26% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 25-35% or 31% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 36 to 46% or 42% total volume of the composition.

[0042] In some aspects, a composition comprises a concentration of bisulfite between or between about 5 and 8 M. In some aspects, a composition comprises a concentration of bisulfite between or between about 5 and 7.5 M. In some aspects, a composition comprises a concentration of bisulfite between or between about 7.3 and 7.5 M. In some aspects, a composition comprises a concentration of bisulfite between or between about 4 and 5.5 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 4.3 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 5.4 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 5.1 M. In some aspects, a pH of the composition is between or between about 5 and 7.5. In some aspects,298000704.1- 20 -ARCD.P0841WO / 24-T-190 a pH of the composition is between or between about 5 and 7. In some aspects, a pH of the composition is between or between about 7 and 7.5. In some aspects, a pH of the composition is or is about 7.0. In some aspects, a pH of the composition is or is about 7.4. In some aspects, a composition is clear at room temperature and / or has little-to-no precipitation. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, a composition does not comprise ammonium salt. In some aspects, a composition further comprises nucleic acids. In some aspects, a nucleic acid comprises ribonucleic acid (RNA). In some aspects, an RNA comprises pseudouridine. In some aspects, an RNA comprises pseudouridine (Ψ) modifications and does not comprise appreciable levels of C-to- U (cytosine to uracil) conversions. In some aspects, an RNA comprises less than 5%, 4%, 3%, 2%, 1%, or 0.5%, or any range derivable therein, C-to-U (cytosine to uracil) conversions. In some aspects, greater than 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%, or 99% of the Ψ in an RNA of a composition are converted to Ψ-BS. In some aspects, an RNA comprises a low level of RNA damage. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer. In some aspects, there is between or between about 5 ng and 50 ng, or 5 ng and 15 ng of RNA in a composition.

[0043] Also disclosed herein, in some aspects, are methods of detecting Ψ modifications in RNA, the methods comprising use of any one or more of the compositions disclosed herein. In some aspects, a method of detecting Ψ modifications in RNA comprises sequencing the RNA that has been incubated with any one of the compositions disclosed herein, or a reverse transcribed copy thereof. In some aspects, a method further comprises a denaturation step and / or desulphonation step.

[0044] Also disclosed herein, in some aspects, are methods for converting Ψ to Ψ-BS in an RNA polynucleotide. In some aspects, disclosed herein are methods for converting Ψ to Ψ-BS in an RNA polynucleotide, the methods comprising incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein: a) the bisulfite concentration is or is about 4 M to 10 M, b) the pH is between or between about 6.0 to 8.5, c)298000704.1- 21 -ARCD.P0841WO / 24-T-190 the temperature is between or between about 50 to 98 °C, and d) the incubation is between 1- 180 minutes. In some aspects, a bisulfite concentration is about 4 M to 6 M, and the temperature is between about 75 to 98 °C. In some aspects, a sulfite solution comprises or does not comprise potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1.66 M to 2.46 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 2.06 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 1.76 M to 2.71 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 2.31 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 2.16 M. In some aspects, a composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0045] Also disclosed herein, in some aspects, are methods for converting Ψ to Ψ-BS in an RNA polynucleotide utilizing compositions and solutions disclosed herein. In some aspects, a base or acid solution comprises or does not comprise potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10-30 M, 15-25 M, 15- 20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises298000704.1- 22 -ARCD.P0841WO / 24-T-190 about 20-30% or 26% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 25-35% or 31% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 36 to 46% or 42% total volume of the composition.

[0046] Also disclosed herein, in some aspects, are methods for converting Ψ to Ψ-BS in an RNA polynucleotide utilizing compositions and solutions disclosed herein. In some aspects, a composition comprises a concentration of bisulfite between about 4 and 5.5 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 4.3 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 5.4 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 5.1 M. In some aspects, pH of the composition is between about 7 and 7.5. In some aspects, pH of the composition is or is about 7.0. In some aspects, pH of the composition is or is about 7.4. In some aspects, a composition is clear at room temperature and / or has little-to-no precipitation. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, an incubation temperature is or is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable therein. In some aspects, an incubation time is 3, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, an incubation time is 6, 8, 10, or 15 minutes, or any range In some aspects, an RNA polynucleotide comprises a low level of damage. In some aspects, an RNA polynucleotide comprises a low level of damage when compared to RNA prepared using a composition comprising an ammonium salt. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer.

[0047] In some aspects, a method results in less than about 5% C-to-U conversion. In some aspects, a method results in less than about 0.5% C-to-U conversion. In some aspects, methods result in 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60- 80%, or about 70-90%, or about 80-100% of the Ψ are converted to Ψ-BS. In some aspects, RNA polynucleotide comprises a total mass between or between about 5 ng and 50 ng, or 5 ng and 15 ng in a composition.

[0048] Also disclosed herein are kits for converting Ψ to Ψ-BS in an RNA polynucleotide. In some aspects, disclosed herein are polynucleotide processing kits comprising: a) a first298000704.1- 23 -ARCD.P0841WO / 24-T-190 component comprising a mixture of a sulfite solution; b) a second component comprising an acid or base solution; and c) optionally instructions for processing a polynucleotide sample. In some aspects, a kit comprises or does not comprise a sulfite solution comprising potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water. In some aspects, a sodium metabisulfite (Na2S2O5) is at a concentration of about 1.66 M to 2.46 M, 2.06 M, 1.76 M to 2.71 M, 2.31 M, or 2.16 M, or any range derivable therein. In some aspects, a composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3). In some aspects, a base or acid solution comprises or does not comprise potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10-30 M, 15-25 M, 15-20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 20-30% or 26% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 25-35% or 31% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 36 to 46% or 42% total volume of the composition. In some aspects, a kit further comprises a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M. In some298000704.1- 24 -ARCD.P0841WO / 24-T-190 aspects, the third component comprises a KOH solution comprising a KOH concentration of about 3.0 M. In some aspects, a kit further comprises a fourth component comprising a bisulfite stabilizer. In some aspects, the bisulfite stabilizer comprises an organic solvent. In some aspects, the bisulfite stabilizer comprises or does not comprise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, the bisulfite stabilizer comprises 1-20% methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, a kit further comprises a fifth component comprising a stabilizer of single stranded nucleic acids. In some aspects, the stabilizer of single stranded nucleic acids comprises or does not comprise methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof. In some aspects, the stabilizer of single stranded nucleic acids comprises THFA. In some aspects, a kit further comprises a sixth component comprising a desulphonation solution. In some aspects, a kit further comprises a seventh component comprising an antioxidant. In some aspects, the antioxidant comprises or does not comprise hydroquinone, vitamin C, glutathione, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), allium sulfur compounds, beta-carotene, or a combination thereof. In some aspects, the antioxidant comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA). In some aspects, the antioxidant comprises 0-25% of Trolox dissolved in THFA. In some aspects, the antioxidant comprises 7.5% of Trolox dissolved in THFA. In some aspects, a kit further comprises desulphonation columns or beads. In some aspects, the first, second, third, fourth, fifth, sixth, and / or seventh components are provided separately. In some aspects, a first and second components are provided as a premixed solution. In some aspects, the first, second, fourth, fifth, and / or seventh components are provided as a premixed solution. In some aspects, a premixed solution is stable at or below room temperature and / or forms little to no precipitate. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, a first and second components are provided separately. In some298000704.1- 25 -ARCD.P0841WO / 24-T-190 aspects, a first and second components are provided as a premixed solution. In some aspects, a premixed solution comprises a concentration of bisulfite between or between about 4 and 5.5 M. In some aspects, a premixed solution comprises a concentration of bisulfite of or of about 4.3 M. In some aspects, a premixed solution comprises a concentration of bisulfite of or of about 5.4 M. In some aspects, a premixed solution comprises a concentration of bisulfite of or of about 5.1 M. In some aspects, the pH of the premixed solution is between or between about 7 and 7.5. In some aspects, the pH of the premixed solution is or is about 7.0. In some aspects, the pH of the premixed solution is or is about 7.4. In some aspects, the premixed solution is clear at room temperature and / or has little-to-no precipitation. In some aspects, kits for converting Ψ to Ψ-BS in an RNA polynucleotide include instructions, wherein the instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at an incubation temperature between or between about 35-95 °C for an incubation time between 1-180 minutes. In some aspects, the incubating temperature is or is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C. In some aspects, incubating time is 3, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the first, second, third, fourth, fifth, sixth, and / or seventh components or a premixed solution of the first, second, fourth, fifth, and / or seventh components at a temperature between about 35-98 °C for a time between 0.5-180 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the third component at about 42 °C for 20 minutes. In some aspects, the instructions comprise instructions for incubating a polynucleotide sample with the sixth component. In some aspects, a kit further comprises one or more buffer solutions and / or enzymes. In some aspects, one or more buffers comprise sequencing reagents. In some aspects, sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0049] Also disclosed herein, in some aspects, are compositions for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine). Also disclosed herein, in some aspects, are compositions comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between or between about 3 M and 6 M, and the pH of the composition is between or between about 5.0 and 6.5. In some aspects, a concentration of bisulfite is between or between about 5 M and 6 M, and the pH is between or between about 5.5 and 6.1.298000704.1- 26 -ARCD.P0841WO / 24-T-190 In some aspects, a bisulfite solution comprises or does not comprise potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a bisulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, or ammonium bisulfite (NH4HSO3) and water, or any combination thereof. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1000 to 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1300 mg per 2000 μL of water, or about 3.42 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.58 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of or of about 1300 mg per 2354 μL of solvent, or about 2.9 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 600 mg per 680 μL of water, or about 3.97 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of or of about 600 mg per 720 μL of solvent, or about 3.75 M In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 50-80% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite solution comprises or comprises about 30-80% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite solution comprises or comprises about 40-80% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 70% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 71% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 72% by weight ammonium bisulfite. In some aspects, a composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added298000704.1- 27 -ARCD.P0841WO / 24-T-190 potassium sulfite (K2SO3). In some aspects, a composition further comprises a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

[0050] Also disclosed herein, in some aspects, are compositions for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) comprises or does not comprise a base or acid solution comprising potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10- 30 M, 15-25 M, 15-20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10-20 M, and comprises or comprises about 5-18% or 17% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 5% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 16 to 18% or 17% total volume of the composition.

[0051] Also disclosed herein, in some aspects, are compositions for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine), wherein a composition comprises a concentration of bisulfite between or between about 4 and 5.5 M, 4.3 M, 5.4 M, or 5.1 M, or any range derivable therein. In some aspects, pH of the composition is between or between about 5.5 and 6.1. In some aspects, pH of the composition is or is about 5.6-5.8. In some aspects, pH of the composition is or is about 5.7. In some aspects, a composition is clear at room temperature and / or has little-to-no precipitation. In some aspects, a composition does not comprise ammonium salt.

[0052] Also disclosed herein, in some aspects, are compositions for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine), comprising nucleic acids. In some aspects, a nucleic acid comprises ribonucleic acid (RNA). In some aspects, an RNA comprises pseudouridine and m5C. In some aspects, an RNA comprises pseudouridine (Ψ) modifications and also comprises appreciable levels of C-to-U (cytosine to uracil) conversions. In some aspects, a C-to-U unconverted rate298000704.1- 28 -ARCD.P0841WO / 24-T-190 is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%, or any range derivable therein. In some aspects, greater than 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%, or 99%, or any range derivable therein, of the Ψ are converted to Ψ-BS. In some aspects, an RNA comprises a low level of RNA damage. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer. In some aspects, there is between about 5 ng and 50 ng, or 5 ng and 15 ng of RNA in a composition.

[0053] In some aspects, also disclosed herein are methods of detecting Ψ and m5C modifications in RNA simultaneously, the methods comprising use of any one or more compositions disclosed herein. In some aspects, a method of detecting Ψ and m5C modifications in RNA simultaneously comprises sequencing an RNA incubated with a composition disclosed herein, or a reverse transcribed copy thereof, after a period of time of incubating the composition. In some aspects, a method further comprises a denaturation step and / or a desulphonation step.

[0054] Also disclosed herein, in some aspects, are methods for converting Ψ to Ψ-BS and C to U in an RNA polynucleotide. In some aspects, methods for converting Ψ to Ψ-BS and C to U in an RNA polynucleotide comprises incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein: a) the bisulfite concentration is or is about 4 M to 6 M, b) the pH is or is about 5-6.1, c) the temperature is between or between about 50 to 98 °C, and d) the incubation is between 1-180 minutes. In some aspects, an incubation temperature is between or between about 65 to 98 °C. In some aspects, a sulfite solution comprises or does not comprise potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, and / or ammonium bisulfite (NH4HSO3) and water. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1000 to298000704.1- 29 -ARCD.P0841WO / 24-T-190 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1300 mg per 2000 μL of water, or about 3.42 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.6 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1300 mg per 2354 μL of solvent, or about 2.9 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 600 mg per 680 μL of water, or about 3.97 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 600 mg per 720 μL of solvent, or about 3.75 M In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 30-80%, 40-80%, or 50-80% by weight ammonium bisulfite.

[0055] In some aspects, methods for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) comprises use of compositions and solutions disclosed herein. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 70% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 71% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 72% by weight ammonium bisulfite. In some aspects, a composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3). In some aspects, a base or acid solution comprises or does not comprise potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0056] In some aspects, methods for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) comprises use of compositions and298000704.1- 30 -ARCD.P0841WO / 24-T-190 solutions disclosed herein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10-30 M, 15-25 M, 15-20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of about 10-20 M, and comprises about 5-18% or about 17% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 20 M, and comprises or comprises about 5% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 16 to 18% or 17% total volume of the composition. In some aspects, a composition comprises a concentration of bisulfite between or between about 4 and 5.5 M. In some aspects, a composition comprises a concentration of bisulfite of or of about 4.3 M, 5.4 M, or 5.1 M, or any range derivable therein. In some aspects, the pH of a composition is between or between about 5.5-6.1, 5.6-5.8, or 5.7, or any range derivable therein. In some aspects, a composition is clear at room temperature and / or has little-to-no precipitation. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, an incubation temperature is or is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable therein. In some aspects, an incubation time is 3, 6, 7, 7.5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, an incubation time is 6, 8, 10, or 15 minutes, or any range derivable therein. In some aspects, an RNA polynucleotide comprises a low level of damage. In some aspects, an RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition comprising an ammonium salt. In some aspects, RNA damage is assessed as RNA fragmentation. In some aspects, RNA damage is measured using gel electrophoresis and / or a fragment analyzer.

[0057] In some aspects, methods for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) result in a C-to-U unconverted rate that is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%, or any range derivable therein. In some aspects, a C-to-U unconverted rate is less than about 3%. In some aspects, greater than 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%,298000704.1- 31 -ARCD.P0841WO / 24-T-190 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range derivable therein, of the Ψ are converted to Ψ-BS. In some aspects, about 20% to 40%, or about 30% to 50%, or about 40- 60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80-100% of the Ψ are converted to Ψ-BS. In some aspects, an RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or 5 ng and 15 ng in the composition.

[0058] In some aspects, also disclosed herein are polynucleotide processing kits for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine). In some aspects, a polynucleotide processing kit comprises: a) a first component comprising a mixture of a sulfite solution; b) a second component comprising an acid or base solution; and c) optionally instructions for processing a polynucleotide sample. In some aspects, a sulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof. In some aspects, a sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, and / or ammonium bisulfite (NH4HSO3) and water. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1000 to 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1300 mg per 2000 μL of water, or about 3.42 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.6 M. In some aspects, sodium metabisulfite (Na2S2O5) is at a concentration of 1300 mg per 2354 μL of solvent, or about 2.9 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 600 mg per 680 μL of water, or about 3.97 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M. In some aspects, potassium metabisulfite (K2S2O5) is at a concentration of 600 mg per 720 μL of solvent, or about 3.75 M. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises about 30-80%, 40-80%, 50-80% by weight ammonium bisulfite. In some aspects, an ammonium bisulfite (NH4HSO3) solution comprises or comprises about 70%, 71%, or 72% by weight ammonium bisulfite. In some aspects, a composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O),298000704.1- 32 -ARCD.P0841WO / 24-T-190 added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0059] In some aspects, disclosed herein are polynucleotide processing kits for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) comprising compositions and solutions disclosed herein. In some aspects, a base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), or sodium hydroxide (NaOH). In some aspects, a base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water. In some aspects, a composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH). In some aspects, a potassium hydroxide (KOH) solution comprises a concentration between or between about 5-15 M, 10-30 M, 15-25 M, 15-20 M, 20 M, or 10 M, or any range derivable therein. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10-20 M, and comprises or comprises about 5-18% or 17% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 5% total volume of the composition. In some aspects, a potassium hydroxide (KOH) solution comprises a concentration of or of about 10 M, and comprises or comprises about 16 to 18% or 17% total volume of the composition. In some aspects, a kit further comprises a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M. . In some aspects, the third component comprises a KOH solution comprising a KOH concentration of about 3.0 M. In some aspects, a kit further comprises a fourth component comprising a bisulfite stabilizer. In some aspects, the bisulfite stabilizer comprises an organic solvent. In some aspects, the bisulfite stabilizer comprises or does not comprise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, dimethoxyethane (DME), or a combination thereof. In some aspects, the bisulfite stabilizer comprises 1-20% methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol,298000704.1- 33 -ARCD.P0841WO / 24-T-190 PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, dimethoxyethane (DME), or a combination thereof. In some aspects, a kit further comprises a fifth component comprising a stabilizer of single stranded nucleic acids. In some aspects, the stabilizer of single stranded nucleic acids comprises or does not comprise methanol, ethanol, 1-propanol, 2- propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, dimethoxyethane (DME), or a combination thereof. In some aspects, the stabilizer of single stranded nucleic acids comprises THFA. In some aspects, a kit further comprises a sixth component comprising a desulphonation solution. In some aspects, a kit further comprises a seventh component comprising an antioxidant. In some aspects, the antioxidant comprises or does not comprise hydroquinone, vitamin C, glutathione, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), allium sulfur compounds, beta-carotene, or a combination thereof. In some aspects, the antioxidant comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA). In some aspects, the antioxidant comprises 0-25% of Trolox dissolved in THFA. In some aspects, the antioxidant comprises 7.5% of Trolox dissolved in THFA. In some aspects, a kit further comprises desulphonation columns or beads. In some aspects, the first, second, third, fourth, fifth, sixth, and / or seventh components are provided separately. In some aspects, the first, second, third, fourth, fifth, sixth, and / or seventh components are provided in suitable containers. In some aspects, a first and second components are provided as a premixed solution. In some aspects, the first, second, fourth, fifth, and / or seventh components are provided as a premixed solution. In some aspects, a premixed solution is stable at or below room temperature and / or forms little to no precipitate. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 1-7 °C. In some aspects, a premixed solution is clear and / or is substantially free of precipitate at about 4 °C. In some aspects, a first and second components are provided separately. In some aspects, a first and second components are provided as a premixed solution. In some aspects, a premixed solution comprises a concentration of bisulfite between or between about 4 and 5.5 M, 4.3 M, 5.4 M, or 5.1 M, or any range derivable therein. In some aspects a pH of the premixed solution is between or between about 5 and 6.1, 5.5 and 6.1, 5.7, 5.8, 5.9, or 6.0, or any range derivable therein. In some aspects, a premixed solution is clear at room temperature and / or has little-to-no precipitation.

[0060] In some aspects, disclosed herein are polynucleotide processing kits for facilitating simultaneous detection of multiple RNA modifications (e.g., pseudouridine and methylcytosine) comprising instructions. In some aspects, instructions comprise instructions298000704.1- 34 -ARCD.P0841WO / 24-T-190 for incubating a polynucleotide sample with the first and second components or the premixed solution at an incubation temperature between about 35-98 °C for an incubation time between 1-180 minutes. In some aspects, an incubation temperature is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C, or any range derivable therein. In some aspects, an incubation time is 3, 6, 7, 7.5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes, or any range derivable therein. In some aspects, kits for facilitating simultaneous detection of multiple RNA modifications further comprise one or more buffer solutions and / or enzymes. In some aspects, one or more buffers comprise sequencing reagents. In some aspects, sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0061] In some aspects, disclosed herein is a composition comprising a mixture of a 60- 70% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and DNA.

[0062] In some aspects, disclosed herein is a composition comprising a mixture of a 60- 70% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and RNA.

[0063] In some aspects, disclosed herein is a kit comprising a 60-70% ammonium bisulfite solution, an about 20 M KOH solution, an about 3 M KOH solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, an antioxidant solution, and / or desulphonation beads or columns.

[0064] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0065] Aspect 1 is a composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 M and 12 M and the pH of the composition is between about 4.0 and 7.

[0066] Aspect 2 is the composition of aspect 1, wherein the concentration of bisulfite in the composition is greater than or equal to about 6 M.

[0067] Aspect 3 is the composition of aspect 1 or 2, wherein the concentration of bisulfite in the composition is between about 9 M and 10 M and the pH of the composition is between about 5 and 6.

[0068] Aspect 4 is the composition of any one of aspects 1-3, wherein the bisulfite solution comprises ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), potassium metabisulfite (K2S2O5), sodium298000704.1- 35 -ARCD.P0841WO / 24-T-190 metabisulfite (Na2S2O5), sodium bisulfite (NaHSO3), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0069] Aspect 5 is the composition of any one of aspects 1-4, comprising ammonium bisulfite, potassium hydroxide (KOH), tetrahydrofurfuryl alcohol, and Trolox, optionally wherein the final concentration of Trolox is between 0.1% and 4% w / v, and optionally further comprising sodium bisulfite.

[0070] Aspect 6 is the composition of any one of aspects 1-5, wherein the bisulfite solution consists essentially of, or consists of ammonium bisulfite and water.

[0071] Aspect 7 is the composition of any one of aspects 1-6, wherein the composition does not comprise added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added potassium bisulfite (KHSO3), added sodium bisulfite (NaHSO3), added sodium sulfite (Na2SO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0072] Aspect 8 is the composition of any one of aspects 1-7, wherein the bisulfite solution comprises about 30-80%, 40-80%, or 50-80% by w / v ammonium bisulfite.

[0073] Aspect 9 is the composition of any one of aspects 1-8, wherein the bisulfite solution comprises about 68-72% by w / v ammonium bisulfite.

[0074] Aspect 10 is the composition of any one of aspects 1-9, wherein the bisulfite solution comprises about 70%, 71%, or 72% by w / v ammonium bisulfite.

[0075] Aspect 11 is the composition of any one of aspects 1-9, wherein the bisulfite solution comprises about 72% by w / v ammonium bisulfite.

[0076] Aspect 12 is the composition of any one of aspects 1-10, wherein the composition comprises ammonium bisulfite at about 68-70% by w / v.

[0077] Aspect 13 is the composition of any one of aspects 1-12, wherein the composition comprises ammonium bisulfite at about 69-69.5% by w / v, or about 69.3% by w / v.

[0078] Aspect 14 is the composition of any one of aspects 1-12, wherein the composition comprises ammonium bisulfite at about 68-68.5% by w / v or about 68.3% by w / v.

[0079] Aspect 15 is the composition of any one of aspects 1-14, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), concentrated hydrochloric acid (HCl), sodium hydroxide (NaOH), lithium hydroxide (LiOH), or ammonium hydroxide (NH4OH).298000704.1- 36 -ARCD.P0841WO / 24-T-190

[0080] Aspect 16 is the composition of any one of aspects 1-15, wherein the base or acid solution comprises, consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0081] Aspect 17 is the composition of any one of aspects 1-16, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0082] Aspect 18 is the composition of any one of aspects 15-17, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 15-30 M, about 15-25 M, about 15-20 M, or about 20 M.

[0083] Aspect 19 is the composition of any one of aspects 15-18, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0084] Aspect 20 is the composition of any one of aspects 1-19, wherein the composition comprises a concentration of bisulfite of greater than or equal to about 9.65 M.

[0085] Aspect 21 is the composition of any one of aspects 1-20, wherein the composition comprises a concentration of bisulfite of about 9.8 M.

[0086] Aspect 22 is the composition of any one of aspects 1-21, wherein the pH of the composition is between about 4.9 and 5.3.

[0087] Aspect 23 is the composition of any one of aspects 1-22, wherein the pH of the composition is about 4.9.

[0088] Aspect 24 is the composition of any one of aspects 1-22, wherein the pH of the composition is about 5.1.

[0089] Aspect 25 is the composition of any one of aspects 1-22, wherein the pH of the composition is about 5.3.

[0090] Aspect 26 is the composition of any one of aspects 1-25, further comprising a bisulfite stabilizer.

[0091] Aspect 27 is the composition of any one of aspects 1-26, further comprising an organic solvent.

[0092] Aspect 28 is the composition of any one of aspects 1-27, further comprising tetrahydrofuran (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof.

[0093] Aspect 29 is the composition of any one of aspects 1-28, further comprising between 1-20% tetrahydrofuran (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1-298000704.1- 37 -ARCD.P0841WO / 24-T-190 butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2- Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof.

[0094] Aspect 30 is the composition of any one of aspects 1-29, wherein the composition is clear and has little-to-no precipitation at or below room temperature.

[0095] Aspect 31 is the composition of any one of aspects 1-30, wherein the composition is clear and / or is substantially free of precipitate at about 1-7 °C.

[0096] Aspect 32 is the composition of any one of aspects 1-31, wherein the composition is clear and / or is substantially free of precipitate at about 4 °C.

[0097] Aspect 33 is the composition of any one of aspects 1-31, further comprising nucleic acids.

[0098] Aspect 34 is the composition of aspect 33, wherein the compositions comprises 20% to 75% w / v bisulfite at a molarity of about 3 M to 11 M.

[0099] Aspect 35 is the composition of any one of aspects 33-34, wherein the compositions comprises 30% to 70% w / v bisulfite at a molarity of about 4 M to 10.5 M.

[0100] Aspect 36 is the composition of any one of aspects 33-35, wherein the compositions comprises 45 to 60% w / v bisulfite at a molarity of about 6.5 to 10.5 M.

[0101] Aspect 37 is the composition of any one of aspects 33-36, wherein the composition comprises 50 to 55 % w / v bisulfite at a molarity of about 7.2 to 7.5 M.

[0102] Aspect 38 is the composition of any one of aspects 33-37, wherein the composition comprises about 52 to 53 % w / v bisulfite at a molarity of about 7.4 M.

[0103] Aspect 39 is the composition of any one of aspects 33-38, wherein the nucleic acid comprises deoxyribonucleic acid (DNA).

[0104] Aspect 40 is the composition of aspect 39, wherein the DNA comprises 5- methylcytosine (5mC) modifications.

[0105] Aspect 41 is the composition of aspect 39 or 40, wherein the DNA comprises 5mC modifications and cytosine to uracil (C-to-U) conversions.

[0106] Aspect 42 is the composition of aspect 41, wherein greater than 90%, 95%, 96%, 97%, 98%, or 99%, of the DNA cytosines are converted to uracil.

[0107] Aspect 43 is the composition of any one of aspects 1-42, further comprising an antioxidant.

[0108] Aspect 44 is the composition of any one of aspects 1-43, further comprising hydroquinone, vitamin C, glutathione, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), allium sulfur compounds, beta-carotene, or a combination thereof.298000704.1- 38 -ARCD.P0841WO / 24-T-190

[0109] Aspect 45 is the composition of any one of aspects 1-44, further comprising Trolox dissolved in tetrahydrofurfuryl alcohol (THFA).

[0110] Aspect 46 is the composition of any one of aspects 1-45, further comprising (w / v) 0-25%, or 0.1-20%, or 5 to 15%, of Trolox dissolved in THFA.

[0111] Aspect 47 is the composition of any one of aspects 1-46, further comprising 7.5% of Trolox dissolved in THFA.

[0112] Aspect 48 is the composition of any one of aspects 39-47, wherein the DNA comprises a low level of DNA damage, optionally wherein the damage is assessed as fragmentation, optionally measured using gel electrophoresis and / or a fragment analyzer.

[0113] Aspect 49 is the composition of any one of aspects 1-48, further comprising a stabilizer of single stranded nucleic acids.

[0114] Aspect 50 is the composition of aspect 49, wherein the stabilizer of single stranded nucleic acids comprises between about 0-30% of the composition.

[0115] Aspect 51 is the composition of any one of aspects 1-50, further comprising methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof.

[0116] Aspect 52 is the composition of any one of aspects 1-51, comprising THFA.

[0117] Aspect 53 is the composition of any one of aspects 1-52, comprising about 0-25% of THFA.

[0118] Aspect 54 is the composition of any one of aspects 1-53, comprising about 10%, 11%, 12%, 13%, 14%, or 15% of THFA.

[0119] Aspect 55 is the composition of any one of aspects 33-54 wherein the majority (greater than 50%) of the nucleic acid molecules exist in a single stranded or denatured state.

[0120] Aspect 56 is the composition of any one of aspects 48-55, wherein the DNA damage is assessed as DNA fragmentation.

[0121] Aspect 57 is a method of detecting 5mC modifications in DNA, the method comprising use of the composition according to any one of aspects 1-56.

[0122] Aspect 58 is a method of detecting 5mC modifications in DNA, the method comprising sequencing the DNA of the composition of any one of aspects 39-56.

[0123] Aspect 59 is the composition of aspect 33-38, wherein the nucleic acid comprises ribonucleic acid (RNA).298000704.1- 39 -ARCD.P0841WO / 24-T-190

[0124] Aspect 60 is the composition of aspect 59, further comprising a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

[0125] Aspect 61 is the composition of aspect 59 or 60, wherein the RNA comprises 5- methylcytosine (m5C) modifications.

[0126] Aspect 62 is the composition of any one of aspects 59-61, wherein the RNA comprises m5C modifications and cytosine to uracil (C-to-U) conversions.

[0127] Aspect 63 is the composition of aspect 62, wherein greater than 90%, 95%, 96%, 97%, 98%, or 99% of the RNA cytosines are converted to uracil.

[0128] Aspect 64 is the composition of any one of aspects 59-63, wherein the RNA comprises a low level of RNA damage.

[0129] Aspect 65 is the composition of aspect 64, wherein the damage is assessed as RNA fragmentation, optionally measured using gel electrophoresis and / or a fragment analyzer.

[0130] Aspect 66 is a method of detecting m5C modifications in RNA, the method comprising use of the composition according to any one of aspects 1-38 or 59-65.

[0131] Aspect 67 is the method of detecting m5C modifications in RNA, the method comprising sequencing the RNA of the composition of any one of aspects 59-66.

[0132] Aspect 68 is a method for converting cytosine to uracil (C-to-U conversion) in a polynucleotide, the method comprising incubating a solution comprising a polynucleotide and bisulfite, wherein the solution is: a) at a bisulfite concentration of about 4 M to 12 M, b) at a pH between about 4.5-6.5, c) at a temperature between about 37 to 98 °C, or 70 to 98 °C, and d) incubated between about 0.5-180 minutes.

[0133] Aspect 69 is the method of aspect 68, wherein the bisulfite comprises, consists essentially of, or consists of ammonium bisulfite.

[0134] Aspect 70 is the method of aspect 68 or 69, wherein the bisulfite concentration is greater than or equal to 6 M.

[0135] Aspect 71 is the method of any one of aspects 68-70, wherein the bisulfite concentration is about 9 M to 10 M, and the pH is between about 4.9 to 5.3.

[0136] Aspect 72 is the method of any one of aspects 68-71, wherein the bisulfite concentration is greater than about 9.65 M.

[0137] Aspect 73 is the method of any one of aspects 68-72, wherein the bisulfite concentration is greater than or equal to about 9.8 M.

[0138] Aspect 74 is the method of any one of aspects 68-73, wherein the bisulfite is provided as a solution at about 70%, 71%, or 72% ammonium bisulfite by w / v.298000704.1- 40 -ARCD.P0841WO / 24-T-190

[0139] Aspect 75 is the method of any one of aspects 68-74, wherein the pH is about 4.9, 5.1, or 5.3.

[0140] Aspect 76 is the method of any one of aspects 68-75, wherein the pH is adjusted using potassium, hydroxide (KOH).

[0141] Aspect 77 is the method of aspect 76, wherein the potassium hydroxide is provided as a solution at a concentration of about 10 M to 25 M, or about 20 M.

[0142] Aspect 78 is the method of any one of aspects 68-77, wherein the temperature is about 37, 55, 70, 75, 80, 85, 90, 95, or 98 °C.

[0143] Aspect 79 is the method of any one of aspects 68-77, wherein the temperature is about 70-98 °C.

[0144] Aspect 80 is the method of any one of aspects 68-79, wherein the temperature is or is about 85 °C.

[0145] Aspect 81 is the method of any one of aspects 68-78, wherein the temperature is or is about 55 °C.

[0146] Aspect 82 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 4.9, and c) temperature about 90 °C.

[0147] Aspect 83 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.1, and c) temperature about 70 °C.

[0148] Aspect 84 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 4.9, and c) temperature about 95 °C.

[0149] Aspect 85 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration greater than or equal to, of about 9.8 M, b) pH about 4.9, and c) temperature about 80 °C.

[0150] Aspect 86 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.1, and c) temperature about 70 °C.

[0151] Aspect 87 is the method of any one of aspects 68-80, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 4.9, and c) temperature about 85 °C.298000704.1- 41 -ARCD.P0841WO / 24-T-190

[0152] Aspect 88 is the method of any one of aspects 68-80, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.1, and c) temperature about 85 °C.

[0153] Aspect 89 is the method of any one of aspects 68-78, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.3, and c) temperature about 50 °C

[0154] Aspect 90 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 4.9, and c) temperature about 98 °C.

[0155] Aspect 91 is the method of any one of aspects 68-79, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.1, and c) temperature about 98 °C.

[0156] Aspect 92 is the method of any one of aspects 68-78, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 4.9, and c) temperature about 55 °C.

[0157] Aspect 93 is the method of any one of aspects 68-78, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.1, and c) temperature about 55 °C.

[0158] Aspect 94 is the method of any one of aspects 68-78, comprising, a) bisulfite concentration of greater than or equal to about 9.8 M, b) pH about 5.3, and c) temperature about 55 °C.

[0159] Aspect 95 is the method of any one of aspects 68-94, wherein the incubation time is, or is between any two of 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 120, or 150 minutes.

[0160] Aspect 96 is the method of aspect 95, wherein the incubation time is 5 to 50 minutes.

[0161] Aspect 97 is the method of aspect 95, wherein the incubation time is 5 to 10 minutes.

[0162] Aspect 98 is the method of aspect 95, wherein the incubation time is 5 to 8 minutes.

[0163] Aspect 99 is the method of aspect 95, wherein the incubation time is 10 minutes.

[0164] Aspect 100 is the method of aspect 95, wherein the incubation time is 20 to 50 minutes.

[0165] Aspect 101 is the method of aspect 95, wherein the incubation time is 80 to 100 minutes.

[0166] Aspect 102 is the method of aspect 95, wherein the incubation time is 90 minutes.298000704.1- 42 -ARCD.P0841WO / 24-T-190

[0167] Aspect 103 is the method of any one of aspects 68-102, wherein the polynucleotide is DNA.

[0168] Aspect 104 is the method of any one of aspects 68-102, wherein the polynucleotide is RNA.

[0169] Aspect 105 is the method of anyone of aspects 68-104, wherein the polynucleotide comprises a low level of damage.

[0170] Aspect 106 is the method of aspect 105, wherein the damage is assessed as RNA and / or DNA fragmentation, optionally measured using gel electrophoresis and / or a fragment analyzer.

[0171] Aspect 107 is a method of detecting 5-methylcytosine in a polynucleotide comprising sequencing the polynucleotide of the composition of any one of aspects 68-106 after incubation.

[0172] Aspect 108 is the method of aspect 107, wherein the polynucleotide comprises a total mass less than about 50 nanograms.

[0173] Aspect 109 is the method of aspect 107 or 108, wherein the polynucleotide comprises a total mass between about 0.01 – 10 nanograms.

[0174] Aspect 110 is the method of aspect 109, wherein the polynucleotide comprises a total mass between about 0.01, 0.05, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nanograms.

[0175] Aspect 111 is the method of any one of aspects 68-110, wherein the C-to-U unconverted rate is less than about 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

[0176] Aspect 112 is the method of any one of aspects 68-111, wherein the C-to-U unconverted rate is or is below about 0.2%.

[0177] Aspect 113 is the method of any one of aspects 68-112, wherein the C-to-U unconverted rate is or is less than about 0.1%.

[0178] Aspect 114 is the method of aspect 113, wherein the C-to-U unconverted rate is less than or about 0.093%, 0.058%, or 0.048%.

[0179] Aspect 115 is the method of any one of aspects 68-114, further comprising a denaturation step that occurs prior to the step of incubating a solution comprising a polynucleotide with bisulfite.

[0180] Aspect 116 is the method of aspect 115, wherein the denaturation step comprises incubating a solution comprising a polynucleotide with a solution comprising KOH, NaOH, LiOH, NH4OH, and / or another strong base.

[0181] Aspect 117 is the method of aspect 116, wherein the solution comprises KOH.298000704.1- 43 -ARCD.P0841WO / 24-T-190

[0182] Aspect 118 is the method of aspect 116 or 117, wherein the KOH solution comprises a concentration of about 1.0-3.0 M.

[0183] Aspect 119 is the method of aspect 118, wherein the composition comprising a polynucleotide and KOH comprises a final KOH concentration between about 0.1-1.0 M.

[0184] Aspect 120 is the method of aspect 118 or 119, wherein the composition comprising a polynucleotide and KOH comprises a final KOH concentration of about 0.3 M.

[0185] Aspect 121 is the method of any one of aspects 116-120, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH at about 35- 55 °C.

[0186] Aspect 122 is the method of any one of aspects 116-121, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH between about 35-55 °C.

[0187] Aspect 123 is the method of any one of aspects 116-122, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH at about 42 °C.

[0188] Aspect 124 is the method of any one of aspects 116-123, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH for 1-40 minutes.

[0189] Aspect 125 is the method of any one of aspects 116-124, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH for 20 minutes.

[0190] Aspect 126 is the method of any one of aspects 116-125, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with a KOH solution at a final concentration of KOH of about 0.3 M at about 42 °C for 20 minutes.

[0191] Aspect 127 is the method of any one of aspects 68-126, further comprising a desulphonation step that occurs after the step of incubating a solution comprising a polynucleotide with bisulfite.

[0192] Aspect 128 is the method of aspect 127, wherein the desulphonation step comprises in-column or on-bead desulphonation.

[0193] Aspect 129 is the method of aspect 127 or 128, wherein the desulphonation step comprises on-bead desulphonation.

[0194] Aspect 130 is a polynucleotide processing kit comprising: a) a first component comprising a first solution comprising ammonium bisulfite and water having a bisulfite concentration between about 60-72% by w / v; b) a second component comprising a potassium hydroxide salt or a second solution comprising potassium hydroxide and water having a298000704.1- 44 -ARCD.P0841WO / 24-T-190 potassium hydroxide concentration of about 10 M, 15 M, or 20 M; and c) optionally instructions for processing a polynucleotide sample.

[0195] Aspect 131 is the kit of aspect 130, wherein the ammonium bisulfite solution has an ammonium bisulfite concentration of about 70%, 71%, or 72% by w / v.

[0196] Aspect 132 is the kit of aspect 130 or 131, further comprising a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M.

[0197] Aspect 133 is the kit aspect 132, wherein the third component comprises a KOH solution comprising a KOH concentration of about 3.0 M.

[0198] Aspect 134 is the kit of any one of aspects 130-133, further comprising a fourth component comprising a bisulfite stabilizer.

[0199] Aspect 135 is the kit of aspect 134, wherein the bisulfite stabilizer comprises an organic solvent.

[0200] Aspect 136 is the kit of aspect 134 or 135, wherein the bisulfite stabilizer comprises methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof.

[0201] Aspect 137 is the kit of any one of aspects 134-136, wherein the bisulfite stabilizer comprises 1-20% methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THFA), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2- Hexanediol, isopropanol, and / or dimethoxyethane (DME), or a combination thereof.

[0202] Aspect 138 is the kit of any one of aspects 130-137, further comprising a fifth component comprising a stabilizer of single stranded nucleic acids.

[0203] Aspect 139 is the kit of aspect 138, wherein the stabilizer of single stranded nucleic acids comprises tetrahydrofuran (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, glycerol, PEG-400 / PEG-600, propylene glycol, 1,2- Hexanediol, isopropanol, dimethoxyethane (DME), or a combination thereof.

[0204] Aspect 140 is the kit of aspect 138 or 139, wherein the stabilizer of single stranded nucleic acids comprises THFA.

[0205] Aspect 141 is the kit of any one of aspects 130-140, further comprising a sixth component comprising a desulphonation solution.298000704.1- 45 -ARCD.P0841WO / 24-T-190

[0206] Aspect 142 is the kit of any one of aspects 130-141, further comprising a seventh component comprising an antioxidant.

[0207] Aspect 143 is the kit of aspect 142, wherein the antioxidant comprises Trolox (6- hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), hydroquinone, vitamin C, glutathione, allium sulfur compounds, beta-carotene, or a combination thereof.

[0208] Aspect 144 is the kit of aspect 142 or 143, wherein the antioxidant comprises Trolox dissolved in tetrahydrofurfuryl alcohol (THFA).

[0209] Aspect 145 is the kit of any one of aspects 142-144, wherein the antioxidant comprises (w / v) 0-25%, or 0.1-20%, or 5 to 15%, of Trolox dissolved in THFA.

[0210] Aspect 146 is the kit of any one of aspects 142-145, wherein the antioxidant comprises 7.5% of Trolox dissolved in THFA.

[0211] Aspect 147 is the kit of any one of aspects 130-146, further comprising desulphonation columns or beads.

[0212] Aspect 148 is the kit of any one of aspects 130-147, wherein the first, second, third, fourth, fifth, sixth, and / or seventh components are provided separately.

[0213] Aspect 149 is the kit of any one of aspects 130-148, wherein the first and second components are provided as a premixed solution.

[0214] Aspect 150 is the kit of any one of aspects 130-149, wherein the first, second, fourth, fifth, and / or seventh components are provided as a premixed solution.

[0215] Aspect 151 is the kit of aspect 149 or 150, wherein the premixed solution is stable at or below room temperature and / or forms little to no precipitate.

[0216] Aspect 152 is the kit of any one of aspects 150 or 151, wherein the composition is clear and / or is substantially free of precipitate at about 4 °C.

[0217] Aspect 153 is the kit of any one of aspects 149-152, wherein the premixed solution comprises a bisulfite concentration between about 6 M to 12 M, or about 9 M to 10 M.

[0218] Aspect 154 is the kit of any one of aspects 149-153, wherein the premixed solution comprises a bisulfite concentration greater than 9.65 M.

[0219] Aspect 155 is the kit of any one of aspects 149-154, wherein the bisulfite concentration is about 9.8 M.

[0220] Aspect 156 is the kit of any one of aspects 149-155, wherein the premixed solution has a pH between about 4.9-5.3.

[0221] Aspect 157 is the kit of any one of aspects 149-156, wherein the premixed solution has a pH about 4.9, 5.1, or 5.3.298000704.1- 46 -ARCD.P0841WO / 24-T-190

[0222] Aspect 158 is the kit of any one of aspects 130-157, wherein the instructions comprise instructions for incubating a polynucleotide sample with the first, second, third, fourth, fifth, sixth, and / or seventh components or a premixed solution of the first, second, fourth, fifth, and / or seventh components at a temperature between about 35-98 °C for a time between 0.5-180 minutes.

[0223] Aspect 159 is the kit of any one of aspects 130-158, wherein the temperature is about 30, 35, 40, 42, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98 °C.

[0224] Aspect 160 is the kit of any one of aspects 130-159, wherein the temperature is about 80, or 85 °C.

[0225] Aspect 161 is the kit of any one of aspects 130-160, wherein the time is between 5 and 100 minutes, or 5, 8, 10, 15, 20, 25, 30, 35, 80, 85, 90, 95, or 100 minutes.

[0226] Aspect 162 is the kit of any one of aspects 130-161, wherein the instructions comprise instructions for incubating a polynucleotide sample with the third component at about 42 °C for 20 minutes.

[0227] Aspect 163 is the kit of any one of aspects 130-162, wherein the instructions comprise instructions for incubating a polynucleotide sample with the sixth component.

[0228] Aspect 164 is the kit of any one of aspects 130-163, further comprising one or more buffer solutions and / or enzymes.

[0229] Aspect 165 is the kit of aspect 164, wherein the one or more buffers comprise sequencing reagents.

[0230] Aspect 166 is the kit of aspect 165, wherein the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0231] Aspect 166.1 is the kit of any one of aspects 130-166, comprising the one or more components in one or more suitable containers.

[0232] Aspect 167 is a composition comprising a mixture of two or more sulfites, wherein the final concentration of bisulfite in the composition is between 4 M and 12 M, and the pH value is between 4.0 to 6.5.

[0233] Aspect 168 is the composition of aspect 167, comprising ammonium bisulfite, sodium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the final concentration of Trolox is between 0.1% and 4% w / v.

[0234] Aspect 169 is the composition of aspect 167 or 168, wherein the bisulfite concentration is greater than or equal to 6 M.298000704.1- 47 -ARCD.P0841WO / 24-T-190

[0235] Aspect 170 is the composition of any one of aspects 167-169, wherein the final concentration of bisulfite in the composition is between about 7 M and 8 M, and the pH value is between about 4.0 to 6.5.

[0236] Aspect 171 is the composition of any one of aspects 167-170, wherein the first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5 also known as sodium pyrosulfite or disodium metabisulfite), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) or ammonium sulfite monohydrate ((NH4)2SO3•H2O).

[0237] Aspect 172 is the composition of aspect 171, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 760 to 950 mg per 1000 μl water, and / or about 4 M to 5 M.

[0238] Aspect 173 is the composition of aspect 171 or 172, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 840 mg per 1000 μl water, and / or about 4.42 M.

[0239] Aspect 174 is the composition of aspect 171 or 172, wherein the ammonium sulfite is hydrated as ammonium sulfite monohydrate ((NH4)2SO3•H2O).

[0240] Aspect 175 is the composition of aspect 174, wherein the ammonium sulfite monohydrate is at a concentration of about 60 to 100 mg per 1000 μl water, and / or about 447 mM to 746.3 mM.

[0241] Aspect 176 is the composition of aspect 174 or 175, wherein the ammonium sulfite monohydrate is at a concentration of about 80 mg per 1000 μl water, and / or about 597 mM.

[0242] Aspect 177 is the composition of any one of aspects 167-176, wherein the bisulfite concentration is about 7.2 M to 7.5 M, or about 7.37 M.

[0243] Aspect 178 is the composition of any one of aspect 167-177, further comprising a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

[0244] Aspect 179 is the composition of any one of aspects 167-178, further comprising nucleic acids.

[0245] Aspect 180 is the composition of aspect 179, wherein the nucleic acid comprises ribonucleic acid (RNA).

[0246] Aspect 181 is the composition of aspect 180, wherein the RNA comprises m5C modifications.

[0247] Aspect 182 is the composition of aspect 179 or 180, wherein the RNA comprises m5C modifications and C-to-U (cytosine to uracil) conversions.298000704.1- 48 -ARCD.P0841WO / 24-T-190

[0248] Aspect 183 is the composition of aspect 182, wherein greater than 90%, 95%, 96%, 97%, 98%, or 99% of the RNA cytosines are converted to uracil.

[0249] Aspect 184 is the composition of any one of aspects 179-184, wherein the RNA comprises a low level of RNA damage.

[0250] Aspect 185 is the composition of aspect 184, wherein the damage is assessed as RNA fragmentation, optionally measured using gel electrophoresis and / or a fragment analyzer.

[0251] Aspect 186 is a method of detecting m5C modifications in RNA, the method comprising use of the composition according to any one of aspects 167-185.

[0252] Aspect 187 is a method of detecting m5C modifications in RNA, the method comprising sequencing the RNA of the composition of any one of aspects 180-186.

[0253] Aspect 188 is a method for converting cytosine to uracil in a polynucleotide, the method comprising incubating a composition comprising a polynucleotide and a mixture of two or more sulfites, wherein the composition is a) at a bisulfite concentration of about 4-12 M, b) at a temperature between about 50 to 98 °C, and c) incubated between about 1-180 minutes.

[0254] Aspect 189 is the method of aspect 188, wherein the bisulfite concentration is greater than or equal to about 6 M.

[0255] Aspect 190 is the method of any one of aspects 188-189, wherein the first sulfite comprises ammonium bisulfite, the second sulfite comprises sodium bisulfite, and the composition further comprises KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the final concentration of Trolox is between 0.1% and 4% w / v.

[0256] Aspect 191 is the method of any one of aspects 188-190, wherein the bisulfite concentration is between about 7 to 8 M, and the temperature is between about 70 to 98 °C.

[0257] Aspect 192 is the method of any one of aspects 188-191, wherein the first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) and / or ammonium sulfite monohydrate ((NH4)2SO3•H2O).

[0258] Aspect 193 is the method of aspect 192, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 760 to 950 mg per 1000 μl water, and / or about 4 M to 5 M.

[0259] Aspect 194 is the method of any one of aspects aspect 192-193, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 840 mg per 1000 μl water, and / or about 4.42 M.298000704.1- 49 -ARCD.P0841WO / 24-T-190

[0260] Aspect 195 is the method of any one of aspects 192-194, wherein the ammonium sulfite monohydrate is at a concentration of about 60 to 100 mg per 1000 μl water, and / or about 447 mM to 746.3 mM.

[0261] Aspect 196 is the method of any one of aspects 192-195, wherein the ammonium sulfite monohydrate is at a concentration of about 80 mg per 1000 μl water, and / or about 597 mM.

[0262] Aspect 197 is the method of any one of aspects 188-196, wherein the bisulfite concentration is about 7.2 M to 7.5 M, or about 7.37 M.

[0263] Aspect 198 is the method of any one of aspects 188-197, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0264] Aspect 199 is the method of any one of aspects 188-198, wherein the temperature is or is about 55 °C.

[0265] Aspect 200 is the method of any one of aspects 188-199, wherein the incubation time is, or is between any two of about 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes.

[0266] Aspect 201 is the method of any one of aspects 188-200, wherein the incubation time is 90 minutes.

[0267] Aspect 202 is the method of any one of aspects 188-201, wherein the polynucleotide is RNA.

[0268] Aspect 203 is the method of any one of aspects 188-202, wherein the polynucleotide comprises a low level of damage.

[0269] Aspect 204 is the method of aspect 203, wherein the damage is assessed as RNA fragmentation, optionally measured using gel electrophoresis and / or a fragment analyzer.

[0270] Aspect 205 is the method of any one of aspects 188-204, further comprising a denaturation step that occurs prior to the step of incubating a solution comprising a polynucleotide with bisulfite.

[0271] Aspect 206 is the method of any one of aspects 188-205, further comprising a desulphonation step that occurs after the step of incubating a solution comprising a polynucleotide with bisulfite.

[0272] Aspect 207 is a method of detecting 5-methylcytosine in a polynucleotide comprising sequencing the polynucleotide of the composition of any one of aspects 188-206, or a reverse transcribed copy thereof, after incubation and / or after desulphonation.

[0273] Aspect 208 is the method of aspect 207, wherein the polynucleotide comprises a total mass less than about 250, 200, 150, 100, or 50 nanograms.298000704.1- 50 -ARCD.P0841WO / 24-T-190

[0274] Aspect 209 is the method of aspect 208, wherein the polynucleotide comprises a total mass between about 50-200 nanograms.

[0275] Aspect 210 is the method of aspect 209, wherein the polynucleotide comprises a total mass about 50, 100, or 200 nanograms.

[0276] Aspect 211 is the method of any one of aspects 188-210, wherein the C-to-U unconverted ratio is less than about 0.4%.

[0277] Aspect 212 is the method of any one of aspects 188-211, wherein the C-to-U unconverted ratio is about 0.4%, or 0.3%.

[0278] Aspect 213 is a polynucleotide processing kit comprising: a) two or more sulfites; and b) optionally instructions for processing a polynucleotide sample.

[0279] Aspect 214 is the kit of aspect 213, wherein the first sulfite comprises, consists essentially of, or consists of ammonium bisulfite, and the second sulfite comprises sodium bisulfite, and the kit further comprises KOH, tetrahydrofurfuryl alcohol, and Trolox.

[0280] Aspect 215 is the kit of aspect 213, wherein the first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) and / or ammonium sulfite monohydrate ((NH4)2SO3•H2O).

[0281] Aspect 216 is the kit of any one of aspects 213-215, wherein the first and second sulfites are provided separately.

[0282] Aspect 217 is the kit of any one of aspects 214-216, wherein the sodium metabisulfite (Na2S2O5) is provided at a concentration of about 760 to 950 mg per 1000 μl water, and / or about 4 M to 5 M.

[0283] Aspect 218 is the kit of any one of aspects 214-217, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 840 mg per 1000 μl water, and / or about 4.42 M.

[0284] Aspect 219 is the kit of any one of aspects 214-218, wherein the ammonium sulfite monohydrate is at a concentration of about 60 to 100 mg per 1000 μl water, and / or about 447 mM to 746.3 mM.

[0285] Aspect 220 is the kit of any one of aspects 214-219, wherein the ammonium sulfite monohydrate is at a concentration of about 80 mg per 1000 μl water, and / or about 597 mM.

[0286] Aspect 221 is the kit of any one of aspects 213-220, wherein the first and second sulfites are provided as a premixed solution.

[0287] Aspect 222 is the kit of aspect 221, wherein the premixed solution comprises a bisulfite concentration between about 7 M to 8 M.298000704.1- 51 -ARCD.P0841WO / 24-T-190

[0288] Aspect 223 is the kit of aspect 222, wherein the premixed solution comprises a bisulfite concentration of about 7.2 M to 7.5 M, or about 7.37 M.

[0289] Aspect 224 is the kit of any one of aspects 213-223, wherein the instructions comprise instructions for incubating the polynucleotide sample with the first and second components or the premixed solution at a temperature between about 50-98 °C for a time between 1-180 minutes.

[0290] Aspect 225 is the kit of aspect 224, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0291] Aspect 226 is the kit of any one of aspects 224-225, wherein the temperature is about 80, or 85 °C.

[0292] Aspect 227 is the kit of any one of aspects 224-226, wherein the time is 5, 8, 10, 15, 20, 25, 30, 35, 80, 85, 90, 95, or 100 minutes.

[0293] Aspect 2280 is the kit of any one of aspects 213-227, further comprising a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, and / or an antioxidant solution.

[0294] Aspect 229 is the kit of aspect 228, wherein one or more of the solutions are provided separately.

[0295] Aspect 230 is the kit of aspect 228, wherein one or more of the solutions are mixed and provided as a premixed solution.

[0296] Aspect 231 is the kit of any one of aspects 213-227, further comprising one or more buffer solutions and / or enzymes.

[0297] Aspect 232 is the kit of aspect 231, wherein the one or more buffers comprise sequencing reagents.

[0298] Aspect 233 is the kit of aspect 232, wherein the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0299] Aspect 233.1 is the kit of any one of aspects 213-233, comprising the one or more components in one or more suitable containers.

[0300] Aspect 234 is a composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 and 10 M, and the pH of the composition is between 6.0 and 8.5.

[0301] Aspect 235 is the composition of aspect 234, wherein the concentration of bisulfite in the composition is between 4 and 6 M.298000704.1- 52 -ARCD.P0841WO / 24-T-190

[0302] Aspect 236 is the composition of aspect 234 or 235, wherein the bisulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0303] Aspect 237 is the composition of aspect 236, wherein the sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, and / or potassium metabisulfite (K2S2O5) and water.

[0304] Aspect 238 is the composition of any one of aspects 236-237, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1.66 M to 2.46 M.

[0305] Aspect 239 is the composition of any one of aspects 236-238, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 2.06 M.

[0306] Aspect 240 is the composition of any one of aspects 236-239, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 1.76 M to 2.71 M.

[0307] Aspect 241 is the composition of any one of aspects 236-240, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.31 M.

[0308] Aspect 242 is the composition of any one of aspects 236-241, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.16 M.

[0309] Aspect 243 is the composition of any one of aspects 236-242, wherein the composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0310] Aspect 244 is the composition of any one of aspects 234-243, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0311] Aspect 245 is the composition of aspect 244, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.298000704.1- 53 -ARCD.P0841WO / 24-T-190

[0312] Aspect 246 is the composition of any one of aspects 234-245, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0313] Aspect 247 is the composition of any one of aspects 234-246, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0314] Aspect 248 is the composition of any one of aspects 244-247, wherein the base or acid solution comprises a potassium hydroxide (KOH) comprising a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0315] Aspect 249 is the composition of any one of aspects 244-248, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 20- 30% or about 26% total volume of the composition.

[0316] Aspect 250 is the composition of any one of aspects 244-248, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 25- 35% or about 31% total volume of the composition.

[0317] Aspect 251 is the composition of any one of aspects 244-248, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 36 to 46% or about 42% total volume of the composition.

[0318] Aspect 252 is the composition of any one of aspects 234-251, wherein the composition comprises a concentration of bisulfite between about 4 and 5.5 M.

[0319] Aspect 253 is the composition of any one of aspects 234-252, wherein the composition comprises a concentration of bisulfite of about 4.3 M.

[0320] Aspect 254 is the composition of any one of aspects 234-252, wherein the composition comprises a concentration of bisulfite of about 5.4 M.

[0321] Aspect 255 is the composition of any one of aspects 234-252, wherein the composition comprises a concentration of bisulfite of about 5.1 M.

[0322] Aspect 256 is the composition of any one of aspects 234-255, wherein the pH of the composition is between about 7 and 7.5.

[0323] Aspect 257 is the composition of any one of aspects 234-256, wherein the pH of the composition is about 7.0.

[0324] Aspect 258 is the composition of any one of aspects 234-256, wherein the pH of the composition is about 7.4.

[0325] Aspect 259 is the composition of any one of aspect 234-258, further comprising a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.298000704.1- 54 -ARCD.P0841WO / 24-T-190

[0326] Aspect 260 is the composition of any one of aspects 234-259, wherein the composition is clear at or below room temperature and / or has little-to-no precipitation.

[0327] Aspect 261 is the composition of any one of aspects 234-260, wherein the composition is clear and / or substantially free of precipitation at about 1-7 °C.

[0328] Aspect 262 is the composition of any one of aspects 234-261, wherein the composition is clear and / or substantially free of precipitation at about 4 °C.

[0329] Aspect 263 is the composition of any one of aspects 234-262, wherein the composition does not comprise ammonium salt.

[0330] Aspect 264 is the composition of any one of aspects 234-263, further comprising nucleic acids.

[0331] Aspect 265 is the composition of aspect 264, wherein the nucleic acid comprises ribonucleic acid (RNA).

[0332] Aspect 266 is the composition of aspect 265, wherein the RNA comprises pseudouridine.

[0333] Aspect 267 is the composition of aspect 265 or 266, wherein the RNA comprises pseudouridine (Ψ) modifications and does not comprise appreciable levels of C-to-U (cytosine to uracil) conversions.

[0334] Aspect 268 is the composition of any one of aspects 265-266, wherein the RNA comprises less than 5%, 4%, 3%, 2%, 1%, or 0.5% C-to-U (cytosine to uracil) conversions.

[0335] Aspect 269 is the composition of aspect any one of aspects 266-268, wherein greater than 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%, or 99% of the Ψ are converted to Ψ-BS.

[0336] Aspect 270 is the composition of any one of aspects 265-269, wherein the RNA comprises a low level of RNA damage.

[0337] Aspect 271 is the composition of aspect 270, wherein the damage is assessed as RNA fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

[0338] Aspect 272 is the composition of any one of aspects 265-271, wherein there is between about 5 ng and 50 ng, or 5 ng and 15 ng of RNA in the composition.

[0339] Aspect 273 is a method of detecting Ψ modifications in RNA, the method comprising use of the composition according to any one of aspects 234-272.298000704.1- 55 -ARCD.P0841WO / 24-T-190

[0340] Aspect 274 is a method of detecting Ψ modifications in RNA, the method comprising sequencing the RNA of the composition of any one of aspects 265-273 or a reverse transcribed copy thereof, after a period of time of incubating the composition.

[0341] Aspect 275 is a method for converting Ψ to Ψ-BS in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein a) the bisulfite concentration is about 4 M to 10 M, b) the pH is between about 6.0 to 8.5, c) the temperature is between about 50 to 98 °C, and d) the incubation is between about 1-180 minutes.

[0342] Aspect 276 is the method of aspect 275, wherein the bisulfite concentration is about 4 M to 6 M, and the temperature is between about 75 to 98 °C.

[0343] Aspect 277 is the method of aspect 275 or 276, wherein the sulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0344] Aspect 278 is the method of any one of aspects 275-277, wherein the sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, and / or potassium metabisulfite (K2S2O5) and water.

[0345] Aspect 279 is the method of any one of aspects 275-278, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1.66 M to 2.46 M.

[0346] Aspect 280 is the method of any one of aspects 275-279, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 2.06 M.

[0347] Aspect 281 is the method of any one of aspects 275-280, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 1.76 M to 2.71 M.

[0348] Aspect 282 is the method of any one of aspects 275-281, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.31 M.

[0349] Aspect 283 is the method of any one of aspects 275-282, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.16 M.

[0350] Aspect 284 is the method of anyone of aspects 275-283, wherein the composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added potassium bisulfite298000704.1- 56 -ARCD.P0841WO / 24-T-190 (KHSO3), added sodium sulfite (Na2SO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0351] Aspect 285 is the method of any one of aspects 275-284, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0352] Aspect 286 is the method of aspect 285, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0353] Aspect 287 is the method of any one of aspects 275-286, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0354] Aspect 288 is the method of any one of aspects 275-287, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0355] Aspect 289 is the method of any one of aspects 275-288, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0356] Aspect 290 is the method of any one of aspects 275-289, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 20- 30% or about 26% total volume of the composition.

[0357] Aspect 291 is the method of any one of aspects 275-290, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 25- 35% or about 31% total volume of the composition.

[0358] Aspect 292 is the method of any one of aspects 275-291, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 36 to 46% or about 42% total volume of the composition.

[0359] Aspect 293 is the method of any one of aspects 275-292, wherein the composition comprises a concentration of bisulfite between about 4 and 5.5 M.

[0360] Aspect 294 is the method of any one of aspects 275-293, wherein the composition comprises a concentration of bisulfite of about 4.3 M.

[0361] Aspect 295 is the method of any one of aspects 275-293, wherein the composition comprises a concentration of bisulfite of about 5.4 M.298000704.1- 57 -ARCD.P0841WO / 24-T-190

[0362] Aspect 296 is the method of any one of aspects 275-293, wherein the composition comprises a concentration of bisulfite of about 5.1 M.

[0363] Aspect 297 is the method of any one of aspects 275-296, wherein the pH of the composition is between about 7 and 7.5.

[0364] Aspect 298 is the method of any one of aspects 275-297, wherein the pH of the composition is about 7.0.

[0365] Aspect 299 is the method of any one of aspects 275-297, wherein the pH of the composition is about 7.4.

[0366] Aspect 300 is the method of any one of aspects 275-299, wherein the composition further comprises a bisulfite stabilizer, a single stranded nucleic acid stabilizer, and / or an antioxidant.

[0367] Aspect 301 is the method of any one of aspects 275-300, wherein the composition is clear at or below room temperature and / or has little-to-no precipitation.

[0368] Aspect 302 is the method of any one of aspects 275-301, wherein the composition is clear and / or is substantially free of precipitate at about 1-7 °C.

[0369] Aspect 303 is the method of any one of aspects 275-302, wherein the composition is clear and / or is substantially free of precipitate at about 4 °C.

[0370] Aspect 303 is the method of any one of aspects 275-303, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0371] Aspect 305 is the method of any one of aspects 275-304, wherein the incubation time is 3, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes.

[0372] Aspect 306 is the method of any one of aspects 275-305, wherein the incubation time is 6, 8, 10, or 15 minutes.

[0373] Aspect 307 is the method of anyone of aspects 275-306, wherein the RNA polynucleotide comprises a low level of damage, optionally wherein the damage is measured as fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

[0374] Aspect 308 is the method of anyone of aspects 275-307, wherein the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition comprising an ammonium salt, optionally wherein the damage is measured as fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

[0375] Aspect 309 is the method of any one of aspects 275-308, further comprising a denaturation step that occurs prior to the step of incubating a composition comprising a polynucleotide with bisulfite.298000704.1- 58 -ARCD.P0841WO / 24-T-190

[0376] Aspect 310 is the method of any one of aspects 275-309, further comprising a desulphonation step that occurs after the step of incubating a composition comprising a polynucleotide with bisulfite.

[0377] Aspect 311 is the method of any one of aspects 307-310, wherein the damage is assessed as RNA fragmentation.

[0378] Aspect 312 is the method of anyone of aspects 275-311, wherein the method comprises less than about 5% C-to-U conversion.

[0379] Aspect 313 is the method of anyone of aspects 275-312, wherein the method comprises less than about 0.5% C-to-U conversion.

[0380] Aspect 314 is the method of anyone of aspects 275-313, wherein about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80-100% of the Ψ are converted to Ψ-BS.

[0381] Aspect 315 is the method of anyone of aspects 275-313, wherein the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or 5 ng and 15 ng in the composition.

[0382] Aspect 316 is a polynucleotide processing kit comprising: a) a first component comprising a mixture of a sulfite solution; b) a second component comprising an acid or base solution; and c) optionally instructions for processing a polynucleotide sample.

[0383] Aspect 317 is the kit of aspect 316, wherein the sulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0384] Aspect 318 is the kit of aspect 316 or 317, wherein the sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water.

[0385] Aspect 319 is the kit of any one of aspects 316-318, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1.66 M to 2.46 M.

[0386] Aspect 320 is the kit of any one of aspects 316-319, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 2.06 M.

[0387] Aspect 321 is the kit of any one of aspects 316-320, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 1.76 M to 2.71 M.298000704.1- 59 -ARCD.P0841WO / 24-T-190

[0388] Aspect 322 is the kit of any one of aspects 316-321, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.31 M.

[0389] Aspect 323 is the kit of any one of aspects 316-321, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 2.16 M.

[0390] Aspect 324 is the kit of anyone of aspects 316-323, wherein the composition does not comprise added ammonium bisulfite (NH4HSO3), added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added potassium bisulfite (KHSO3), added sodium sulfite (Na2SO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0391] Aspect 325 is the kit of any one of aspects 316-324, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0392] Aspect 326 is the kit of aspect 325, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0393] Aspect 327 is the kit of any one of aspects 316-326, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0394] Aspect 328 is the kit of any one of aspects 316-327, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0395] Aspect 329 is the kit of any one of aspects 316-328, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0396] Aspect 330 is the kit of any one of aspects 316-329, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 20- 30% or about 26% total volume of the composition.

[0397] Aspect 331 is the kit of any one of aspects 316-330, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 25- 35% or about 31% total volume of the composition.298000704.1- 60 -ARCD.P0841WO / 24-T-190

[0398] Aspect 332 is the kit of any one of aspects 316-331, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 36 to 46% or about 42% total volume of the composition.

[0399] Aspect 333 is the kit of aspect 316-332, wherein the first and second components are provided separately.

[0400] Aspect 334 is the kit of any one of aspects 316-332, wherein the first and second components are provided as a premixed solution.

[0401] Aspect 335 is the kit of aspect 334, wherein the premixed solution comprises a concentration of bisulfite between about 4 and 5.5 M.

[0402] Aspect 336 is the kit of aspect 334 or 335, wherein the premixed solution comprises a concentration of bisulfite of about 4.3 M.

[0403] Aspect 337 is the kit of any one of aspects 334-335, wherein the premixed solution comprises a concentration of bisulfite of about 5.4 M.

[0404] Aspect 338 is the kit of any one of aspects 334-335, wherein the premixed solution comprises a concentration of bisulfite of about 5.1 M.

[0405] Aspect 339 is the kit of any one of aspects 334-338, wherein the pH of the premixed solution is between about 7 and 7.5.

[0406] Aspect 340 is the kit of any one of aspects 334-339, wherein the pH of the premixed solution is about 7.0.

[0407] Aspect 341 is the kit of any one of aspects 334-339, wherein the pH of the premixed solution is about 7.4.

[0408] Aspect 342 is the kit of any one of aspects 334-341, further comprising a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, and / or an antioxidant solution.

[0409] Aspect 343 is the kit of aspect 342, wherein one or more of the solutions are provided separately.

[0410] Aspect 344 is the kit of aspect 342, wherein one or more of the solutions are mixed and provided as a premixed solution.

[0411] Aspect 345 is the kit of any one of aspects 334-344, wherein the premixed solution is clear at or below room temperature and / or has little-to-no precipitation.

[0412] Aspect 346 is the kit of any one of aspects 334-345, wherein the premixed solution is clear or is substantially free of precipitate at about 1-7 °C.

[0413] Aspect 347 is the kit of any one of aspects 334-346, wherein the premixed solution is clear or is substantially free of precipitate at about 4 °C.298000704.1- 61 -ARCD.P0841WO / 24-T-190

[0414] Aspect 348 is the kit of any one of aspects 316-347, wherein the instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at a temperature between about 50-95 °C for a time between 1-180 minutes.

[0415] Aspect 349 is the kit of aspect 348, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0416] Aspect 350 is the kit of aspect 348 or 349, wherein the time is 3, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes.

[0417] Aspect 351 is the kit of any one of aspects 316-350, further comprising one or more buffer solutions and / or enzymes.

[0418] Aspect 352 is the kit of aspect 351, wherein the one or more buffers comprise sequencing reagents.

[0419] Aspect 353 is the kit of aspect 352, wherein the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0420] Aspect 353.1 is the kit of any one of aspects 316-353, comprising the one or more components in one or more suitable containers.

[0421] Aspect 354 is a composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between about 3 M and 6 M, and the pH of the composition is between about 5.0 and 6.5

[0422] Aspect 355 is the composition of aspect 354, wherein the concentration of bisulfite is between about 5 M and 6 M, and the pH is between about 5.5 and 6.1.

[0423] Aspect 356 is the composition of aspect 354 or 355, wherein the bisulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), potassium bisulfite (KHSO3), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0424] Aspect 357 is the composition of aspect 356, wherein the bisulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, or ammonium bisulfite (NH4HSO3) and water, or any combination thereof.298000704.1- 62 -ARCD.P0841WO / 24-T-190

[0425] Aspect 358 is the composition of any one of aspects 354-357, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1000 to 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M.

[0426] Aspect 359 is the composition of any one of aspects 354-358, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2000 μL of water, or about 3.42 M.

[0427] Aspect 360 is the composition of any one of aspects 354-359, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.6 M.

[0428] Aspect 361 is the composition of any one of aspects 354-360, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2354 μL of solvent, or about 2.9 M.

[0429] Aspect 362 is the composition of any one of aspects 354-361, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M.

[0430] Aspect 363 is the composition of any one of aspects 354-362, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 680 μL of water, or about 3.9 M.

[0431] Aspect 364 is the composition of any one of aspects 354-363, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M.

[0432] Aspect 365 is the composition of any one of aspects 354-364, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 720 μL of solvent, or about 3.75 M.

[0433] Aspect 366 is the composition of any one of aspects 354-365, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 30-80%, 40-80%, or 50-80% by w / v ammonium bisulfite.

[0434] Aspect 367 is the composition of any one of aspects 354-366, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 70%, 71%, or 72% by w / v ammonium bisulfite.

[0435] Aspect 368 is the composition of any one of aspects 354-367, wherein the composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite298000704.1- 63 -ARCD.P0841WO / 24-T-190 (KHSO3), added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0436] Aspect 369 is the composition of any one of aspects 354-368, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0437] Aspect 370 is the composition of aspect 369, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0438] Aspect 371 is the composition of any one of aspects 354-370, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0439] Aspect 372 is the composition of any one of aspects 354-371, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0440] Aspect 373 is the composition of any one of aspects 369-372, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0441] Aspect 374 is the composition of any one of aspects 369-373, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10-20 M, and comprises about 5-18% or about 17% total volume of the composition.

[0442] Aspect 375 is the composition of any one of aspects 369-373, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 5% total volume of the composition.

[0443] Aspect 376 is the composition of any one of aspects 369-373, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 16 to 18% or about 17% total volume of the composition.

[0444] Aspect 377 is the composition of any one of aspects 354-376, wherein the composition comprises a concentration of bisulfite between about 4 and 5.5 M.

[0445] Aspect 378 is the composition of any one of aspects 354-377, wherein the composition comprises a concentration of bisulfite of about 4.3 M.

[0446] Aspect 379 is the composition of any one of aspects 354-377, wherein the composition comprises a concentration of bisulfite of about 5.4 M.298000704.1- 64 -ARCD.P0841WO / 24-T-190

[0447] Aspect 380 is the composition of any one of aspects 354-377, wherein the composition comprises a concentration of bisulfite of about 5.1 M.

[0448] Aspect 381 is the composition of any one of aspects 354-380, wherein the pH of the composition is between about 5.5 and 6.1.

[0449] Aspect 382 is the composition of any one of aspects 354-381, wherein the pH of the composition is about 5.6-5.8.

[0450] Aspect 383 is the composition of any one of aspects 354-382, wherein the pH of the composition is about 5.7.

[0451] Aspect 384 is the composition of any one of aspects 354-383, further comprising a bisulfite stabilizer, a single stranded nucleic acid stabilizer, and / or an antioxidant.

[0452] Aspect 385 is the composition of any one of aspects 354-384, wherein the composition is clear at or below room temperature and / or has little-to-no precipitation.

[0453] Aspect 386 is the composition of any one of aspects 354-385, wherein the composition is clear and / or is substantially free of precipitate at about 1-7 °C.

[0454] Aspect 387 is the composition of any one of aspects 354-386, wherein the composition is clear and / or is substantially free of precipitate at about 4 °C.

[0455] Aspect 388 is the composition of any one of aspects 354-387, wherein the composition does not comprise ammonium salt.

[0456] Aspect 389 is the composition of any one of aspects 354-388, further comprising nucleic acids.

[0457] Aspect 390 is the composition of aspect 389, wherein the nucleic acid comprises ribonucleic acid (RNA).

[0458] Aspect 391 is the composition of aspect 390, wherein the RNA comprises pseudouridine and m5C.

[0459] Aspect 392 is the composition of aspect 390 or 391, wherein the RNA comprises pseudouridine (Ψ) modifications and also comprises appreciable levels of C-to-U (cytosine to uracil) conversions.

[0460] Aspect 393 is the composition of any one of aspects 390-392, wherein the C-to-U unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%.

[0461] Aspect 394 is the composition of aspect any one of aspects 390-393, wherein greater than 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%,298000704.1- 65 -ARCD.P0841WO / 24-T-190 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the Ψ are converted to Ψ-BS.

[0462] Aspect 395 is the composition of any one of aspects 390-394, wherein the RNA comprises a low level of RNA damage.

[0463] Aspect 396 is the composition of aspect 395, wherein the damage is assessed as RNA fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

[0464] Aspect 397 is the composition of any one of aspects 390-396, wherein there is between about 5 ng and 50 ng, or 5 ng and 15 ng of RNA in the composition.

[0465] Aspect 398 is a method of detecting Ψ and m5C modifications in RNA simultaneously, the method comprising use of the composition according to any one of aspects 354-397.

[0466] Aspect 399 is a method of detecting Ψ and m5C modifications in RNA simultaneously, the method comprising sequencing the RNA of the composition of any one of aspects 390-397 or a reverse transcribed copy thereof, after a period of time of incubating the composition.

[0467] Aspect 400 is a method for converting Ψ to Ψ-BS and C to U in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein a) the bisulfite concentration is about 4 M to 6 M, b) the pH is about 5-6.1, c) the temperature is between about 50 to 98 °C, and d) the incubation is between about 1-180 minutes.

[0468] Aspect 401 is the method of aspect 400, wherein the temperature is between about 65 to 98 °C.

[0469] Aspect 402 is the method of aspect 400 or 401, wherein the sulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

[0470] Aspect 403 is the method of any one of aspects 400-402, wherein the sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, and / or ammonium bisulfite (NH4HSO3) and water.298000704.1- 66 -ARCD.P0841WO / 24-T-190

[0471] Aspect 404 is the method of any one of aspects 400-403, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about of about 1000 to 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M.

[0472] Aspect 405 is the method of any one of aspects 400-404, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2000 μL of water, or about 3.42 M.

[0473] Aspect 406 is the method of any one of aspects 400-405, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about of about 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.36 M.

[0474] Aspect 407 is the method of any one of aspects 400-406, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2354 μL of solvent, or about 2.9 M.

[0475] Aspect 408 is the method of any one of aspects 400-407, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M.

[0476] Aspect 409 is the method of any one of aspects 400-408, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 680 μL of water, or about 3.97 M.

[0477] Aspect 410 is the method of any one of aspects 400-409, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M.

[0478] Aspect 411 is the method of any one of aspects 400-410, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 720 μL of solvent, or about 3.75 M.

[0479] Aspect 412 is the method of any one of aspects 400-411, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 30-80%, 40-80%, or 50-80% by w / v ammonium bisulfite.

[0480] Aspect 413 is the method of any one of aspects 400-412, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 70%, 71%, or 72% by w / v ammonium bisulfite.

[0481] Aspect 414 is the method of anyone of aspects 400-413, wherein the composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added potassium bisulfite (KHSO3), added sodium sulfite (Na2SO3),298000704.1- 67 -ARCD.P0841WO / 24-T-190 added guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0482] Aspect 415 is the method of any one of aspects 400-414, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0483] Aspect 416 is the composition of aspect 415, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0484] Aspect 417 is the method of any one of aspects 400-416, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0485] Aspect 418 is the method of any one of aspects 400-417, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0486] Aspect 419 is the method of any one of aspects 400-418, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0487] Aspect 420 is the method of any one of aspects 400-419, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10-20 M, and comprises about 5-18% or about 17% total volume of the composition.

[0488] Aspect 421 is the method of any one of aspects 400-420, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 5% total volume of the composition.

[0489] Aspect 422 is the method of any one of aspects 400-421, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 16 to 18% or about 17% total volume of the composition.

[0490] Aspect 423 is the method of any one of aspects 400-422, wherein the composition comprises a concentration of bisulfite between about 4 and 5.5 M.

[0491] Aspect 424 is the method of any one of aspects 400-423, wherein the composition comprises a concentration of bisulfite of about 4.3 M.

[0492] Aspect 425 is the method of any one of aspects 400-423, wherein the composition comprises a concentration of bisulfite of about 5.4 M.298000704.1- 68 -ARCD.P0841WO / 24-T-190

[0493] Aspect 426 is the method of any one of aspects 400-423, wherein the composition comprises a concentration of bisulfite of about 5.1 M.

[0494] Aspect 427 is the method of any one of aspects 400-426, wherein the pH of the composition is between about 5.5 and 6.1.

[0495] Aspect 428 is the method of any one of aspects 400-427, wherein the pH of the composition is about 5.6-5.8.

[0496] Aspect 429 is the method of any one of aspects 400-428, wherein the pH of the composition is about 5.7.

[0497] Aspect 430 is the method of any one of aspects 400-429, wherein the composition further comprises a bisulfite stabilizer, a single stranded nucleic acid stabilizer, and / or an antioxidant.

[0498] Aspect 431 is the method of any one of aspects 400-430, wherein the composition is clear at or below room temperature and / or has little-to-no precipitation.

[0499] Aspect 432 is the method of any one of aspects 400-431, wherein the composition is clear and / or is substantially free of precipitate at about 1-7 °C.

[0500] Aspect 433 is the method of any one of aspects 400-432, wherein the composition is clear and / or is substantially free of precipitate at about 4 °C.

[0501] Aspect 434 is the method of any one of aspects 400-433, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0502] Aspect 435 is the method of any one of aspects 400-434, wherein the incubation time is about 3, 6, 7, 7.5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes.

[0503] Aspect 436 is the method of any one of aspects 400-435, wherein the incubation time is about 6, 8, 10, or 15 minutes.

[0504] Aspect 437 is the method of anyone of aspects 400-436, wherein the RNA polynucleotide comprises a low level of damage.

[0505] Aspect 438 is the method of anyone of aspects 400-437, wherein the RNA polynucleotide comprises a low level of damage compared to a method comprising the use of a composition comprising an ammonium salt.

[0506] Aspect 439 is the method of aspect 437 or 438, wherein the damage is assessed as RNA fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

[0507] Aspect 440 is the method of anyone of aspects 400-439, wherein the wherein the C-to-U unconverted rate is less than about 5%, 4%, 3%, 2%, 1%, or 0.5%.298000704.1- 69 -ARCD.P0841WO / 24-T-190

[0508] Aspect 441 is the method of anyone of aspects 400-440, wherein the C-to-U unconverted rate is less than about 3%.

[0509] Aspect 442 is the method of any one of aspects 400-441, wherein greater than 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%, or 99% of the Ψ are converted to Ψ-BS.

[0510] Aspect 443 is the method of anyone of aspects 400-442, wherein about 20% to 40%, or about 30% to 50%, or about 40-60%, or about 50-70%, or about 60-80%, or about 70-90%, or about 80-100% of the Ψ are converted to Ψ-BS.

[0511] Aspect 444 is the method of anyone of aspects 400-443, wherein the RNA polynucleotide comprises a total mass between about 5 ng and 50 ng, or 5 ng and 15 ng in the composition.

[0512] Aspect 445 is the method of any one of aspects 400-444, further comprising a denaturation step that occurs prior to the step of incubating a composition comprising a polynucleotide with bisulfite.

[0513] Aspect 446 is the method of any one of aspects 400-445, further comprising a desulphonation step that occurs after the step of incubating a composition comprising a polynucleotide with sulfite.

[0514] Aspect 447 is a polynucleotide processing kit comprising: a) a first component comprising a mixture of a sulfite solution; b) a second component comprising an acid or base solution; and c) optionally instructions for processing a polynucleotide sample.

[0515] Aspect 448 is the kit of aspect 447, wherein the sulfite solution comprises potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium sulfite (Na2SO3), potassium sulfite (K2SO3), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof

[0516] Aspect 449 is the kit of aspect 447 or 448, wherein the sulfite solution consists essentially of, or consists of sodium metabisulfite (Na2S2O5) and water, or potassium metabisulfite (K2S2O5) and water, and / or ammonium bisulfite (NH4HSO3) and water.298000704.1- 70 -ARCD.P0841WO / 24-T-190

[0517] Aspect 450 is the kit of any one of aspects 447-449, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1000 to 1500 mg per 2000 μL of water, or about 2.63 M to 3.95 M.

[0518] Aspect 451 is the kit of any one of aspects 447-450, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2000 μL of water, or about 3.42 M.

[0519] Aspect 452 is the kit of any one of aspects 447-451, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1000 to 1500 mg per 2354 μL of solvent, or about 2.23 M to 3.36 M.

[0520] Aspect 453 is the kit of any one of aspects 447-452, wherein the sodium metabisulfite (Na2S2O5) is at a concentration of about 1300 mg per 2354 μL of solvent, or about 2.9 M.

[0521] Aspect 454 is the kit of any one of aspects 447-453, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 680 μL of water, or about 3.64 M to 4.3 M.

[0522] Aspect 455 is the kit of any one of aspects 447-454, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 680 μL of water, or about 3.97 M.

[0523] Aspect 456 is the kit of any one of aspects 447-455, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 550 to 650 mg per 720 μL of solvent, or about 3.44 M to 4.06 M.

[0524] Aspect 457 is the kit of any one of aspects 447-456, wherein the potassium metabisulfite (K2S2O5) is at a concentration of about 600 mg per 720 μL of solvent, or about 3.75 M.

[0525] Aspect 458 is the composition of any one of aspects 447-457, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 30-80%, 40-80%, or 50-80% by w / v ammonium bisulfite.

[0526] Aspect 459 is the composition of any one of aspects 447-458, wherein the ammonium bisulfite (NH4HSO3) solution comprises about 70%, 71%, or 72% by w / v ammonium bisulfite.

[0527] Aspect 460 is the kit of anyone of aspects 447-458, wherein the composition does not comprise, added ammonium sulfite ((NH4)2SO3), added ammonium sulfite monohydrate ((NH4)2SO3•H2O), added potassium metabisulfite (K2S2O5), added sodium metabisulfite (Na2S2O5), added sodium sulfite (Na2SO3), added potassium bisulfite (KHSO3), added298000704.1- 71 -ARCD.P0841WO / 24-T-190 guanidinium bisulfite, added guanidinium hydrogen sulfite, added guanidinium ammonium bisulfite, and / or added potassium sulfite (K2SO3).

[0528] Aspect 461 is the kit of any one of aspects 447-460, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), sodium sulfite (Na2SO3), ammonium bisulfite (NH4HSO3), sodium bisulfite (NaHSO3), concentrated hydrochloric acid (HCl), lithium hydroxide (LiOH), ammonium hydroxide (NH4OH), and / or sodium hydroxide (NaOH).

[0529] Aspect 462 is the composition of aspect 461, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.5.

[0530] Aspect 463 is the method of any one of aspects 447-462, wherein the base or acid solution consists essentially of, or consists of potassium hydroxide (KOH) and water.

[0531] Aspect 464 is the kit of any one of aspects 447-463, wherein the composition does not comprise a base or acid solution comprising added sulfuric acid (H2SO4), added sodium sulfite (Na2SO3), added ammonium bisulfite (NH4HSO3), added sodium bisulfite (NaHSO3), added concentrated hydrochloric acid (HCl), or added sodium hydroxide (NaOH).

[0532] Aspect 465 is the kit of any one of aspects 447-464, wherein the potassium hydroxide (KOH) solution comprises a concentration between about 5-15 M, about 10-30 M, about 15-25 M, about 15-20 M, about 20 M, or about 10 M.

[0533] Aspect 466 is the kit of any one of aspects 447-465, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10-20 M, and comprises about 5-18% or about 17% total volume of the composition.

[0534] Aspect 467 is the kit of any one of aspects 447-466, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 20 M, and comprises about 5% total volume of the composition.

[0535] Aspect 468 is the kit of any one of aspects 447-467, wherein the potassium hydroxide (KOH) solution comprises a concentration of about 10 M, and comprises about 16 to 18% or about 17% total volume of the composition.

[0536] Aspect 469 is the kit of aspect 447-468, wherein the first and second components are provided separately.

[0537] Aspect 470 is the kit of any one of aspects 447-468, wherein the first and second components are provided as a premixed solution.

[0538] Aspect 471 is the kit of aspect 470, wherein the premixed solution comprises a concentration of bisulfite between about 4 and 5.5 M.298000704.1- 72 -ARCD.P0841WO / 24-T-190

[0539] Aspect 472 is the kit of any one of aspects 470-471, wherein the premixed solution comprises a concentration of bisulfite of about 4.3 M.

[0540] Aspect 473 is the kit of any one of aspects 470-471, wherein the premixed solution comprises a concentration of bisulfite of about 5.4 M.

[0541] Aspect 474 is the kit of any one of aspects 470-471, wherein the premixed solution comprises a concentration of bisulfite of about 5.1 M.

[0542] Aspect 475 is the kit of any one of aspects 470-474, wherein the pH of the premixed solution is between about 5 and 6.1.

[0543] Aspect 476 is the kit of any one of aspects 470-475, wherein the pH of the premixed solution is about 5.5 and 6.1.

[0544] Aspect 477 is the kit of any one of aspects 470-476, wherein the pH of the premixed solution is about 5.7, 5.8, 5.9, or 6.0.

[0545] Aspect 478 is the kit of any one of aspects 447-477, further comprising a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, and / or an antioxidant solution.

[0546] Aspect 479 is the kit of aspect 478, wherein one or more of the solutions are provided separately.

[0547] Aspect 480 is the kit of aspect 478, wherein one or more of the solutions are mixed and provided as a premixed solution.

[0548] Aspect 481 is the kit of any one of aspects 447-480, wherein the premixed solution is clear at or below room temperature and / or has little-to-no precipitation.

[0549] Aspect 482 is the kit of any one of aspects 447-481, wherein the premixed solution is clear or is substantially free of precipitate at about 1-7 °C.

[0550] Aspect 483 is the kit of any one of aspects 470-482, wherein the premixed solution is clear or is substantially free of precipitate at about 4 °C.

[0551] Aspect 484 is the kit of any one of aspects 447-481, wherein the instructions comprise instructions for incubating a polynucleotide sample with the first and second components or the premixed solution at a temperature between about 50-98 °C for a time between 1-180 minutes.

[0552] Aspect 485 is the kit of aspect 484, wherein the temperature is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 98 °C.

[0553] Aspect 486 is the kit of aspect 484 or 485, wherein the time is 3, 6, 7, 7.5, 8, 10, 15, 20, 25, 30, 35, 40, 45, 90, 120, or 150 minutes.298000704.1- 73 -ARCD.P0841WO / 24-T-190

[0554] Aspect 487 is the kit of any one of aspects 447-486, further comprising one or more buffer solutions and / or enzymes.

[0555] Aspect 488 is the kit of aspect 487, wherein the one or more buffers comprise sequencing reagents.

[0556] Aspect 489 is the kit of aspect 488, wherein the sequencing reagents comprise one or more primers, one or more dNTPs, one or more probes, one or more adaptors, one or more fluorophores, one or more enzymes, one or more polymerases, or a combination thereof.

[0557] Aspect 489.1 is the kit of any one of aspects 447-489, comprising the one or more components in one or more suitable containers.

[0558] Aspect 490 is a composition comprising a mixture of a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and optionally DNA.

[0559] Aspect 491 is a composition comprising a mixture of a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and optionally RNA.

[0560] Aspect 492 is a kit comprising a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, an about 3 M KOH solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, an antioxidant solution, and / or desulphonation beads or columns.

[0561] Aspect 493 is a composition comprising ammonium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the concentration of Trolox is between 0.1% and 4% w / v.

[0562] Aspect 494 is a composition comprising ammonium bisulfite, sodium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the concentration of Trolox is between 0.1% and 4% w / v.

[0563] Aspect 495 is the use of the composition, kit, or method of any one of the preceding aspects in diagnosing, identifying, and / or treating a disease and / or disorder in an individual.

[0564] Aspect 496 is the kit of any one of the preceding claims wherein the one or more solutions are comprised in one or more suitable containers.

[0565] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0566] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”298000704.1- 74 -ARCD.P0841WO / 24-T-190

[0567] The phrase “and / or” means “and” or “or”. To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and / or” operates as an inclusive or.

[0568] 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 additional, unrecited elements or method steps.

[0569] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of” any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of” any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed invention.

[0570] A person of ordinary skill in the art would understand that a solution that does not contain a particular chemical (e.g., ammonium sulfite, sodium bisulfite, etc.) does not contain an added quantity 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 minute amounts.

[0571] As understood by the skilled artisan, a solution of the present disclosure may be described in terms of molarity (M), percent by weight (also “weight percent”; w / w), or any other units. When described in terms of one unit (e.g., w / w) equivalent solutions expressed by other units (e.g., M) are also contemplated herein.

[0572] As utilized herein, “added” has a traditional meaning, to include, put in, or join, a component to another component, e.g., a chemical can be added to a composition or method. A component that is added can be described as being included in a form substantially as described through an active step. A component that is added may also occur without specific inclusion, for example, a component may come into existence through an in situ chemical reaction without being specifically added. A component that arises through a passive in situ chemical reaction is not considered added.

[0573] It is specifically contemplated that any limitation discussed with respect to one aspect of the invention may apply to any other aspect of the invention. Furthermore, 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 to utilize any composition of the invention. Any aspect298000704.1- 75 -ARCD.P0841WO / 24-T-190 discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0574] 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 the specific examples, while indicating specific aspects of the invention, are given by way of illustration only, since various changes and modifications 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

[0575] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The 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.

[0576] FIG. 1 shows results of matrix-assisted laser desorption / ionization (MALDI) time of flight (TOF) spectrometry for the conversion rate of cytosine (C) (peak at m / z ~1589) to C- bisulfite (peak at m / z ~1671) in a DNA DO-20-3 C-5mer test oligonucleotide using composition R7-4.9 (200 μL NH4HSO3 + 2 μL 20 M KOH; final pH 4.9; final bisulfite concentration ~9.8 M) at 90 °C for 1 minute (bottom row). The results showed that R7-4.9 produced higher C to C-bisulfite conversion than control composition A7 (70% ammonium bisulfite 710 μL + 50% ammonium bisulfite 71 μL; final bisulfite concentration ~9.65 M) at 90 °C for 1 minute (middle row). An untreated C-5mer DNA was added as a loading control (top row).

[0577] FIG. 2 shows results of MALDI-TOF spectrometry for the conversion rate of C to C-bisulfite in a DNA DO-20-3 C-5mer test oligonucleotide using composition R7-5.1 (200 μL NH4HSO3+ 5.5 μL 20 M KOH; final pH ~5.1; final bisulfite concentration ~9.8 M) at 70 °C for 3 minutes (bottom row) compared to composition A7 at 70 °C for 3 minutes (middle row). The results showed that R7-5.1 produced higher C to C-bisulfite conversion than control A7. An untreated C-5mer DNA was added as a loading control (top row).298000704.1- 76 -ARCD.P0841WO / 24-T-190

[0578] FIG. 3 shows results from MALDI-TOF spectrometry for the conversion rate of C to C-bisulfite in a DNA DO-20-3 C-5mer test oligonucleotide using composition R7-5.3 (200 μL NH4HSO3 + 9.5 μL 20 M KOH; final pH ~5.3; final bisulfite concentration ~9.8 M) at 37 °C for 30 minutes (bottom row) compared to composition A7 at 37 °C for 30 minutes (middle row). The results showed that R7-5.3 produced higher C to C-bisulfite conversion than control A7. An untreated C-5mer DNA was added as a loading control (top row).

[0579] FIG. 4 shows DNA damage assessment by gel electrophoresis of lambda DNA treated with A7, R7-4.9, R7-5.1, or R7-5.3, at 98 °C for 8 minutes. The results showed that R7- 4.9, R7-5.1, and R7-5.3 induced less DNA damage than A7. The results also indicated that higher pH induced less DNA damage.

[0580] FIG.5 shows the cytosine to uracil (C-to-U) conversion rate of lambda DNA using different compositions (A7, Zymo kit, R7-4.9, R7-5.1, and R6b, as separated by dashed vertical lines), at different temperatures (ranging from 37 °C to 98 °C, noted on X axis), and for different length of time (from 8 minutes to 3 hours, noted on X axis). The C-to-U conversion rate was assessed by next generation sequencing (Y axis). The results showed that the optimal temperature range for C-to-U conversion was between 70°C to 90 °C, under which the background noise is lower than A7 at 98 °C for 10 minutes. The results also showed that temperature higher than 90 °C or lower than 70 °C gave higher background noise. The results also indicated that longer incubation time can give higher conversion efficiency.

[0581] FIG. 6 shows results from a fragmentation assay of lambda DNA treated with various bisulfite compositions (ST = no bisulfite treatment; A7 = 98 °C, 10 min; Zymo = commercially available kit, used following manufacturer’s instructions; R7-4.9 (“C”): C1 = 98 °C, 8 min., C2 = 95 °C, 10 min, C3 = 90 °C, 10 min., C4 = 90 °C, 15 min., C5 = 85 °C, 15 min., C6 = 85 °C, 20 min., C7 = 85 °C, 25 min., C8 = 85 °C, 30 min., C9 = 80 °C, 25 min., C10 = 80 °C, 30 min., C11 = 80 °C, 35 min.; R7-5.1 (“D”): D1 = 75 °C, 30 min., D2 = 75 °C, 35 min., D3 = 75 °C, 40 min., D4 = 70 °C, 40 min., D5 = 70 °C, 45 min., D6 = 70 °C, 50 min.; R6b (“E”): E1 = 70 °C, 90 min., E2 = 70 °C, 120 min., E3 = 70 °C, 150 min.). All samples were denatured at 95 °C for 6 min in 10 mM Tris-HCl 7.5 before bisulfite treatment. The results showed that the temperature played a more important role in DNA damage than treatment time.

[0582] FIG.7 shows MALDI-TOF results for the C-to-C-bisulfite conversion rate in a DO- 9-1 C-5mer RNA using control composition R1-G (100 mg ammonium sulfite dissolved in 1 mL of 50% ammonium bisulfite; second from top row), R5f (Na2S2O5760 μL + K2SO380 μL + H2O 880 μL; final bisulfite concentration ~7.9 M; middle row), R6b (Na2S2O5(MW=190)298000704.1- 77 -ARCD.P0841WO / 24-T-190 840 mg + (NH4)2SO3.H2O, (MW=134) 80 mg + H2O 1000 μL; final bisulfite concentration ~7.37 M; 2ndfrom bottom row), and R7-4.9 (bottom row), each tested at 90 °C for 1 minute. The results showed that R7-4.9 produced the highest C to C-bisulfite conversion rate. The results also showed that R6b produced a similar conversion rate to R1-G. An untreated C-5mer RNA was added as a loading control (top row).

[0583] FIG.8 shows MALDI-TOF results for the C-to-C-bisulfite conversion rate in RNA using composition R1-G (second from top row), R5f (middle row), R6b, and R7-5.1, each tested at 70 °C for 2 minutes. The results showed that, at 70 °C, R7-5.1 showed the highest C to C-bisulfite conversion rate. The results also showed that R6b showed a similar conversion rate to R1-G. An untreated C-5mer RNA was added as a loading control (top row).

[0584] FIG. 9 shows a fragmentation assay of total HeLa RNA after treatment with the specified conditions. The results showed that R6b (3rdcolumn) and R7-4.9 (columns 4-16) showed less RNA damage than R1-G under many reaction conditions, indicating that the new compositions were suitable for treating low input samples.

[0585] FIG. 10 shows percentage of mapped reads from HeLa RNA samples treated with R6b in triplicates. Starting HeLa RNA (A = 200 ng, B =100 ng, or C = 50 ng, each one in triplicates, i.e., A1-A3 is a set of triplicates, B1-B3 is a set of triplicates, and C1-C3 is a set of triplicates) was treated with DNA probes designed to anneal with rRNA and RNase H to digest rRNA while keeping all the non-rRNA intact, followed by next generation sequencing. The results showed that the rRNA reads percentages were low (< 13%).

[0586] FIG. 11 shows unconverted ration of C-to-U in RNA samples treated with R6b, as described in FIG.10 (i.e., A = 200 ng input, B = 100 ng input, and C = 50 ng input). The results showed that the average unconverted ratios for all the C sites in rRNA was very low in all cases (< 0.4%).

[0587] FIG. 12 shows percentage of mapped reads from total Zebrafish embryo RNA samples treated with R6b. The results showed that rRNA reads percentages were low (< 6%).

[0588] FIG.13 shows conversion and unconverted ratios for total Zebrafish embryo RNA samples treated with R6b. The results showed that the average unconverted ratio for all the C sites in rRNA was very low (< 0.3%).

[0589] FIG.14 shows the number and type of m5C sites detected by low depth sequencing (10 million reads) from total Zebrafish RNA treated with R6b. The results showed that R6b allowed for detection of more than 2000 m5C sites and their categorization, most of which were located in coding sequences.298000704.1- 78 -ARCD.P0841WO / 24-T-190

[0590] FIG. 15 shows number of m5C sites mapped to NSUN2 and NSUN6 known sites. The results showed that about 20% of the detected m5C sites showed very strong conserved motif (CUCCA; top row) which was consistent with them being NSUN6 substrates, while the remaining 80% of m5C sites showed GC-enriched motifs which was consistent with reported NSUN2 substrates.

[0591] FIG. 16 shows C-to-U conversion rate in HeLa total RNA as measured by next generation sequencing without clustered read filtration. The results showed that under certain conditions (i.e., temperature and incubation time), new compositions (i.e., R7-4.9 and R6b) gave better conversion efficiency of unmethylated cytosines and higher unconverted rates of methylated cytosines than controls (i.e., R1-G). The two large dots in each plot represent the two known m5C sites in 28S rRNA.

[0592] FIG. 17 shows C-to-U conversion rate in HeLa total RNA as measured by next generation sequencing with clustered read filtration. The results showed that under certain conditions (i.e., temperature and incubation time), new compositions (i.e., R7-4.9 and R6b) gave better conversion efficiency and lower unconverted rates than controls (i.e., R1-G).

[0593] FIG.18 shows MALDI TOF results for pseudouridine (Ψ) to Ψ-bisulfite conversion in an RNA DO-9-3 AGΨGA 5-mer using novel compositions R1-R5 (see table below) at 90 °C for 3 minutes. The results showed higher conversion efficiency (1671 Ψ BS peak) with all recipes R1-R5 (R5 conversion efficiency ~80%) compared to untreated and BID-seq controls (BID-seq conversion efficiency ~<4%). The results also showed that R5 displayed the highest efficiency (~>20 fold than BID-seq recipe).

[0594] FIG. 19 shows an RNA degradation assay using total HeLa RNA treated with control and R1-R5 compositions. The results showed that that treatment of HeLa RNA with R1, R2 or R3 resulted in higher RNA degradation than the control BID-seq recipe, whereas treatment of HeLa RNA with R4 or R5 produced lower levels RNA degradation.298000704.1- 79 -ARCD.P0841WO / 24-T-190

[0595] FIG. 20 shows assessment of Ψ to Ψ-bisulfite conversion and C-to-U conversion in a AGΨGA 5-mer (left panels) or in a C-5mer (right panel) treated with R5 (at pH 7, 98 °C for 8 min; middle rows) or R6 (at pH 7.4, 98 °C for 8 min; bottom rows) compositions, control samples, i.e., 5mer oligo without BS treatment, are represented in the top row. For inducing a good rate of Ψ conversion with a low rate of C-to-U conversion, R6 was superior relative to R5.

[0596] FIG.21 shows sequencing mapping results for screening various compositions and conditions (i.e., incubation time and temperature) for high Ψ to Ψ-bisulfite conversion rate (efficiency) and low C-to-U conversion rate (background) in 18S, 5.8S, and 28S rRNA. The results showed that higher temperatures, less time, and higher pH produced higher efficiency and lower background. The results also suggested that optimal conditions were R6, pH 7.2, 98 °C for 8 min. RBA7.2 = R4, and RBK7.2 = R5.

[0597] FIG. 22 shows box-and-whisker plots for data of FIG. 21.

[0598] FIG.23 shows C-to-U conversion rates (background) for various compositions and conditions (i.e., incubation time and temperature; RBA7.2 = R4, and RBK7.2 = R5). The results showed that higher temperature, lower incubation time, and higher pH produced lower background.

[0599] FIG. 24 shows the number of Ψ sites detected from libraries starting with 10 ng or 5 ng of HeLa total RNA when treated with R6 (pH 7.4) at 98 °C for 8 min. The results showed a high degree of overlap between libraries starting from 10 ng and 5 ng.

[0600] FIG. 25 shows correlation of mapped Ψ sites detected from libraries starting with 10 ng or 5 ng of HeLa total RNA when treated with R6. The results showed a high degree of correlation between libraries starting from 10 ng and 5 ng.

[0601] FIG. 26 shows plotting of pH to C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R1 at 98 °C for 7.5 minutes. The results showed that the optimal pH for dual conversion by R1 was between about 5.5 to 5.9.

[0602] FIG. 27 shows plotting of pH to C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R2 at 98 °C for 7.5 minutes. The results showed that the optimal pH for dual conversion by R2 was about 5.8.

[0603] FIG. 28 shows plotting of pH to C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R3 at 98 °C for 7.5 minutes. The results showed that the optimal pH for dual conversion by R3 was about 5.9.298000704.1- 80 -ARCD.P0841WO / 24-T-190

[0604] FIG. 29 shows plotting of pH to C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R4 at 98 °C for 3 minutes. The results showed that the optimal pH for dual conversion by R4 was about 5.75.

[0605] FIG. 30 shows plotting of pH to C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R5 at 98 °C for 6 minutes. The results showed that the optimal pH for dual conversion by R5 was about 6.0.

[0606] FIG.31 shows a comparison of C to C-bisulfite and Ψ to Ψ-bisulfite conversion for R1-R5 at pH 5.7. The results showed that R5 showed the highest efficiency for both C and Ψ conversion.

[0607] FIG. 32 shows an RNA degradation assay for R1-R3 at pH 5.7 under various temperature and incubation time conditions. The results showed that most of the conditions gave acceptable levels of RNA degradation and did not significantly inhibit library construction.

[0608] FIG. 33 shows conversion and unconverted rates of m5C in RNA using R1-R5 at pH 5.7 under various temperature and incubation time conditions. The results showed that some conditions produced high conversion rates for detection of m5C in RNA.

[0609] FIGs.34A-34B show conversion and unconverted rates of Ψ in RNA using R1-R5 at pH 5.7 under various temperature and incubation time conditions. FIG. 34A shows the results for R1, R2, and R3. FIG.34B shows the results for R4, and R5. The results showed that some conditions produced high conversion rates for detection of Ψ sites in RNA.

[0610] FIGs. 35A-35B show MALDI TOF mass spectrometry data showing the conversion rate of cytosine to uracil in no treatment conditions, and when treated with UBS-1 or UMBS (New BS) bisulfite reagents. The data suggest that the new BS recipe (e.g., UMBS) combining 70% ammonium bisulfite and 20 M KOH achieved higher BS conversion efficiency and speed than the UBS recipe.

[0611] FIG. 36 shows MALDI TOF mass spectrometry data showing the conversion rate of cytosine to uracil in no treatment conditions, and when treated with UMBS bisulfite reagent, or commercial bisulfite reagents, including ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), ZYMO EZ DNA METHYLATION LIGHTNING kit (ZYMO ®), EPIGENTEK ®, QIAGEN ®, PROMEGA ®, DIAGENODE ®, or RAYBIOTECH ®.

[0612] FIGs. 37A-37B show electrophoresis results to measure fragmentation of DNA after treatment with different bisulfite reagents at various treatment temperatures and times. The RNA Pico Bioanalyzer assay suggested that lower temperatures with longer reaction times298000704.1- 81 -ARCD.P0841WO / 24-T-190 consistently achieved less DNA damage.55 °C for 90 minutes was identified as an optimal BS treatment condition to induce minimal DNA damage.

[0613] FIG. 38 shows qPCR results to assess desulphonation efficiency of ZYMO’s L- and M-Desulphonation buffers.

[0614] FIG. 39 shows qPCR results to assess desulphonation efficiency on-beads and in- column desulphonation methods.

[0615] FIG. 40 shows electrophoresis results to measure fragmentation of λDNA after treatment with different bisulfite reagents including UMBS, ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®), and TAPS.

[0616] FIG. 41 shows average qPCR Ct values for samples ranging from of 5.0 ng to 0.01 ng of DNA each treated with NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), or UMBS. Each group shows three bars representing, from left to right, NEBNEXT ENZYMATIC METHYL- SEQ™ (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and UMBS.

[0617] FIGs. 42A-42B. FIG. 42A shows concentration of λDNA libraries (ng / µL) at different input amounts (0.01, 0.05, 0.5, and 5.0 ng) after treatment with different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and UMBS. FIG. 42B shows DNA concentrations from various libraries with different λDNA input amounts (5 ng, 500 pg, 50 pg, and 10 pg), after different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®) (CBS), and UMBS. UMBS demonstrated higher library concentrations and DNA yields compared to EM (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®) and CBS (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) across all λDNA input amounts. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0618] FIGs. 43A-43B show average size of DNA fragments in sequenced libraries as a readout of DNA damage after treatment with different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®) (CBS), and UMBS. FIG. 43A shows a bar graph of the averaged size of insert DNA over treatment after different bisulfite reagents. FIG. 43B shows density over DNA insert (bp) for each treatment group. The results suggest that UMBS298000704.1- 82 -ARCD.P0841WO / 24-T-190 achieved the largest DNA insert fragment sizes of λDNA in the sequenced library (NGS) compared to other two methods, including EM-seq (NEBNEXT ENZYMATIC METHYL- SEQ™ kit (NEW ENGLAND BIOLABS®)) and EZ-Gold (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)), indicating it causes the least DNA damage.

[0619] FIGs. 44A-44B show percentage of unconverted cytosines after treatment with different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®) (CBS), and UMBS. Shown in FIG. 44A, each group was tested with four different amounts of input DNA labeled as “A” for 5 ng, “B” for 500 pg, “C” for 50 pg, and “D” for 10 pg. Shown in FIG. 44B, each group was tested with four different amounts of input DNA (5 ng, 500 pg, 50 pg, and 10 pg). Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq. The results suggest that UMBS achieved the lowest unconverted C ratio compared to other two other 5mC conversion methods, including EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)) and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)), reflecting higher conversion efficiency and lower background noise.

[0620] FIGs. 45A-45B. FIG. 45A shows percentage of detected methylated cytosines in DNA samples of different inputs amounts (“A” for 5 ng, “B” for 500 pg, “C” for 50 pg, and “D” for 10 pg) treated with different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and UMBS. FIG. 45B shows percentage of detected methylated cytosines in CpG, CHG, and CHH contexts on methylated pUC19 DNA treated with different bisulfite reagents including NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and UMBS. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS- seq. The results suggest that UMBS-seq exhibited comparable average DNA methylation levels to CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) and EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit v1 (NEW ENGLAND BIOLABS®)) on methylated pUC19 DNA, demonstrating its robustness for low-input DNA samples.

[0621] FIGs.46A-46B show percentage of mapped methylated cytosines in DNA samples of different inputs amounts (“A” for 5 ng, “B” for 500 pg, “C” for 50 pg, and “D” for 10 pg (FIG. 46A) and 5 ng, 0.5 ng, 0.05 ng, and 0.01 ng (FIG. 46B)) treated with different bisulfite reagents including, NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®), ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and UMBS. In298000704.1- 83 -ARCD.P0841WO / 24-T-190 FIG. 46B, left columns are UMBS-seq; middle columns are EM-seq; right columns are EZ- Gold BS-seq. The results suggest that UMBS-seq exhibited the most consistent mapping ratios across all input scales compared to EZ-Gold (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)), highlighting its robustness for low-input DNA samples. In contrast, the mapping ratios obtained with EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)) were less consistent. A, B, and C represent different DNA input amounts.

[0622] FIG. 47 shows MALDI-TOF mass spectrometry data of untreated and bisulfite- treated 5-mer DNA oligonucleotides. The results suggest that UMBS treatment selectively converts cytosines on the unmethylated DNA oligonucleotide, while 5-methylcytosines (5mC) on the methylated oligonucleotide remain unconverted. UMBS may achieve complete conversion of a C-containing model DNA oligonucleotide while preserving 5mC integrity.

[0623] FIG. 48 shows duplication rates of sequencing libraries generated from varying lambda DNA amounts converted by the three methods, UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)), highlighting the superior complexity of UMBS-seq libraries. Statistical significance was assessed using Student’s t-test (n = 3); p- values are indicated. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0624] FIG. 49 shows scatterplots showing the unconverted cytosine ratios at individual CpG sites in 5 ng of lambda DNA treated with the three different conversion methods, UMBS, ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®). The average unconverted CpG ratio for each method is indicated. The results suggest that UMBS-seq demonstrates the lowest background signal and false-positive rate among the methods tested. Unreliable CpG sites are defined as CpG sites with over 1 unconverted ratios.

[0625] FIG. 50 shows mapping ratios of sequencing libraries generated from varying inputs of lambda DNA (5 ng, 0.5 ng, 0.05 ng, 0.01 ng) treated with different conversion methods including UMBS, NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®) and ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®). UMBS-seq libraries exhibit consistent mapping ratios across input amounts, highlighting the robustness of the method. Left columns are UMBS-seq; middle columns are EM-seq; right columns are EZ- Gold BS-seq.298000704.1- 84 -ARCD.P0841WO / 24-T-190

[0626] FIG. 51 shows the concentration of sequencing libraries generated from varying cfDNA amounts (5 ng, 500 pg, 50 pg, 10 pg) treated with the three different 5mC conversion methods UMBS, EM (NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®)) and CBS (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)). Concentrations were measured after the indicated number of PCR cycles with a 15^µL elution volume. Statistical significance was assessed using Student’s t-test (n = 3); p-values are indicated. The results suggest that UMBS-seq achieves the highest DNA yields from libraries prepared with varying cfDNA inputs using three 5mC conversion methods. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0627] FIG. 52 shows a comparison of cfDNA library yields using three 5mC conversion methods measured by real-time PCR. Amplification curves from real-time PCR quantification of cfDNA libraries were prepared with 5 ng, 0.5 ng, 0.05 ng, and 0.01 ng of cfDNA input using UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ (NEW ENGLAND BIOLABS®)), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) 5mC conversion methods. The results suggest that UMBS-seq consistently produces the highest cfDNA library yields across all input amounts.

[0628] FIG. 53 shows a comparison of library complexities for sequencing libraries generated from varying cfDNA inputs using three 5mC conversion methods. Duplication rates of sequencing libraries were generated from varying input amounts of cfDNA. The results suggest that superior library complexity can be achieved with UMBS-seq. Statistical significance was assessed using Student’s t-test (n = 3); p-values are indicated. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0629] FIGs. 54A-54B shows preservation levels of various DNA insert sizes in cfDNA libraries across varying input amounts. FIG.54A shows a bar plot of the averaged DNA insert size of cfDNA libraries that were prepared using each conversion method at each input. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq. FIG. 54B shows DNA insert size density distribution of 10 pg cfDNA libraries. These results suggest that UMBS-seq and EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), preserve larger insert sizes and better maintain cfDNA integrity compared to CBS-seq.

[0630] FIG. 55 shows genomic coverage (Mb; ≥1X) of cfDNA libraries across various genomic features. Libraries were prepared using 5 ng of cfDNA with each method. The results suggest that cfDNA libraries prepared with UMBS-seq exhibit the best genomic coverage298000704.1- 85 -ARCD.P0841WO / 24-T-190 across different features at equivalent sequencing depth. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0631] FIG.56 shows heatmaps showing read coverage distribution within a 2 kb window around transcription start sites (TSS) (left) and a 1 kb window around CpG islands (right). The results suggest that UMBS-seq and EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)) libraries exhibit greater and more uniform coverage compared to CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) libraries in both regions. The results suggest enhanced representation of transcriptional control features (promoters and CpG Islands) using UMBS-seq and EM-seq.

[0632] FIG. 57 shows CpGs covered (millions) and CHGs covered (millions) over observed coverage depth of cfDNA libraries prepared by using UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) methods. Number of identified CpG sites in cfDNA libraries prepared from different amount of cfDNA input show that both UMBS-seq and EM-seq can detect more CpG sites than CBS-seq at higher sequencing coverage depth and at lower sequencing depths.

[0633] FIG. 58 shows a UCSC Genome Browser snapshot showing DNA methylation levels at individual CpG sites across a representative genomic region. cfDNA libraries were prepared with UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) methods using varying cfDNA input amounts. The results suggest that cfDNA libraries prepared with UMBS-seq show the best CpG coverage compared to EM-seq and CBS- seq under different cfDNA input amount.

[0634] FIG. 59 shows GC-bias plot, normalized coverage over GC content (%), for UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) cfDNA libraries prepared from 5 ng of cfDNA input. The results suggest that UMBS-seq and EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®) exhibited superior GC coverage uniformity than CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)).

[0635] FIGs. 60A-60C shows estimated average DNA methylation levels at CpG (FIG. 60A), CHG (FIG. 60B) and CHH (FIG. 60C) contexts in cfDNA libraries prepared with varying cfDNA input amounts. UMBS-seq was compared to EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)) and CBS-seq (ZYMO298000704.1- 86 -ARCD.P0841WO / 24-T-190 EZ DNA METHYLATION GOLD® kit (ZYMO ®)) as benchmarks. Statistical significance was assessed using Student’s t-test (n = 3); p-values are indicated. The results suggest that UMBS-seq provides more accurate DNA methylation estimates Than EM-seq and CBS-seq across three cytosine contexts and various cfDNA input levels. Left columns are UMBS-seq; middle columns are EM-seq; right columns are CBS-seq.

[0636] FIG. 61 shows DNA methylation percentages across gene bodies in cfDNA libraries prepared from 5 ng of input using UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) treatments. Methylation levels were calculated separately for CpG, CHG, and CHH contexts. The results suggest that UMBS-seq more accurately reconstructed known DNA methylation patterns within promoter regions and gene bodies.

[0637] FIGs. 62A-62C show scatter plots of DNA methylation levels at individual CpG sites in 5 ng cfDNA libraries between technical replications of UMBS-seq (FIG.62A), UMBS- seq and CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) libraries (FIG. 62B) and UMBS-seq and EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS)) libraries (FIG. 62C). The results suggest that UMBS-seq demonstrate high reproducibility between replicates and compared to EM-seq and CBS-seq.

[0638] FIG.63 shows depth of CpG coverage over genes when targeted capture is coupled with UMBS-seq, EM-seq (NEBNEXT ENZYMATIC METHYL-SEQ™ kit (NEW ENGLAND BIOLABS®)), or CBS-seq (ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®)) methods. The combination of the Nanodigmbio μCaler capture kit with UMBS- seq on 5 ng cfDNA from healthy individuals resulted in enhanced CpG coverage compared to its combination with EM-seq and CBS-seq. DETAILED DESCRIPTION

[0639] Aspects of the present disclosure relate to compositions, methods, and kits for detection and analysis of methylated DNA, methylated RNA, pseudouridine in RNA, or methylated RNA and pseudouridine simultaneously. Certain aspects are directed to compositions for bisulfite treatment of methylated DNA, methylated RNA, RNA comprising pseudouridine, or methylated RNA comprising pseudouridine. Also disclosed, in some aspects, are methods for bisulfite treatment of methylated DNA, methylated RNA, RNA comprising pseudouridine, or methylated RNA comprising pseudouridine including methods comprising incubation for short time periods (e.g., < 45 minutes) at moderate temperatures (e.g., 70-90 °C)298000704.1- 87 -ARCD.P0841WO / 24-T-190 using the disclosed bisulfite solutions. Kits including the disclosed compositions are also described herein, along with instructions for analysis of methylated DNA, methylated RNA, RNA comprising pseudouridine, or methylated RNA comprising pseudouridine. Aspects of the disclosure provide bisulfite sequencing methods comprising rapid bisulfite treatment, low background noise, and high sensitivity, enabling highly accurate sequencing of m5C in RNA, 5mC in DNA, Ψ in RNA, and m5C and Ψ simultaneously in RNA starting from low-input biological RNA or DNA samples.

[0640] In some aspects, a nucleic acid comprises or is cell free DNA (cfDNA) and / or from a formalin-fixed paraffin-embedded (FFPE) sample. In some aspects, a nucleic acid is greater than or is , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572,298000704.1- 88 -ARCD.P0841WO / 24-T-190 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, or 1000 bases, or longer, or any range or value derivable therein. I. DNA Processing Methods

[0641] Aspects of the present disclosure relate to compositions and methods for DNA processing. Particular aspects relate to compositions comprising ammonium bisulfite and methods for use of such compositions in bisulfite treatment of DNA. Accordingly, disclosed herein, in some aspects, are methods for DNA processing comprising incubating a solution comprising a DNA molecule and ammonium bisulfite under conditions sufficient to deaminate a cytosine residue of the DNA molecule, where the solution optionally does not comprise sodium bisulfite or added sodium bisulfite. Such methods may further comprise subjecting the DNA molecule to acidic conditions. As disclosed herein, incubating one or more DNA298000704.1- 89 -ARCD.P0841WO / 24-T-190 molecules in a bisulfite solution of the disclosure under appropriate conditions results in extremely rapid deamination of cytosines with low DNA degradation while preserving 5- methylcytosine (5mC), leading to identification of methylated nucleotides with very low false positive rate. In some aspects, methods provided herein provide BS treatments suitable for accurately distinguishing 5mC from N4-methylcytosine (4mC). In some aspects, methods provided herein facilitate deamination of 4mC at greater rate relative to canonical-BS treatments. In some aspects, methods provided herein facilitate conversion of 4mC to uracil at greater rate relative to canonical-BS treatments. In some aspects methods provided herein quantitatively deaminates 4mC. In some aspects, methods provided herein facilitate deamination of 4mC at an efficiency of greater than about 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, methods provided herein substantially avoid BS treatment false positives generated by the existence of 4mC in the genome.

[0642] In some aspects, provided herein are methods and compositions for DNA and / or RNA processing comprising incubating a solution comprising a DNA and / or RNA molecule and ammonium bisulfite under conditions sufficient to deaminate a cytosine residue of the DNA and / or RNA molecule. In some aspects, the solution also comprises a second bisulfite. In some aspects, the solution comprises sodium bisulfite, added sodium bisulfite, other bisulfite salts, and / or other added bisulfite salts. In some aspects, solutions provided herein comprise ammonium bisulfite at a concentration of 2 to 10 M (or any range or value derivable therein, e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, etc.) and other bisulfite salts (for example but not limited to Na+, K+, Li+, etc. bisulfite salts) at a concentration of 0.5 to 8 M (or any range or value derivable therein, e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc.), providing a total bisulfite concentration of 4 to 12 M (or any range or value derivable therein, e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, etc.) at a NH4+ total bisulfite molar ratio of 0.3 to 1 (or any range derivable therein, e.g., 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, etc.). In some aspects, provided herein are methods and compositions comprising two or more sources of bisulfite, wherein at least one source comprises ammonium bisulfite. In some aspects, provided herein are methods and compositions comprising two or more sources of bisulfite, wherein a first source of bisulfite comprises, consists essentially of, or consists of ammonium bisulfite, and the ratio of ammonium bisulfite to the one or more additional source of bisulfite is 0.1:1, 0.15:1, 0.2:1, 0.25:1, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1, 0.55:1, 0.6:1, 0.65:1, 0.7:1, 0.75:1,298000704.1- 90 -ARCD.P0841WO / 24-T-190 0.8:1, 0.85:1, 0.9:1, 0.95:1, 1:1, 1:0.95, 1:0.85, 1:0.8, 1:0.75, 1:0.7, 1:0.65, 1:0.6, 1:0.55, 1:0.5, 1:0.45, 1:0.4, 1:0.35, 1:0.3, 1:0.25, 1:0.2, 1:0.15, 1:0.1, or any range or ratio derivable therein.

[0643] In some aspects, provided herein are compositions and methods for high methyl conversion efficiency with low DNA damage comprising a weight / volume (w / v) of 50% to 75% (w / v) ammonium bisulfite solution with a molarity of 6.5 M to 10.5 M. In some aspects, provided herein are compositions and methods with suitable methyl conversion efficiency (e.g., which may have a lower-stringency requirement) that comprise a weight / volume (w / v) of 30% to 80% (w / v) ammonium bisulfite solution with a molarity of about 4 M to about 11 M.

[0644] In some aspects, conventional or canonical-BS treatments comprise BS concentrations below 6.0 M. In some aspects, conventional BS treatments comprise incubation at 98 ˚C for 10 min followed by 64 ˚C for 150 min. In some aspects, conventional BS- treatments, e.g., ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®) are thought to comprise about 4.5 M BS concentration based on titration experiments. In some aspects, the inventors measured the BS concentration of conventional BS-treatments, e.g., ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), using two methods: (1) weighing the BS reagents and calculating the BS concentration to be 4.35 M assuming it is NaHSO3, and 4.76 M assuming it is Na2S2O5; and (2) conducting an iodine titration and finding that BS concentration is around 4.5 M. In some aspects, the titration procedure comprises preparing a 10 μL BS solution prepared from a conventional BS-treatment, e.g., ZYMO EZ DNA METHYLATION GOLD® kit (ZYMO ®), and adding it to 490 μL of water containing 14 mg starch as an indicator, followed by adding a solution of 72 mg iodine and 608 mg sodium iodide dissolved in 8 mL water in a dropwise manner to the BS solution until the blue color is sustained; 1125 μL were used and the BS concentration was calculated to be 4.43 M. In some aspects, the highest BS concentration know previously known in the art using NaHSO3and NaOH is 5.7 M. In some aspects, NaHSO3 dissolved in water cannot reach this high concentration but adding NaOH to adjust pH to around 5.1 can reach a concentration of 5.7 M (see e.g., C Grunau 1, S J Clark, A Rosenthal, Bisulfite genomic sequencing: systematic investigation of critical experimental parameters, Nucleic Acids Res, 2001 Jul 1;29(13):E65-5, which is incorporated herein by reference in its entirety).

[0645] In some aspects, DNA processing methods of the disclosure include incubating one or more DNA molecules in a bisulfite solution, where the bisulfite solution comprises ammonium bisulfite and optionally does not comprise sodium bisulfite or added sodium bisulfite. In some aspects, the bisulfite solution comprises sodium at a concentration of, or of at most 1 M, 0.1 M, 0.01 M, 1x10-3M, 1x10-4M, 1x10-5M, 1x10-6M, 1x10-7M, 1x10-8M,298000704.1- 91 -ARCD.P0841WO / 24-T-190 1x10-9M, 1x10-10M, 1x10-11M, 1x10-12M, 1x10-13M, 1x10-14M, 1x10-15M, 1x10-16M, 1x10-17M, 1x10-18M, 1x10-19M, 1x10-20M or less. In some aspects, the solution does not comprise sodium. In some aspects, the solution does not comprise ammonium salt. In some aspects, the solution comprises ammonium sulfite at a concentration of, or of at most 10 M, 1 M, 0.1 M, 0.01 M, 1x10-3M, 1x10-4M, 1x10-5M, 1x10-6M, 1x10-7M, 1x10-8M, 1x10-9M, 1x10-10M, or less. In certain aspects the solution does not comprise ammonium sulfite or added ammonium sulfite.

[0646] In certain aspects a solution (e.g., bisulfite solution) of the disclosure comprises between 30% and 80%, 40% and 80%, 50% and 80%, or 50% and 72% ammonium bisulfite by weight, including any range or value derivable therein. In some aspects, the solution comprises at least, at most, or about 30%, 30.1%, 30.2%, 30.3%, 30.4%, 30.5%, 30.6%, 30.7%, 30.8%, 30.9%, 31%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.6%, 31.7%, 31.8%, 31.9%, 32%, 32.1%, 32.2%, 32.3%, 32.4%, 32.5%, 32.6%, 32.7%, 32.8%, 32.9%, 33%, 33.1%, 33.2%, 33.3%, 33.4%, 33.5%, 33.6%, 33.7%, 33.8%, 33.9%, 34%, 34.1%, 34.2%, 34.3%, 34.4%, 34.5%, 34.6%, 34.7%, 34.8%, 34.9%, 35%, 35.1%, 35.2%, 35.3%, 35.4%, 35.5%, 35.6%, 35.7%, 35.8%, 35.9%, 36%, 36.1%, 36.2%, 36.3%, 36.4%, 36.5%, 36.6%, 36.7%, 36.8%, 36.9%, 37%, 37.1%, 37.2%, 37.3%, 37.4%, 37.5%, 37.6%, 37.7%, 37.8%, 37.9%, 38%, 38.1%, 38.2%, 38.3%, 38.4%, 38.5%, 38.6%, 38.7%, 38.8%, 38.9%, 39%, 39.1%, 39.2%, 39.3%, 39.4%, 39.5%, 39.6%, 39.7%, 39.8%, 39.9%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 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%, 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%, 55.1%, 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%, 56.7%, 56.8%, 56.9%, 57%, 57.1%, 57.2%, 57.3%, 57.4%, 57.5%, 57.6%, 57.7%, 57.8%, 57.9%, 58%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.6%, 58.7%, 58.8%,298000704.1- 92 -ARCD.P0841WO / 24-T-190 58.9%, 59%, 59.1%, 59.2%, 59.3%, 59.4%, 59.5%, 59.6%, 59.7%, 59.8%, 59.9%, 60%, 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%, 61.6%, 61.7%, 61.8%, 61.9%, 62%, 62.1%, 62.2%, 62.3%, 62.4%, 62.5%, 62.6%, 62.7%, 62.8%, 62.9%, 63%, 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%, 64.7%, 64.8%, 64.9%, 65%, 65.1%, 65.2%, 65.3%, 65.4%, 65.5%, 65.6%, 65.7%, 65.8%, 65.9%, 66%, 66.1%, 66.2%, 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%, 70%, 70.1%, 70.2%, 70.3%, 70.4%, 70.5%, 70.6%, 70.7%, 70.8%, 70.9%, 71%, 71.1%, 71.2%, 71.3%, 71.4%, 71.5%, 71.6%, 71.7%, 71.8%, 71.9%, 72%, 72.1%, 72.2%, 72.3%, 72.4%, 72.5%, 72.6%, 72.7%, 72.8%, 72.9%, or 73% ammonium bisulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises at least, at most, or about 69%, 69.01%, 69.02%, 69.03%, 69.04%, 69.05%, 69.06%, 69.07%, 69.08%, 69.09%, 69.1%, 69.11%, 69.12%, 69.13%, 69.14%, 69.15%, 69.16%, 69.17%, 69.18%, 69.19%, 69.2%, 69.21%, 69.22%, 69.23%, 69.24%, 69.25%, 69.26%, 69.27%, 69.28%, 69.29%, 69.3%, 69.31%, 69.32%, 69.33%, 69.34%, 69.35%, 69.36%, 69.37%, 69.38%, 69.39%, 69.4%, 69.41%, 69.42%, 69.43%, 69.44%, 69.45%, 69.46%, 69.47%, 69.48%, 69.49%, 69.5%, 69.51%, 69.52%, 69.53%, 69.54%, 69.55%, 69.56%, 69.57%, 69.58%, 69.59%, 69.6%, 69.61%, 69.62%, 69.63%, 69.64%, 69.65%, 69.66%, 69.67%, 69.68%, 69.69%, 69.7%, 69.71%, 69.72%, 69.73%, 69.74%, 69.75%, 69.76%, 69.77%, 69.78%, 69.79%, 69.8%, 69.81%, 69.82%, 69.83%, 69.84%, 69.85%, 69.86%, 69.87%, 69.88%, 69.89%, 69.9%, 69.91%, 69.92%, 69.93%, 69.94%, 69.95%, 69.96%, 69.97%, 69.98%, 69.99%, 70%, 70.01%, 70.02%, 70.03%, 70.04%, 70.05%, 70.06%, 70.07%, 70.08%, 70.09%, 70.1%, 70.11%, 70.12%, 70.13%, 70.14%, 70.15%, 70.16%, 70.17%, 70.18%, 70.19%, 70.2%, 70.21%, 70.22%, 70.23%, 70.24%, 70.25%, 70.26%, 70.27%, 70.28%, 70.29%, 70.3%, 70.31%, 70.32%, 70.33%, 70.34%, 70.35%, 70.36%, 70.37%, 70.38%, 70.39%, 70.4%, 70.41%, 70.42%, 70.43%, 70.44%, 70.45%, 70.46%, 70.47%, 70.48%, 70.49%, 70.5%, 70.51%, 70.52%, 70.53%, 70.54%, 70.55%, 70.56%, 70.57%, 70.58%, 70.59%, 70.6%, 70.61%, 70.62%, 70.63%, 70.64%, 70.65%, 70.66%, 70.67%, 70.68%, 70.69%, 70.7%, 70.71%, 70.72%, 70.73%, 70.74%, 70.75%, 70.76%, 70.77%, 70.78%, 70.79%, 70.8%, 70.81%, 70.82%, 70.83%, 70.84%, 70.85%, 70.86%, 70.87%, 70.88%, 70.89%, 70.9%, 70.91%, 70.92%, 70.93%, 70.94%, 70.95%, 70.96%, 70.97%, 70.98%, 70.99%, 71%, 71.01%, 71.02%, 71.03%, 71.04%, 71.05%, 71.06%, 71.07%, 71.08%, 71.09%, 71.1%, 71.11%, 71.12%,298000704.1- 93 -ARCD.P0841WO / 24-T-190 71.13%, 71.14%, 71.15%, 71.16%, 71.17%, 71.18%, 71.19%, 71.2%, 71.21%, 71.22%, 71.23%, 71.24%, 71.25%, 71.26%, 71.27%, 71.28%, 71.29%, 71.3%, 71.31%, 71.32%, 71.33%, 71.34%, 71.35%, 71.36%, 71.37%, 71.38%, 71.39%, 71.4%, 71.41%, 71.42%, 71.43%, 71.44%, 71.45%, 71.46%, 71.47%, 71.48%, 71.49%, 71.5%, 71.51%, 71.52%, 71.53%, 71.54%, 71.55%, 71.56%, 71.57%, 71.58%, 71.59%, 71.6%, 71.61%, 71.62%, 71.63%, 71.64%, 71.65%, 71.66%, 71.67%, 71.68%, 71.69%, 71.7%, 71.71%, 71.72%, 71.73%, 71.74%, 71.75%, 71.76%, 71.77%, 71.78%, 71.79%, 71.8%, 71.81%, 71.82%, 71.83%, 71.84%, 71.85%, 71.86%, 71.87%, 71.88%, 71.89%, 71.9%, 71.91%, 71.92%, 71.93%, 71.94%, 71.95%, 71.96%, 71.97%, 71.98%, 71.99%, 72%, 72.01%, 72.02%, 72.03%, 72.04%, 72.05%, 72.06%, 72.07%, 72.08%, 72.09%, 72.1%, 72.11%, 72.12%, 72.13%, 72.14%, 72.15%, 72.16%, 72.17%, 72.18%, 72.19%, 72.2%, 72.21%, 72.22%, 72.23%, 72.24%, 72.25%, 72.26%, 72.27%, 72.28%, 72.29%, 72.3%, 72.31%, 72.32%, 72.33%, 72.34%, 72.35%, 72.36%, 72.37%, 72.38%, 72.39%, 72.4%, 72.41%, 72.42%, 72.43%, 72.44%, 72.45%, 72.46%, 72.47%, 72.48%, 72.49%, 72.5%, 72.51%, 72.52%, 72.53%, 72.54%, 72.55%, 72.56%, 72.57%, 72.58%, 72.59%, 72.6%, 72.61%, 72.62%, 72.63%, 72.64%, 72.65%, 72.66%, 72.67%, 72.68%, 72.69%, 72.7%, 72.71%, 72.72%, 72.73%, 72.74%, 72.75%, 72.76%, 72.77%, 72.78%, 72.79%, 72.8%, 72.81%, 72.82%, 72.83%, 72.84%, 72.85%, 72.86%, 72.87%, 72.88%, 72.89%, 72.9%, 72.91%, 72.92%, 72.93%, 72.94%, 72.95%, 72.96%, 72.97%, 72.98%, 72.99%, or 73% ammonium bisulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises about 70% ammonium bisulfite by weight. In some aspects, the solution comprises about 71% ammonium bisulfite by weight. In some aspects, the solution comprises about 72% ammonium bisulfite by weight. In some aspects, the solution comprises about 73% ammonium bisulfite by weight. In some aspects, a bisulfite solution does not comprise ammonium sulfite or added ammonium sulfite. In some aspects, a bisulfite solution comprises ammonium sulfite.

[0647] In some aspects, a novel bisulfite solution disclosed herein is at a bisulfite concentration greater than about 6 M. In some aspects, the bisulfite solution can reach a bisulfite concentration of up to about 12 M. In some aspects, the bisulfite solution is at a bisulfite concentration of between 6 M and 12 M, including any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of between 9 M and 12 M, including any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of between 9 M and 10 M, including any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of at least, at most, or about 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7.0 M, 7.1 M,298000704.1- 94 -ARCD.P0841WO / 24-T-190 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8.0 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9.0 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10 M, 10.1 M, 10.2 M, 10.3 M, 10.4 M, 10.5 M, 10.6 M, 10.7 M, 10.8 M, 10.9 M, 11.0 M, 11.1 M, 11.2 M, 11.3 M, 11.4 M, 11.5 M, 11.6 M, 11.7 M, 11.8 M, 11.9 M, or 12.0 M, or any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of about 9.8 M. In some aspects, the bisulfite solution is at a bisulfite concentration of about 9.71 M, 7.72 M.9.73 M, 9.74 M, 9.75 M, 9.76 M, 9.77 M, 9.78 M, 9.79 M, 9.8 M, 9.81 M, 7.82 M. 9.83 M, 9.84 M, 9.85 M, 9.86 M, 9.87 M, 9.88 M, 9.89 M, or 9.9 M, or any range derivable therein.

[0648] In some aspects, a DNA processing method comprises incubating one or more DNA molecules in a bisulfite solution of the disclosure at a set temperature for a set period of time (e.g., a methyl conversion step). In some aspects, the method comprises incubating one or more DNA molecules in a bisulfite solution at a temperature of, of at least, of at most, or of about 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 70.1 °C, 70.2 °C, 70.3 °C, 70.4 °C, 70.5 °C, 70.6 °C, 70.7 °C, 70.8 °C, 70.9 °C, 71 °C, 71.1 °C, 71.2 °C, 71.3 °C, 71.4 °C, 71.5 °C, 71.6 °C, 71.7 °C, 71.8 °C, 71.9 °C, 72 °C, 72.1 °C, 72.2 °C, 72.3 °C, 72.4 °C, 72.5 °C, 72.6 °C, 72.7 °C, 72.8 °C, 72.9 °C, 73 °C, 73.1 °C, 73.2 °C, 73.3 °C, 73.4 °C, 73.5 °C, 73.6 °C, 73.7 °C, 73.8 °C, 73.9 °C, 74 °C, 74.1 °C, 74.2 °C, 74.3 °C, 74.4 °C, 74.5 °C, 74.6 °C, 74.7 °C, 74.8 °C, 74.9 °C, 75 °C, 75.1 °C, 75.2 °C, 75.3 °C, 75.4 °C, 75.5 °C, 75.6 °C, 75.7 °C, 75.8 °C, 75.9 °C, 76 °C, 76.1 °C, 76.2 °C, 76.3 °C, 76.4 °C, 76.5 °C, 76.6 °C, 76.7 °C, 76.8 °C, 76.9 °C, 77 °C, 77.1 °C, 77.2 °C, 77.3 °C, 77.4 °C, 77.5 °C, 77.6 °C, 77.7 °C, 77.8 °C, 77.9 °C, 78 °C, 78.1 °C, 78.2 °C, 78.3 °C, 78.4 °C, 78.5 °C, 78.6 °C, 78.7 °C, 78.8 °C, 78.9 °C, 79 °C, 79.1 °C, 79.2 °C, 79.3 °C, 79.4 °C, 79.5 °C, 79.6 °C, 79.7 °C, 79.8 °C, 79.9 °C, 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 °C, 84 °C, 84.1 °C, 84.2 °C, 84.3 °C, 84.4 °C, 84.5 °C, 84.6 °C, 84.7 °C, 84.8 °C, 84.9 °C, 85 °C, 85.1 °C, 85.2 °C, 85.3 °C, 85.4 °C, 85.5 °C, 85.6 °C, 85.7 °C, 85.8 °C, 85.9 °C, 86 °C, 86.1 °C, 86.2 °C, 86.3 °C, 86.4 °C, 86.5 °C, 86.6 °C, 86.7 °C, 86.8 °C, 86.9 °C, 87 °C, 87.1 °C, 87.2 °C, 87.3 °C, 87.4 °C, 87.5 °C, 87.6 °C, 87.7 °C, 87.8 °C, 87.9 °C, 88 °C, 88.1 °C, 88.2 °C, 88.3 °C, 88.4 °C, 88.5 °C, 88.6 °C, 88.7 °C, 88.8 °C, 88.9 °C, 89 °C, 89.1 °C, 89.2 °C, 89.3 °C, 89.4 °C, 89.5 °C, 89.6 °C, 89.7 °C, 89.8 °C, 89.9 °C, 90 °C, 90.1 °C, 90.2 °C, 90.3 °C, 90.4 °C, 90.5 °C, 90.6 °C, 90.7 °C, 90.8 °C, 90.9 °C, 91 °C, 91.1 °C, 91.2 °C, 91.3 °C, 91.4298000704.1- 95 -ARCD.P0841WO / 24-T-190 °C, 91.5 °C, 91.6 °C, 91.7 °C, 91.8 °C, 91.9 °C, 92 °C, 92.1 °C, 92.2 °C, 92.3 °C, 92.4 °C, 92.5 °C, 92.6 °C, 92.7 °C, 92.8 °C, 92.9 °C, 93 °C, 93.1 °C, 93.2 °C, 93.3 °C, 93.4 °C, 93.5 °C, 93.6 °C, 93.7 °C, 93.8 °C, 93.9 °C, 94 °C, 94.1 °C, 94.2 °C, 94.3 °C, 94.4 °C, 94.5 °C, 94.6 °C, 94.7 °C, 94.8 °C, 94.9 °C, 95 °C, 95.1 °C, 95.2 °C, 95.3 °C, 95.4 °C, 95.5 °C, 95.6 °C, 95.7 °C, 95.8 °C, 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 range or value derivable therein) for at most or about 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 44, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14.9, 14.8, 14.7, 14.6, 14.5, 14.4, 14.3, 14.2, 14.1, 14, 13.9, 13.8, 13.7, 13.6, 13.5, 13.4, 13.3, 13.2, 13.1, 13, 12.9, 12.8, 12.7, 12.6, 12.5, 12.4, 12.3, 12.2, 12.1, 12, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1 minutes (or any range or value derivable therein). Any combination of the preceding incubation times and temperatures may be used in a DNA processing method of the present disclosure. Any combination of the preceding incubation times and temperatures may be expressly excluded from a DNA processing method of the present disclosure.

[0649] In some aspects, a DNA processing method comprises incubating one or more DNA molecules in a bisulfite solution of the disclosure at a temperature of at least 70 °C for at most 500 minutes, 70 °C for at most 30 minutes, of at least 75 °C for at most 30 minutes, of at least 80 °C for at most 30 minutes, of at least 85 °C for at most 30 minutes, of at least 90 °C for at most 30 minutes, of at least 95 °C for at most 30 minutes, at a temperature of at least 96 °C for at most 30 minutes, at a temperature of at least 97 °C for at most 30 minutes, at a temperature of at least 98 °C for at most 30 minutes, at a temperature of at least 99 °C for at most 30 minutes, of at least 70 °C for at most 25 minutes, of at least 75 °C for at most 25 minutes, of at least 80 °C for at most 25 minutes, of at least 85 °C for at most 25 minutes, of at least 90 °C for at most 25 minutes, of at least 95 °C for at most 25 minutes, at a temperature of at least 96 °C for at298000704.1- 96 -ARCD.P0841WO / 24-T-190 most 25 minutes, at a temperature of at least 97 °C for at most 25 minutes, at a temperature of at least 98 °C for at most 25 minutes, at a temperature of at least 99 °C for at most 25 minutes, of at least 70 °C for at most 20 minutes, of at least 75 °C for at most 20 minutes, of at least 80 °C for at most 20 minutes, of at least 85 °C for at most 20 minutes, of at least 90 °C for at most 20 minutes, of at least 95 °C for at most 20 minutes, at a temperature of at least 96 °C for at most 20 minutes, at a temperature of at least 97 °C for at most 20 minutes, at a temperature of at least 98 °C for at most 20 minutes, at a temperature of at least 99 °C for at most 20 minutes, of at least 70 °C for at most 20 minutes, of at least 75 °C for at most 20 minutes, of at least 80 °C for at most 20 minutes, of at least 85 °C for at most 20 minutes, of at least 90 °C for at most 20 minutes, of at least 95 °C for at most 20 minutes, at a temperature of at least 96 °C for at most 20 minutes, at a temperature of at least 97 °C for at most 20 minutes, at a temperature of at least 98 °C for at most 20 minutes, at a temperature of at least 99 °C for at most 20 minutes, of at least 70 °C for at most 12 minutes, of at least 75 °C for at most 12 minutes, of at least 80 °C for at most 12 minutes, of at least 85 °C for at most 12 minutes, of at least 90 °C for at most 12 minutes, of at least 95 °C for at most 12 minutes, at a temperature of at least 96 °C for at most 12 minutes, at a temperature of at least 97 °C for at most 12 minutes, at a temperature of at least 98 °C for at most 12 minutes, at a temperature of at least 99 °C for at most 12 minutes, at a temperature of at least 70 °C for at most 10 minutes, at a temperature of at least 75 °C for at most 10 minutes, at a temperature of at least 80 °C for at most 10 minutes, at a temperature of at least 85 °C for at most 10 minutes, at a temperature of at least 90 °C for at most 10 minutes, at a temperature of at least 95 °C for at most 11 minutes, at a temperature of at least 96 °C for at most 11 minutes, at a temperature of at least 97 °C for at most 11 minutes, at a temperature of at least 98 °C for at most 11 minutes, at a temperature of at least 99 °C for at most 11 minutes, at a temperature of at least 70 °C for at most 10 minutes, at a temperature of at least 75 °C for at most 10 minutes, at a temperature of at least 80 °C for at most 10 minutes, at a temperature of at least 85 °C for at most 10 minutes, at a temperature of at least 90 °C for at most 10 minutes, at a temperature of at least 95 °C for at most 10 minutes, at a temperature of at least 96 °C for at most 10 minutes, at a temperature of at least 97 °C for at most 10 minutes, at a temperature of at least 98 °C for at most 10 minutes, at a temperature of at least 99 °C for at most 10 minutes, at a temperature of at least 70 °C for at most 9 minutes, at a temperature of at least 75 °C for at most 9 minutes, at a temperature of at least 80 °C for at most 9 minutes, at a temperature of at least 85 °C for at most 9 minutes, at a temperature of at least 90 °C for at most 9 minutes, at a temperature of at least 95 °C for at most 9 minutes, at a temperature of at least 96 °C for at most 9 minutes, at a temperature of at least 97 °C for at most 9 minutes, at a temperature of at298000704.1- 97 -ARCD.P0841WO / 24-T-190 least 98 °C for at most 9 minutes, at a temperature of at least 99 °C for at most 9 minutes, at a temperature of at least 70 °C for at most 8 minutes, at a temperature of at least 75 °C for at most 8 minutes, at a temperature of at least 80 °C for at most 8 minutes, at a temperature of at least 85 °C for at most 8 minutes, at a temperature of at least 90 °C for at most 8 minutes, at a temperature of at least 95 °C for at most 8 minutes, at a temperature of at least 96 °C for at most 8 minutes, at a temperature of at least 97 °C for at most 8 minutes, at a temperature of at least 98 °C for at most 8 minutes, at a temperature of at least 99 °C for at most 8 minutes, at a temperature of at least 70 °C for at most 7 minutes, at a temperature of at least 75 °C for at most 7 minutes, at a temperature of at least 80 °C for at most 7 minutes, at a temperature of at least 85 °C for at most 7 minutes, at a temperature of at least 90 °C for at most 7 minutes, at a temperature of at least 95 °C for at most 7 minutes, at a temperature of at least 96 °C for at most 7 minutes, at a temperature of at least 97 °C for at most 7 minutes, at a temperature of at least 98 °C for at most 7 minutes, at a temperature of at least 99 °C for at most 7 minutes, at a temperature of at least 70 °C for at most 6 minutes, at a temperature of at least 75 °C for at most 6 minutes, at a temperature of at least 80 °C for at most 6 minutes, at a temperature of at least 85 °C for at most 6 minutes, at a temperature of at least 90 °C for at most 6 minutes, at a temperature of at least 95 °C for at most 6 minutes, at a temperature of at least 96 °C for at most 6 minutes, at a temperature of at least 97 °C for at most 6 minutes, at a temperature of at least 98 °C for at most 6 minutes, at a temperature of at least 99 °C for at most 6 minutes, at a temperature of at least 70 °C for at most 5 minutes, at a temperature of at least 75 °C for at most 5 minutes, at a temperature of at least 80 °C for at most 5 minutes, at a temperature of at least 85 °C for at most 5 minutes, at a temperature of at least 90 °C for at most 5 minutes, at a temperature of at least 95 °C for at most 5 minutes, at a temperature of at least 96 °C for at most 5 minutes, at a temperature of at least 97 °C for at most 5 minutes, at a temperature of at least 98 °C for at most 5 minutes, or at a temperature of at least 99 °C for at most 5 minutes.

[0650] In some aspects, for ultrafast BS sequencing, recipe R7-4.9 (BS concentration of 9.8 M; pH 7.4), is used at 90 ˚C for 10 minutes, or 98 ˚C for 5-8 minutes. In some aspects, for low DNA damage, a BS solution comprising a bisulfite concentration between 6 M to 12 M, with a pH ranging from 4.5 to 6.0, is used at a temperature between 50-90 ˚C.

[0651] In some aspects, the BS concentration of a bisulfite solution is or can be the primary consideration for achieving the results disclosed herein or for practicing the methods disclosed herein. In some aspects, the pH, the components in the BS solution, and the treatment conditions, such as temperature and time, may also be important. In some aspects, BS concentrations may be as high as 12.0 M.298000704.1- 98 -ARCD.P0841WO / 24-T-190

[0652] As disclosed herein, incubating DNA molecules with a bisulfite solution of the present disclosure (e.g., a solution comprising ammonium bisulfite such as 50%-72% ammonium bisulfite which optionally does not comprise sodium bisulfite, or added sodium bisulfite) under appropriate conditions (e.g., at a temperature of at least 70 °C for at most 30 minutes) is sufficient to deaminate a majority of cytosine residues in the DNA molecules. In some aspects, after incubating DNA molecules with a bisulfite solution of the present disclosure under appropriate conditions, greater than 90% of the DNA molecules comprise no cytosine residue. In some aspects, greater than or equal to 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%, or 99.9% (or any range or value derivable therein) of the DNA molecules comprise no cytosine residue. In some aspects, greater than 99% of the DNA molecules comprise no cytosine residue.

[0653] DNA processing methods of the disclosure may be useful in, for example, preparing DNA molecules for sequencing in order to detect, quantify, and / or analyze DNA cytosine methylation. In some aspects, DNA processing methods of the disclosure provide DNA molecules for sequencing analysis that result in a reduced level of false positives, increased level of true positives, reduced level of false negatives, and / or increased level of true negatives relative to canonical-BS treatments.

[0654] In some aspects, methods described herein comprise enriching for non-rRNA species. In some aspects, enrichment comprises annealing DNA probes complementary to rRNA and digesting the rRNA with RNase H, which digests RNA in RNA / DNA hybrids. II. RNA Processing Methods

[0655] Aspects of the present disclosure relate to compositions and methods for RNA processing. Particular aspects relate to compositions comprising ammonium bisulfite and methods for use of such compositions in bisulfite treatment of RNA. Accordingly, disclosed herein, in some aspects, are methods for RNA processing comprising incubating a solution comprising an RNA molecule, ammonium bisulfite, and ammonium sulfite under conditions298000704.1- 99 -ARCD.P0841WO / 24-T-190 sufficient to deaminate a cytosine residue of the RNA molecule or ring-opening in Ψ-BS adducts. Such methods may further comprise subjecting the RNA molecule to acidic conditions, or neutral conditions. As disclosed herein, incubating one or more RNA molecules in a bisulfite solution of the disclosure under appropriate conditions results in extremely rapid deamination of cytosines or ring-opening in Ψ-BS adducts with low RNA degradation, leading to identification of methylated nucleotides or Ψ with very low false positive rate. In some aspects, methods disclosed herein result in a reduced level of background noise (e.g., unconverted cytosines or Ψ) relative to canonical-BS treatments.

[0656] In some aspects, RNA processing methods of the disclosure include incubating one or more RNA molecules in a bisulfite solution, where the bisulfite solution comprises ammonium bisulfite or ammonium sulfite. In some aspects, the bisulfite solution comprises sodium bisulfite, sodium sulfite, sodium metabisulfite, potassium metabisulfite, potassium bisulfite, guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, and / or potassium sulfite.

[0657] In certain aspects a solution (e.g., bisulfite solution) of the disclosure comprises between 30% and 80%, 40% and 80%, 50% and 80%, or 50% and 72% ammonium bisulfite by weight, including any range or value derivable therein. In some aspects, the solution comprises at least, at most, or about 30%, 30.1%, 30.2%, 30.3%, 30.4%, 30.5%, 30.6%, 30.7%, 30.8%, 30.9%, 31%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.6%, 31.7%, 31.8%, 31.9%, 32%, 32.1%, 32.2%, 32.3%, 32.4%, 32.5%, 32.6%, 32.7%, 32.8%, 32.9%, 33%, 33.1%, 33.2%, 33.3%, 33.4%, 33.5%, 33.6%, 33.7%, 33.8%, 33.9%, 34%, 34.1%, 34.2%, 34.3%, 34.4%, 34.5%, 34.6%, 34.7%, 34.8%, 34.9%, 35%, 35.1%, 35.2%, 35.3%, 35.4%, 35.5%, 35.6%, 35.7%, 35.8%, 35.9%, 36%, 36.1%, 36.2%, 36.3%, 36.4%, 36.5%, 36.6%, 36.7%, 36.8%, 36.9%, 37%, 37.1%, 37.2%, 37.3%, 37.4%, 37.5%, 37.6%, 37.7%, 37.8%, 37.9%, 38%, 38.1%, 38.2%, 38.3%, 38.4%, 38.5%, 38.6%, 38.7%, 38.8%, 38.9%, 39%, 39.1%, 39.2%, 39.3%, 39.4%, 39.5%, 39.6%, 39.7%, 39.8%, 39.9%, 40%, 40.1%, 40.2%, 40.3%, 40.4%, 40.5%, 40.6%, 40.7%, 40.8%, 40.9%, 41%, 41.1%, 41.2%, 41.3%, 41.4%, 41.5%, 41.6%, 41.7%, 41.8%, 41.9%, 42%, 42.1%, 42.2%, 42.3%, 42.4%, 42.5%, 42.6%, 42.7%, 42.8%, 42.9%, 43%, 43.1%, 43.2%, 43.3%, 43.4%, 43.5%, 43.6%, 43.7%, 43.8%, 43.9%, 44%, 44.1%, 44.2%, 44.3%, 44.4%, 44.5%, 44.6%, 44.7%, 44.8%, 44.9%, 45%, 45.1%, 45.2%, 45.3%, 45.4%, 45.5%, 45.6%, 45.7%, 45.8%, 45.9%, 46%, 46.1%, 46.2%, 46.3%, 46.4%, 46.5%, 46.6%, 46.7%, 46.8%, 46.9%, 47%, 47.1%, 47.2%, 47.3%, 47.4%, 47.5%, 47.6%, 47.7%, 47.8%, 47.9%, 48%, 48.1%, 48.2%, 48.3%, 48.4%, 48.5%, 48.6%, 48.7%, 48.8%, 48.9%, 49%, 49.1%, 49.2%, 49.3%, 49.4%, 49.5%, 49.6%, 49.7%, 49.8%, 49.9%, 50%, 50.1%, 50.2%, 50.3%,298000704.1- 100 -ARCD.P0841WO / 24-T-190 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%, 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%, 55.1%, 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%, 56.7%, 56.8%, 56.9%, 57%, 57.1%, 57.2%, 57.3%, 57.4%, 57.5%, 57.6%, 57.7%, 57.8%, 57.9%, 58%, 58.1%, 58.2%, 58.3%, 58.4%, 58.5%, 58.6%, 58.7%, 58.8%, 58.9%, 59%, 59.1%, 59.2%, 59.3%, 59.4%, 59.5%, 59.6%, 59.7%, 59.8%, 59.9%, 60%, 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%, 61.6%, 61.7%, 61.8%, 61.9%, 62%, 62.1%, 62.2%, 62.3%, 62.4%, 62.5%, 62.6%, 62.7%, 62.8%, 62.9%, 63%, 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%, 64.7%, 64.8%, 64.9%, 65%, 65.1%, 65.2%, 65.3%, 65.4%, 65.5%, 65.6%, 65.7%, 65.8%, 65.9%, 66%, 66.1%, 66.2%, 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%, 70%, 70%, 70.1%, 70.2%, 70.3%, 70.4%, 70.5%, 70.6%, 70.7%, 70.8%, 70.9%, 71%, 71.1%, 71.2%, 71.3%, 71.4%, 71.5%, 71.6%, 71.7%, 71.8%, 71.9%, 72%, 72.1%, 72.2%, 72.3%, 72.4%, 72.5%, 72.6%, 72.7%, 72.8%, 72.9%, or 73% ammonium bisulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises at least, at most, 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%, 66.2%, 66.21%, 66.22%, 66.23%, 66.24%, 66.25%, 66.26%, 66.27%, 66.28%, 66.29%, 66.3%, 66.31%, 66.32%, 66.33%, 66.34%, 66.35%, 66.36%, 66.37%, 66.38%, 66.39%, 66.4%, 66.41%, 66.42%, 66.43%, 66.44%, 66.45%, 66.46%, 66.47%, 66.48%, 66.49%, 66.5%, 66.51%, 66.52%, 66.53%, 66.54%, 66.55%, 66.56%, 66.57%, 66.58%, 66.59%, 66.6%, 66.61%, 66.62%, 66.63%, 66.64%, 66.65%, 66.66%, 66.67%, 66.68%, 66.69%, 66.7%, 66.71%, 66.72%, 66.73%, 66.74%, 66.75%, 66.76%, 66.77%, 66.78%, 66.79%, 66.8%, 66.81%, 66.82%, 66.83%, 66.84%, 66.85%, 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% ammonium bisulfite by weight, or any range or value derivable therein.

[0658] In some aspects, a solution (e.g., bisulfite solution) of the disclosure comprises bisulfite, ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium metabisulfite (Na2S2O5), sodium bisulfite (NaHSO3),298000704.1- 101 -ARCD.P0841WO / 24-T-190 sodium sulfite (Na2SO3), sodium thiosulfate (Na2S2O3), potassium thiosulfate (K2S2O3), potassium metabisulfite (K2S2O5), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, and / or potassium sulfite (K2SO3), at a concentration between 4 M and 5 M, including any range or value derivable therein. In some aspects, solutions of the disclosure do not comprise added sodium thiosulfate (Na2S2O3) and / or added potassium thiosulfate (K2S2O3). In some aspects, the solution comprises a concentration of bisulfite, ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), sodium metabisulfite (Na2S2O5), sodium bisulfite (NaHSO3), sodium sulfite (Na2SO3), potassium metabisulfite (K2S2O5), potassium bisulfite (KHSO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, and / or potassium sulfite (K2SO3), of, of at least, of at most, or of about 1.61 M, 1.62 M, 1.63 M, 1.64 M, 1.65 M, 1.66 M, 1.67 M, 1.68 M, 1.69 M, 1.7 M, 1.71 M, 1.72 M, 1.73 M, 1.74 M, 1.75 M, 1.76 M, 1.77 M, 1.78 M, 1.79 M, 1.8 M, 1.81 M, 1.82 M, 1.83 M, 1.84 M, 1.85 M, 1.86 M, 1.87 M, 1.88 M, 1.89 M, 1.9 M, 1.91 M, 1.92 M, 1.93 M, 1.94 M, 1.95 M, 1.96 M, 1.97 M, 1.98 M, 1.99 M, 2 M, 2.01 M, 2.02 M, 2.03 M, 2.04 M, 2.05 M, 2.06 M, 2.07 M, 2.08 M, 2.09 M, 2.1 M, 2.11 M, 2.12 M, 2.13 M, 2.14 M, 2.15 M, 2.16 M, 2.17 M, 2.18 M, 2.19 M, 2.2 M, 2.21 M, 2.22 M, 2.23 M, 2.24 M, 2.25 M, 2.26 M, 2.27 M, 2.28 M, 2.29 M, 2.3 M, 2.31 M, 2.32 M, 2.33 M, 2.34 M, 2.35 M, 2.36 M, 2.37 M, 2.38 M, 2.39 M, 2.4 M, 2.41 M, 2.42 M, 2.43 M, 2.44 M, 2.45 M, 2.46 M, 2.47 M, 2.48 M, 2.49 M, 2.5 M, 2.51 M, 2.52 M, 2.53 M, 2.54 M, 2.55 M, 2.56 M, 2.57 M, 2.58 M, 2.59 M, 2.6 M, 2.61 M, 2.62 M, 2.63 M, 2.64 M, 2.65 M, 2.66 M, 2.67 M, 2.68 M, 2.69 M, 2.7 M, 2.71 M, 2.72 M, 2.73 M, 2.74 M, 2.75 M, 2.76 M, 2.77 M, 2.78 M, 2.79 M, 2.8 M, 2.81 M, 2.82 M, 2.83 M, 2.84 M, 2.85 M, 2.86 M, 2.87 M, 2.88 M, 2.89 M, 2.9 M, 2.91 M, 2.92 M, 2.93 M, 2.94 M, 2.95 M, 2.96 M, 2.97 M, 2.98 M, 2.99 M, 3 M, 3.01 M, 3.02 M, 3.03 M, 3.04 M, 3.05 M, 3.06 M, 3.07 M, 3.08 M, 3.09 M, 3.1 M, 3.11 M, 3.12 M, 3.13 M, 3.14 M, 3.15 M, 3.16 M, 3.17 M, 3.18 M, 3.19 M, 3.2 M, 3.21 M, 3.22 M, 3.23 M, 3.24 M, 3.25 M, 3.26 M, 3.27 M, 3.28 M, 3.29 M, 3.3 M, 3.31 M, 3.32 M, 3.33 M, 3.34 M, 3.35 M, 3.36 M, 3.37 M, 3.38 M, 3.39 M, 3.4 M, 3.41 M, 3.42 M, 3.43 M, 3.44 M, 3.45 M, 3.46 M, 3.47 M, 3.48 M, 3.49 M, 3.5 M, 3.51 M, 3.52 M, 3.53 M, 3.54 M, 3.55 M, 3.56 M, 3.57 M, 3.58 M, 3.59 M, 3.6 M, 3.61 M, 3.62 M, 3.63 M, 3.64 M, 3.65 M, 3.66 M, 3.67 M, 3.68 M, 3.69 M, 3.7 M, 3.71 M, 3.72 M, 3.73 M, 3.74 M, 3.75 M, 3.76 M, 3.77 M, 3.78 M, 3.79 M, 3.8 M, 3.81 M, 3.82 M, 3.83 M, 3.84 M, 3.85 M, 3.86 M, 3.87 M, 3.88 M, 3.89 M, 3.9 M, 3.91 M, 3.92 M, 3.93 M, 3.94 M, 3.95 M, 3.96 M, 3.97 M, 3.98 M, 3.99 M, 4.0 M, 4.01 M, 4.02 M, 4.03 M, 4.04 M, 4.05 M, 4.06 M, 4.07 M, 4.08 M, 4.09 M, 4.1 M, 4.11 M, 4.12 M, 4.13 M, 4.14 M, 4.15 M, 4.16 M,298000704.1- 102 -ARCD.P0841WO / 24-T-190 4.17 M, 4.18 M, 4.19 M, 4.2 M, 4.21 M, 4.22 M, 4.23 M, 4.24 M, 4.25 M, 4.26 M, 4.27 M, 4.28 M, 4.29 M, 4.3 M, 4.31 M, 4.32 M, 4.33 M, 4.34 M, 4.35 M, 4.36 M, 4.37 M, 4.38 M, 4.39 M, 4.4 M, 4.41 M, 4.42 M, 4.43 M, 4.44 M, 4.45 M, 4.46 M, 4.47 M, 4.48 M, 4.49 M, 4.5 M, 4.51 M, 4.52 M, 4.53 M, 4.54 M, 4.55 M, 4.56 M, 4.57 M, 4.58 M, 4.59 M, 4.6 M, 4.61 M, 4.62 M, 4.63 M, 4.64 M, 4.65 M, 4.66 M, 4.67 M, 4.68 M, 4.69 M, 4.7 M, 4.71 M, 4.72 M, 4.73 M, 4.74 M, 4.75 M, 4.76 M, 4.77 M, 4.78 M, 4.79 M, 4.8 M, 4.81 M, 4.82 M, 4.83 M, 4.84 M, 4.85 M, 4.86 M, 4.87 M, 4.88 M, 4.89 M, 4.9 M, 4.91 M, 4.92 M, 4.93 M, 4.94 M, 4.95 M, 4.96 M, 4.97 M, 4.98 M, 4.99 M, 5 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7.0 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8.0 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9.0 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10.0 M, 10.1 M, 10.2 M, 10.3 M, 10.4 M, 10.5 M, 10.6 M, 10.7 M, 10.8 M, 10.9 M, 11.0 M, 11.1 M, 11.2 M, 11.3 M, 11.4 M, 11.5 M, 11.6 M, 11.7 M, 11.8 M, 11.9 M, 12.0 M, or any concentration or range of concentrations derivable therein. In some aspects, a solution comprises both a source of bisulfite (e.g., potassium metabisulfite, sodium metabisulfite, etc.) and a buffer, such as an acidic or a basic buffer (e.g., a KOH buffer, etc.). In some aspects, the solution comprises a concentration sodium metabisulfite of, of at least, of at most, or of about 4.42 M.

[0659] In some aspects, the solution comprises a concentration of bisulfite greater than or equal to 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7.0 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8.0 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9.0 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10.0 M, 10.1 M, 10.2 M, 10.3 M, 10.4 M, 10.5 M, 10.6 M, 10.7 M, 10.8 M, 10.9 M, 11.0 M, 11.1 M, 11.2 M, 11.3 M, 11.4 M, 11.5 M, 11.6 M, 11.7 M, 11.8 M, 11.9 M, or 12.0 M, or any concentration or range of concentrations derivable therein.

[0660] In some aspects, a solution (e.g., bisulfite solution) of the disclosure comprises ammonium sulfite monohydrate at a concentration between 447 mM and 747 mM, including any range or value derivable therein. In some aspects, the solution comprises a concentration of ammonium sulfite monohydrate of, of at least, of at most, or of about 447 mM, 448 mM, 449 mM, 450 mM, 451 mM, 452 mM, 453 mM, 454 mM, 455 mM, 456 mM, 457 mM, 458 mM, 459 mM, 460 mM, 461 mM, 462 mM, 463 mM, 464 mM, 465 mM, 466 mM, 467 mM, 468 mM, 469 mM, 470 mM, 471 mM, 472 mM, 473 mM, 474 mM, 475 mM, 476 mM, 477 mM, 478 mM, 479 mM, 480 mM, 481 mM, 482 mM, 483 mM, 484 mM, 485 mM, 486 mM, 487 mM, 488 mM, 489 mM, 490 mM, 491 mM, 492 mM, 493 mM, 494 mM, 495 mM, 496298000704.1- 103 -ARCD.P0841WO / 24-T-190 mM, 497 mM, 498 mM, 499 mM, 500 mM, 501 mM, 502 mM, 503 mM, 504 mM, 505 mM, 506 mM, 507 mM, 508 mM, 509 mM, 510 mM, 511 mM, 512 mM, 513 mM, 514 mM, 515 mM, 516 mM, 517 mM, 518 mM, 519 mM, 520 mM, 521 mM, 522 mM, 523 mM, 524 mM, 525 mM, 526 mM, 527 mM, 528 mM, 529 mM, 530 mM, 531 mM, 532 mM, 533 mM, 534 mM, 535 mM, 536 mM, 537 mM, 538 mM, 539 mM, 540 mM, 541 mM, 542 mM, 543 mM, 544 mM, 545 mM, 546 mM, 547 mM, 548 mM, 549 mM, 550 mM, 551 mM, 552 mM, 553 mM, 554 mM, 555 mM, 556 mM, 557 mM, 558 mM, 559 mM, 560 mM, 561 mM, 562 mM, 563 mM, 564 mM, 565 mM, 566 mM, 567 mM, 568 mM, 569 mM, 570 mM, 571 mM, 572 mM, 573 mM, 574 mM, 575 mM, 576 mM, 577 mM, 578 mM, 579 mM, 580 mM, 581 mM, 582 mM, 583 mM, 584 mM, 585 mM, 586 mM, 587 mM, 588 mM, 589 mM, 590 mM, 591 mM, 592 mM, 593 mM, 594 mM, 595 mM, 596 mM, 597 mM, 598 mM, 599 mM, 600 mM, 601 mM, 602 mM, 603 mM, 604 mM, 605 mM, 606 mM, 607 mM, 608 mM, 609 mM, 610 mM, 611 mM, 612 mM, 613 mM, 614 mM, 615 mM, 616 mM, 617 mM, 618 mM, 619 mM, 620 mM, 621 mM, 622 mM, 623 mM, 624 mM, 625 mM, 626 mM, 627 mM, 628 mM, 629 mM, 630 mM, 631 mM, 632 mM, 633 mM, 634 mM, 635 mM, 636 mM, 637 mM, 638 mM, 639 mM, 640 mM, 641 mM, 642 mM, 643 mM, 644 mM, 645 mM, 646 mM, 647 mM, 648 mM, 649 mM, 650 mM, 651 mM, 652 mM, 653 mM, 654 mM, 655 mM, 656 mM, 657 mM, 658 mM, 659 mM, 660 mM, 661 mM, 662 mM, 663 mM, 664 mM, 665 mM, 666 mM, 667 mM, 668 mM, 669 mM, 670 mM, 671 mM, 672 mM, 673 mM, 674 mM, 675 mM, 676 mM, 677 mM, 678 mM, 679 mM, 680 mM, 681 mM, 682 mM, 683 mM, 684 mM, 685 mM, 686 mM, 687 mM, 688 mM, 689 mM, 690 mM, 691 mM, 692 mM, 693 mM, 694 mM, 695 mM, 696 mM, 697 mM, 698 mM, 699 mM, 700 mM, 701 mM, 702 mM, 703 mM, 704 mM, 705 mM, 706 mM, 707 mM, 708 mM, 709 mM, 710 mM, 711 mM, 712 mM, 713 mM, 714 mM, 715 mM, 716 mM, 717 mM, 718 mM, 719 mM, 720 mM, 721 mM, 722 mM, 723 mM, 724 mM, 725 mM, 726 mM, 727 mM, 728 mM, 729 mM, 730 mM, 731 mM, 732 mM, 733 mM, 734 mM, 735 mM, 736 mM, 737 mM, 738 mM, 739 mM, 740 mM, 741 mM, 742 mM, 743 mM, 744 mM, 745 mM, 746 mM, 747 mM. In some aspects, the solution comprises a concentration of ammonium sulfite monohydrate of, of at least, of at most, or of about 597 mM.

[0661] In some aspects, the bisulfite solution is at a bisulfite concentration of between 4 M and 12 M, including any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of between 4 M and 8 M, including any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of, of at least, of at most, or of about 4.0 M, 4.1 M, 4.2 M, 4.3 M, 4.4 M, 4.5 M, 4.6 M, 4.7 M, 4.8 M, 4.9 M, 5.0298000704.1- 104 -ARCD.P0841WO / 24-T-190 M, 5.1 M, 5.2 M, 5.3 M, 5.4 M, 5.5 M, 5.6 M, 5.7 M, 5.8 M, 5.9 M, 6.0 M, 6.1 M, 6.2 M, 6.3 M, 6.4 M, 6.5 M, 6.6 M, 6.7 M, 6.8 M, 6.9 M, 7.0 M, 7.1 M, 7.2 M, 7.3 M, 7.4 M, 7.5 M, 7.6 M, 7.7 M, 7.8 M, 7.9 M, 8.0 M, 8.1 M, 8.2 M, 8.3 M, 8.4 M, 8.5 M, 8.6 M, 8.7 M, 8.8 M, 8.9 M, 9.0 M, 9.1 M, 9.2 M, 9.3 M, 9.4 M, 9.5 M, 9.6 M, 9.7 M, 9.8 M, 9.9 M, 10.0 M, 10.1 M, 10.2 M, 10.3 M, 10.4 M, 10.5 M, 10.6 M, 10.7 M, 10.8 M, 10.9 M, 11.0 M, 11.1 M, 11.2 M, 11.3 M, 11.4 M, 11.5 M, 11.6 M, 11.7 M, 11.8 M, 11.9 M, 12.0 M or any range or value derivable therein. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 4.3 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 5.1 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 5.4 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 5.5 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 7.0 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 7.2 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 7.37 M. In some aspects, the bisulfite solution is at a bisulfite concentration of or of about 7.5 M.

[0662] In some aspects, a bisulfite solution of the disclosure used for RNA processing comprises between 50% and 80% ammonium sulfite by weight, or any range or value derivable therein. In some aspects, the solution comprises at least, at most, or about 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% ammonium sulfite by weight, or any range or value derivable therein.

[0663] In some aspects, an RNA processing method comprises incubating one or more RNA molecules in a bisulfite solution of the disclosure at a set temperature for a set period of time. In some aspects, the method comprises incubating one or more RNA molecules in a bisulfite solution at a temperature of, of at least, of at most, or of about 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70.0 °C, 70.1 °C, 70.2 °C, 70.3 °C, 70.4 °C, 70.5 °C, 70.6 °C, 70.7 °C, 70.8 °C, 70.9 °C, 71.0 °C, 71.1 °C, 71.2 °C, 71.3 °C, 71.4 °C, 71.5 °C, 71.6 °C, 71.7 °C, 71.8 °C, 71.9 °C, 72.0 °C, 72.1 °C, 72.2 °C, 72.3 °C, 72.4 °C, 72.5 °C, 72.6 °C, 72.7 °C, 72.8 °C, 72.9 °C, 73.0 °C, 73.1 °C, 73.2 °C, 73.3 °C, 73.4 °C, 73.5 °C, 73.6 °C, 73.7 °C, 73.8 °C, 73.9 °C, 74.0 °C, 74.1 °C, 74.2 °C, 74.3 °C, 74.4 °C, 74.5 °C, 74.6 °C, 74.7 °C, 74.8 °C, 74.9 °C, 75.0 °C, 75.1 °C, 75.2 °C, 75.3 °C, 75.4 °C, 75.5 °C, 75.6 °C, 75.7 °C, 75.8 °C, 75.9 °C, 76.0 °C, 76.1 °C, 76.2 °C, 76.3 °C, 76.4 °C, 76.5 °C, 76.6 °C, 76.7 °C, 76.8 °C, 76.9 °C, 77.0 °C, 77.1 °C, 77.2 °C, 77.3 °C, 77.4 °C, 77.5 °C, 77.6 °C, 77.7 °C, 77.8 °C, 77.9 °C, 78.0 °C, 78.1 °C, 78.2 °C, 78.3 °C, 78.4 °C, 78.5 °C, 78.6 °C, 78.7 °C, 78.8 °C, 78.9 °C, 79.0 °C, 79.1 °C,298000704.1- 105 -ARCD.P0841WO / 24-T-190 79.2 °C, 79.3 °C, 79.4 °C, 79.5 °C, 79.6 °C, 79.7 °C, 79.8 °C, 79.9 °C, 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 °C, 84 °C, 84.1 °C, 84.2 °C, 84.3 °C, 84.4 °C, 84.5 °C, 84.6 °C, 84.7 °C, 84.8 °C, 84.9 °C, 85 °C, 85.1 °C, 85.2 °C, 85.3 °C, 85.4 °C, 85.5 °C, 85.6 °C, 85.7 °C, 85.8 °C, 85.9 °C, 86 °C, 86.1 °C, 86.2 °C, 86.3 °C, 86.4 °C, 86.5 °C, 86.6 °C, 86.7 °C, 86.8 °C, 86.9 °C, 87 °C, 87.1 °C, 87.2 °C, 87.3 °C, 87.4 °C, 87.5 °C, 87.6 °C, 87.7 °C, 87.8 °C, 87.9 °C, 88 °C, 88.1 °C, 88.2 °C, 88.3 °C, 88.4 °C, 88.5 °C, 88.6 °C, 88.7 °C, 88.8 °C, 88.9 °C, 89 °C, 89.1 °C, 89.2 °C, 89.3 °C, 89.4 °C, 89.5 °C, 89.6 °C, 89.7 °C, 89.8 °C, 89.9 °C, 90 °C, 90.1 °C, 90.2 °C, 90.3 °C, 90.4 °C, 90.5 °C, 90.6 °C, 90.7 °C, 90.8 °C, 90.9 °C, 91 °C, 91.1 °C, 91.2 °C, 91.3 °C, 91.4 °C, 91.5 °C, 91.6 °C, 91.7 °C, 91.8 °C, 91.9 °C, 92 °C, 92.1 °C, 92.2 °C, 92.3 °C, 92.4 °C, 92.5 °C, 92.6 °C, 92.7 °C, 92.8 °C, 92.9 °C, 93 °C, 93.1 °C, 93.2 °C, 93.3 °C, 93.4 °C, 93.5 °C, 93.6 °C, 93.7 °C, 93.8 °C, 93.9 °C, 94 °C, 94.1 °C, 94.2 °C, 94.3 °C, 94.4 °C, 94.5 °C, 94.6 °C, 94.7 °C, 94.8 °C, 94.9 °C, 95 °C, 95.1 °C, 95.2 °C, 95.3 °C, 95.4 °C, 95.5 °C, 95.6 °C, 95.7 °C, 95.8 °C, 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 range or value derivable therein) for at most or about 20, 19.9, 19.8, 19.7, 19.6, 19.5, 19.4, 19.3, 19.2, 19.1, 19.0, 18.9, 18.8, 18.7, 18.6, 18.5, 18.4, 18.3, 18.2, 18.1, 18.0, 17.9, 17.8, 17.7, 17.6, 17.5, 17.4, 17.3, 17.2, 17.1, 17.0, 16.9, 16.8, 16.7, 16.6, 16.5, 16.4, 16.3, 16.2, 16.1, 16.0, 15.9, 15.8, 15.7, 15.6, 15.5, 15.4, 15.3, 15.2, 15.1, 15, 14.9, 14.8, 14.7, 14.6, 14.5, 14.4, 14.3, 14.2, 14.1, 14, 13.9, 13.8, 13.7, 13.6, 13.5, 13.4, 13.3, 13.2, 13.1, 13, 12.9, 12.8, 12.7, 12.6, 12.5, 12.4, 12.3, 12.2, 12.1, 12, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, 10, 9.9, 9.8, 9.7, 9.6, 9.5, 9.4, 9.3, 9.2, 9.1, 9, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 7, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, or 1 minutes (or any range or value derivable therein). Any combination of the preceding incubation times and temperatures may be used in an RNA processing method of the present disclosure. In some aspects, specific incubation times and / or temperatures may be expressly excluded in an RNA processing method of the present disclosure298000704.1- 106 -ARCD.P0841WO / 24-T-190

[0664] In some aspects, an RNA processing method comprises incubating one or more RNA molecules in a bisulfite solution of the disclosure at a temperature of at least 70 °C for at most 30 minutes, of at least 75 °C for at most 30 minutes, of at least 80 °C for at most 30 minutes, of at least 85 °C for at most 30 minutes, of at least 90 °C for at most 30 minutes, of at least 95 °C for at most 30 minutes, at a temperature of at least 96 °C for at most 30 minutes, at a temperature of at least 97 °C for at most 30 minutes, at a temperature of at least 98 °C for at most 30 minutes, at a temperature of at least 99 °C for at most 30 minutes, of at least 70 °C for at most 25 minutes, of at least 75 °C for at most 25 minutes, of at least 80 °C for at most 25 minutes, of at least 85 °C for at most 25 minutes, of at least 90 °C for at most 25 minutes, of at least 95 °C for at most 25 minutes, at a temperature of at least 96 °C for at most 25 minutes, at a temperature of at least 97 °C for at most 25 minutes, at a temperature of at least 98 °C for at most 25 minutes, at a temperature of at least 99 °C for at most 25 minutes, of at least 70 °C for at most 20 minutes, of at least 75 °C for at most 20 minutes, of at least 80 °C for at most 20 minutes, of at least 85 °C for at most 20 minutes, of at least 90 °C for at most 20 minutes, of at least 95 °C for at most 20 minutes, at a temperature of at least 96 °C for at most 20 minutes, at a temperature of at least 97 °C for at most 20 minutes, at a temperature of at least 98 °C for at most 20 minutes, at a temperature of at least 99 °C for at most 20 minutes, of at least 70 °C for at most 20 minutes, of at least 75 °C for at most 20 minutes, of at least 80 °C for at most 20 minutes, of at least 85 °C for at most 20 minutes, of at least 90 °C for at most 20 minutes, of at least 95 °C for at most 20 minutes, at a temperature of at least 96 °C for at most 20 minutes, at a temperature of at least 97 °C for at most 20 minutes, at a temperature of at least 98 °C for at most 20 minutes, at a temperature of at least 99 °C for at most 20 minutes, of at least 70 °C for at most 12 minutes, of at least 75 °C for at most 12 minutes, of at least 80 °C for at most 12 minutes, of at least 85 °C for at most 12 minutes, of at least 90 °C for at most 12 minutes, of at least 95 °C for at most 12 minutes, at a temperature of at least 96 °C for at most 12 minutes, at a temperature of at least 97 °C for at most 12 minutes, at a temperature of at least 98 °C for at most 12 minutes, at a temperature of at least 99 °C for at most 12 minutes, at a temperature of at least 70 °C for at most 10 minutes, at a temperature of at least 75 °C for at most 10 minutes, at a temperature of at least 80 °C for at most 10 minutes, at a temperature of at least 85 °C for at most 10 minutes, at a temperature of at least 90 °C for at most 10 minutes, at a temperature of at least 95 °C for at most 11 minutes, at a temperature of at least 96 °C for at most 11 minutes, at a temperature of at least 97 °C for at most 11 minutes, at a temperature of at least 98 °C for at most 11 minutes, at a temperature of at least 99 °C for at most 11 minutes, at a temperature of at least 70 °C for at most 10 minutes, at a temperature of at least 75 °C for at most 10 minutes,298000704.1- 107 -ARCD.P0841WO / 24-T-190 at a temperature of at least 80 °C for at most 10 minutes, at a temperature of at least 85 °C for at most 10 minutes, at a temperature of at least 90 °C for at most 10 minutes, at a temperature of at least 95 °C for at most 10 minutes, at a temperature of at least 96 °C for at most 10 minutes, at a temperature of at least 97 °C for at most 10 minutes, at a temperature of at least 98 °C for at most 10 minutes, at a temperature of at least 99 °C for at most 10 minutes, at a temperature of at least 70 °C for at most 9 minutes, at a temperature of at least 75 °C for at most 9 minutes, at a temperature of at least 80 °C for at most 9 minutes, at a temperature of at least 85 °C for at most 9 minutes, at a temperature of at least 90 °C for at most 9 minutes, at a temperature of at least 95 °C for at most 9 minutes, at a temperature of at least 96 °C for at most 9 minutes, at a temperature of at least 97 °C for at most 9 minutes, at a temperature of at least 98 °C for at most 9 minutes, at a temperature of at least 99 °C for at most 9 minutes, at a temperature of at least 70 °C for at most 8 minutes, at a temperature of at least 75 °C for at most 8 minutes, at a temperature of at least 80 °C for at most 8 minutes, at a temperature of at least 85 °C for at most 8 minutes, at a temperature of at least 90 °C for at most 8 minutes, at a temperature of at least 95 °C for at most 8 minutes, at a temperature of at least 96 °C for at most 8 minutes, at a temperature of at least 97 °C for at most 8 minutes, at a temperature of at least 98 °C for at most 8 minutes, at a temperature of at least 99 °C for at most 8 minutes, at a temperature of at least 70 °C for at most 7 minutes, at a temperature of at least 75 °C for at most 7 minutes, at a temperature of at least 80 °C for at most 7 minutes, at a temperature of at least 85 °C for at most 7 minutes, at a temperature of at least 90 °C for at most 7 minutes, at a temperature of at least 95 °C for at most 7 minutes, at a temperature of at least 96 °C for at most 7 minutes, at a temperature of at least 97 °C for at most 7 minutes, at a temperature of at least 98 °C for at most 7 minutes, at a temperature of at least 99 °C for at most 7 minutes, at a temperature of at least 70 °C for at most 6 minutes, at a temperature of at least 75 °C for at most 6 minutes, at a temperature of at least 80 °C for at most 6 minutes, at a temperature of at least 85 °C for at most 6 minutes, at a temperature of at least 90 °C for at most 6 minutes, at a temperature of at least 95 °C for at most 6 minutes, at a temperature of at least 96 °C for at most 6 minutes, at a temperature of at least 97 °C for at most 6 minutes, at a temperature of at least 98 °C for at most 6 minutes, at a temperature of at least 99 °C for at most 6 minutes, at a temperature of at least 70 °C for at most 5 minutes, at a temperature of at least 75 °C for at most 5 minutes, at a temperature of at least 80 °C for at most 5 minutes, at a temperature of at least 85 °C for at most 5 minutes, at a temperature of at least 90 °C for at most 5 minutes, at a temperature of at least 95 °C for at most 5 minutes, at a temperature of at least 96 °C for at most 5 minutes, at a298000704.1- 108 -ARCD.P0841WO / 24-T-190 temperature of at least 97 °C for at most 5 minutes, at a temperature of at least 98 °C for at most 5 minutes, or at a temperature of at least 99 °C for at most 5 minutes.

[0665] As disclosed herein, incubating RNA molecules with a bisulfite solution of the present disclosure under appropriate conditions is sufficient to deaminate a majority of cytosine residues in the RNA molecules or cause ring-opening in Ψ-BS adducts. In some aspects, after incubating RNA molecules with a bisulfite solution of the present disclosure under appropriate conditions, greater than 90% of the RNA molecules comprise no cytosine residue. In some aspects, greater than 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%, or 99.9% (or any range or value derivable therein) of the RNA molecules comprise no cytosine residue. In some aspects, greater than 99% of the RNA molecules comprise no cytosine residue. In some aspects, after incubating RNA molecules with a bisulfite solution of the present disclosure under appropriate conditions, about 14 to 45% of the RNA molecules comprise no Ψ residue. In some aspects, about 20% of the RNA molecules comprise no Ψ residue. In some aspects, about 25% of the RNA molecules comprise no Ψ residue. In some aspects, about 30% of the RNA molecules comprise no Ψ residue. In some aspects, about 35% of the RNA molecules comprise no Ψ residue. In some aspects, about 40% of the RNA molecules comprise no Ψ residue. In some aspects, about 45% of the RNA molecules comprise no Ψ residue. In some aspects, about 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%, 14.7%, 14.8%, 14.9%, 15.0%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%, 15.9%, 16.0%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17.0%, 17.1%, 17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18.0%, 18.1%, 18.2%, 18.3%, 18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19.0%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%, 19.6%, 19.7%, 19.8%, 19.9%, 20.0%, 20.1%, 20.2%, 20.3%, 20.4%, 20.5%, 20.6%, 20.7%, 20.8%, 20.9%, 21.0%, 21.1%, 21.2%, 21.3%, 21.4%, 21.5%, 21.6%, 21.7%, 21.8%, 21.9%, 22.0%, 22.1%, 22.2%, 22.3%, 22.4%, 22.5%, 22.6%, 22.7%, 22.8%, 22.9%, 23.0%, 23.1%, 23.2%, 23.3%, 23.4%, 23.5%, 23.6%, 23.7%, 23.8%, 23.9%, 24.0%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25.0%, 25.1%, 25.2%, 25.3%, 25.4%,298000704.1- 109 -ARCD.P0841WO / 24-T-190 25.5%, 25.6%, 25.7%, 25.8%, 25.9%, 26.0%, 26.1%, 26.2%, 26.3%, 26.4%, 26.5%, 26.6%, 26.7%, 26.8%, 26.9%, 27.0%, 27.1%, 27.2%, 27.3%, 27.4%, 27.5%, 27.6%, 27.7%, 27.8%, 27.9%, 28.0%, 28.1%, 28.2%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29.0%, 29.1%, 29.2%, 29.3%, 29.4%, 29.5%, 29.6%, 29.7%, 29.8%, 29.9%, 30.0%, 30.1%, 30.2%, 30.3%, 30.4%, 30.5%, 30.6%, 30.7%, 30.8%, 30.9%, 31.0%, 31.1%, 31.2%, 31.3%, 31.4%, 31.5%, 31.6%, 31.7%, 31.8%, 31.9%, 32.0%, 32.1%, 32.2%, 32.3%, 32.4%, 32.5%, 32.6%, 32.7%, 32.8%, 32.9%, 33.0%, 33.1%, 33.2...

Claims

ARCD.P0841WO / 24-T-190 WHAT IS CLAIMED IS:

1. A composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 M and 12 M and the pH of the composition is between about 4.0 and 8.

0.

2. The composition of claim 1, wherein the concentration of bisulfite in the composition is greater than or about 6 M, greater than or about 9.65 M, greater than or about 9.8 M, or between about 9 M and 10.5 M, and the pH of the composition is between about 5.0 and 7.

0.

3. The composition of claim 1, wherein the pH of the composition is between about 4.9 and 5.3, is about 4.9, is about 5.1, or is about 5.

3.

4. The composition of claim 1, wherein the bisulfite solution comprises ammonium bisulfite (NH4HSO3), ammonium sulfite ((NH4)2SO3), ammonium sulfite monohydrate ((NH4)2SO3•H2O), potassium metabisulfite (K2S2O5), sodium metabisulfite (Na2S2O5), sodium bisulfite (NaHSO3), sodium sulfite (Na2SO3), potassium bisulfite (KHSO3), potassium sulfite (K2SO3), guanidinium bisulfite, guanidinium hydrogen sulfite, guanidinium ammonium bisulfite, or a combination thereof.

5. The composition of claims 1, comprising ammonium bisulfite, potassium hydroxide (KOH), tetrahydrofurfuryl alcohol, and Trolox, optionally wherein the final concentration of Trolox is between 0.1% and 4% w / v, and optionally further comprising sodium bisulfite.

6. The composition of claim 1, wherein the bisulfite solution comprises, consists essentially of, or consists of ammonium bisulfite and water.

7. The composition of claim 1, wherein the bisulfite solution comprises about 30-80%, 40-80%, 50-80%, 68-72%, 68%, 69%, 70%, 71%, or 72%, w / v ammonium bisulfite.

8. The composition of claim 1, wherein the composition comprises ammonium bisulfite at about 69-69.5% w / v, or about 69.3% w / v, or about 68-68.5% w / v, or about 68.3% w / v.

9. The composition of claim 1, wherein the base or acid solution comprises potassium hydroxide (KOH), sulfuric acid (H2SO4), concentrated hydrochloric acid (HCl), sodium hydroxide (NaOH), lithium hydroxide (LiOH), or ammonium hydroxide (NH4OH).298000704.1170ARCD.P0841WO / 24-T-190 10. The composition of claim 1, wherein the base or acid solution comprises, consists essentially of, or consists of potassium hydroxide (KOH) and water, at a KOH concentration between about 15-30 M, about 15-25 M, about 15-20 M, or about 20 M.

11. The composition of claim 9, wherein the base or acid solution comprises a pH of about 11.0 to 14.0, about 11.5 to 14.0, or about 12.0 to 13.

5.

12. The composition of claim 1, further comprising a bisulfite stabilizer.

13. The composition of claim 1, further comprising an organic solvent.

14. The composition of claim 1, further comprising tetrahydrofuran (THF), tetrahydrofurfuryl alcohol (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof.

15. The composition of claim 1, further comprising between 0.1-20% tetrahydrofuran (THF), tetrahydrofurfuryl alcohol (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1- butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof.

16. The composition of claim 1, wherein the composition is clear and has little-to-no precipitation at or below room temperature, and / or the composition is clear and / or is substantially free of precipitate at about 1-7 °C or at about 4 °C.

17. The composition of claim 1, further comprising nucleic acids.

18. The composition of claim 17, wherein the composition comprises 20% to 75% w / v bisulfite at a molarity of about 3 M to 11 M.

19. The composition of claim 17, wherein the composition comprises 30% to 72% w / v bisulfite at a molarity of about 4 M to 10.5 M, 45 to 60% w / v bisulfite at a molarity of about 6.5 to 10.5 M, 50 to 55 % w / v bisulfite at a molarity of about 7.2 to 7.5 M, or 52 to 53 % w / v bisulfite at a molarity of about 7.4 M.298000704.1171ARCD.P0841WO / 24-T-190 20. The composition of claim 17, wherein the nucleic acid comprises deoxyribonucleic acid (DNA).

21. The composition of claim 20, wherein the DNA comprises 5-methylcytosine (5mC) modifications and / or the DNA comprises 5mC modifications and cytosine to uracil (C-to-U) conversions.

22. The composition of claim 21, wherein greater than 90%, 95%, 96%, 97%, 98%, or 99%, of the DNA cytosines are converted to uracil.

23. The composition of claim 1, further comprising an antioxidant.

24. The composition of claim 1, further comprising Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), hydroquinone, vitamin C, glutathione, allium sulfur compounds, beta-carotene, or a combination thereof.

25. The composition of claim 1, further comprising Trolox dissolved in tetrahydrofurfuryl alcohol (THFA).

26. The composition of claim 1, further comprising (w / v) 0.01-25%, or 0.1-20%, or 5 to 15% w / v of Trolox dissolved in THFA.

27. The composition of claim 20, wherein the DNA comprises a low level of DNA damage, optionally wherein the DNA damage is measured as DNA fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

28. The composition of claim 1, further comprising a stabilizer of single stranded nucleic acids.

29. The composition of claim 28, wherein the stabilizer of single stranded nucleic acids comprises between about 0-30% of the composition.

30. The composition of claim 29, wherein the stabilizer of single stranded nucleic acids comprises methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofuran (THF), tetrahydrofurfuryl alcohol (THFA), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof.298000704.1172ARCD.P0841WO / 24-T-190 31. The composition of claim 1, further comprising THFA at about 0.01-25%, 10%, 11%, 12%, 13%, 14%, or 15% w / v.

32. The composition of claim 17 wherein the majority (greater than 50%) of the nucleic acid molecules exist in a single stranded or denatured state.

33. A method of detecting 5mC modifications in DNA, the method comprising incubation of the DNA with the composition according to any one of claims 1-32, and then sequencing the DNA.

34. The composition of claim 17, wherein the nucleic acid comprises ribonucleic acid (RNA).

35. The composition of claim 34, further comprising a bisulfite stabilizer, a stabilizer of single stranded nucleic acids, and / or an antioxidant.

36. The composition of claim 34, wherein the RNA comprises 5-methylcytosine (m5C) modifications and / or wherein the RNA comprises m5C modifications and cytosine to uracil (C-to-U) conversions.

37. The composition of claim 36, wherein greater than 90%, 95%, 96%, 97%, 98%, or 99% of the RNA cytosines are converted to uracil.

38. A method of detecting m5C modifications in RNA, the method comprising incubation of the RNA with the composition according to any one of claims 1-32 or 34-37, and then sequencing the RNA.

39. A method for converting cytosine to uracil (C-to-U conversion) in a polynucleotide, the method comprising incubating a solution comprising a polynucleotide and bisulfite, wherein the solution is a) at a bisulfite concentration of about 4 M to 12 M, b) at a pH between about 4.5-6.5, c) at a temperature between about 37 to 98 °C, or 70 to 98 °C, and d) incubated between about 0.5-180 minutes.

40. The method of claim 39, wherein the bisulfite comprises ammonium bisulfite.298000704.1173ARCD.P0841WO / 24-T-190 41. The method of claim 39, wherein the bisulfite concentration is greater than or equal to 6 M, about or greater than 9.65 M, about or greater than 9.8 M, or between about 9 M to 10 M, and the pH is between about 4.5 to 6.5 or about 4.9 to 5.

3.

42. The method of claim 39, wherein the pH is adjusted using potassium, hydroxide (KOH), optionally wherein the potassium hydroxide is provided as a solution at a concentration of about 10 M to 25 M, or about 20 M.

43. The method of claim 39, wherein the temperature is about 37, 55, 70, 75, 80, 85, 90, 95, or 98 °C, or is about 70-98 °C.

44. The method of claim 39, wherein the incubation time is, or is between any two of 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 120, or 150 minutes.

45. The method of claim 39, wherein the polynucleotide is DNA and / or RNA, and wherein the polynucleotide comprises a low level of damage, optionally wherein the damage is assessed as RNA and / or DNA fragmentation, optionally using gel electrophoresis and / or a fragment analyzer.

46. A method of detecting 5-methylcytosine in a polynucleotide comprising sequencing the polynucleotide of the composition of claim 39 after the incubation.

47. The method of claim 46, wherein the polynucleotide comprises a total mass less than about 50 nanograms, or between about 0.01 – 10 nanograms, or about 0.01, 0.05, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nanograms.

48. The method of claim 39, wherein the C-to-U unconverted rate is less than about 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

49. The method of claim 39, further comprising a denaturation step that occurs prior to the step of incubating a solution comprising a polynucleotide with bisulfite.

50. The method of claim 49, wherein the denaturation step comprises incubating a solution comprising a polynucleotide with a solution comprising KOH, NaOH, LiOH, NH4OH, and / or another strong base.

51. The method of claim 50, wherein the solution comprising KOH comprises KOH at a starting concentration of about 1.0-3.0 M.298000704.1174ARCD.P0841WO / 24-T-190 52. The method of claim 51, wherein the composition comprising a polynucleotide and KOH comprises a final KOH concentration between about 0.1-1.0 M, or about 0.3 M.

53. The method of claim 50, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH at or between about 35-55 °C, or about 42 °C.

54. The method of claim 50, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with KOH for 1-40 minutes, or for 20 minutes.

55. The method of claim 50, wherein the denaturation step comprises incubating the solution comprising a polynucleotide with a KOH solution at a final concentration of KOH of about 0.3 M at about 42 °C for 20 minutes.

56. The method of claim 39, further comprising a desulphonation step that occurs after the step of incubating a solution comprising a polynucleotide with bisulfite.

57. The method of claim 56, wherein the desulphonation step comprises in-column or on- bead desulphonation.

58. A polynucleotide processing kit comprising: a) a first component comprising a first solution comprising ammonium bisulfite and water having a bisulfite concentration between about 30-80%, 40-80%, 50-80%, or 60- 72% w / v; b) a second component comprising a potassium hydroxide salt or a second solution comprising potassium hydroxide and water having a potassium hydroxide concentration of about 10 M, 15 M, or 20 M; and c) optionally instructions for processing a polynucleotide sample.

59. The kit of claim 58, wherein the ammonium bisulfite solution has an ammonium bisulfite concentration of about 70%, 71%, or 72% w / v.

60. The kit of claim 58, further comprising a third component comprising a KOH solution comprising a KOH concentration of about 1.0-5.0 M, or about 3.0 M.

61. The kit of claim 58, further comprising a component comprising a bisulfite stabilizer, optionally wherein the bisulfite stabilizer comprises an organic solvent.298000704.1175ARCD.P0841WO / 24-T-190 62. The kit of claim 61, wherein the bisulfite stabilizer comprises 0.01-20% methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), tetrahydrofurfuryl alcohol (THFA), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2- Hexanediol, isopropanol, or a combination thereof.

63. The kit of claim 58, further comprising a component comprising a stabilizer of single stranded nucleic acids.

64. The kit of claim 63, wherein the stabilizer of single stranded nucleic acids comprises tetrahydrofurfuryl alcohol (THFA), methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- butanol, acetone, acetonitrile, dimethyl sulfoxide (DMSO), formamide, dimethylformamide (DMF), dioxane, dimethoxyethane (DME), glycerol, PEG-400 / PEG-600, propylene glycol, 1,2-Hexanediol, isopropanol, or a combination thereof.

65. The kit of claim 63, wherein the stabilizer of single stranded nucleic acids comprises THFA.

66. The kit of claim 58, further comprising a component comprising a desulphonation solution.

67. The kit of claim 58, further comprising a component comprising an antioxidant.

68. The kit of claim 67, wherein the antioxidant comprises Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), hydroquinone, vitamin C, glutathione, allium sulfur compounds, beta-carotene, or a combination thereof.

69. The kit of claim 67, wherein the antioxidant comprises Trolox dissolved in THFA.

70. The kit of claim 67, wherein the antioxidant comprises 0.01-25%, or 0.1-20%, or 5 to 15% w / v of Trolox dissolved in THFA.

71. The kit of claim 58, further comprising desulphonation columns or beads.

72. The kit of claim 58, wherein the first and second components are provided as a premixed solution and the premixed solution is stable at or below room temperature and / or forms little to no precipitate.298000704.1176ARCD.P0841WO / 24-T-190 73. The kit of claim 72, wherein the premixed solution comprises a bisulfite concentration between about 6 M to 12 M, or about 9 M to 10 M, or a bisulfite concentration greater than 9.65 M, or is about 9.8 M.

74. The kit of claim 72, wherein the premixed solution has a pH between about 4 to 8, between about 4.5 to 6, between about 4.9 to 5.3, or about 4.9, 5.1, or 5.

3.

75. The kit of claim 58, further comprising one or more buffer solutions and / or enzymes.

76. A composition comprising a mixture of two or more sulfites, wherein the final concentration of bisulfite in the composition is between 4 M and 12 M, and the pH value is between 4.0 to 6.

5.

77. The composition of claim 76, comprising ammonium bisulfite, sodium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the final concentration of Trolox is between 0.1% and 4% w / v.

78. The composition of claim 76, wherein the bisulfite concentration is greater than or equal to 6 M.

79. The composition of claim 76, wherein the first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5 also known as sodium pyrosulfite or disodium metabisulfite), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) or ammonium sulfite monohydrate ((NH4)2SO3•H2O).

80. A method of detecting m5C modifications in RNA, the method comprising use of the composition according to claim 76.

81. A method for converting cytosine to uracil in a polynucleotide, the method comprising incubating a composition comprising a polynucleotide and a mixture of two or more sulfites, wherein the composition is a) at a bisulfite concentration of about 4-12 M, b) at a temperature between about 50 to 98 °C, and d) incubated between about 1-180 minutes.

82. A polynucleotide processing kit comprising: a) two or more sulfites; and298000704.1177ARCD.P0841WO / 24-T-190 b) optionally instructions for processing a polynucleotide sample.

83. The kit of claim 82, wherein the first sulfite comprises, consists essentially of, or consists of sodium metabisulfite (Na2S2O5), and the second sulfite comprises, consists essentially of, or consists of ammonium sulfite ((NH4)2SO3) and / or ammonium sulfite monohydrate ((NH4)2SO3•H2O).

84. The kit of claim 82, wherein the first sulfite comprises ammonium bisulfite, and the second sulfite comprises sodium bisulfite, and the kit further comprises KOH, tetrahydrofurfuryl alcohol, and Trolox.

85. The kit of claim 82, further comprising a denaturation solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, and / or an antioxidant solution.

86. A composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between 4 and 10 M, and the pH of the composition is between 6.0 and 8.

5.

87. A method for converting Ψ to Ψ-BS in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein a) the bisulfite concentration is about 4 M to 10 M, b) the pH is between about 6.0 to 8.5, c) the temperature is between about 50 to 98 °C, and d) the incubation is between about 1-180 minutes.

88. A polynucleotide processing kit comprising: a) a first component comprising a mixture of a sulfite solution; b) a second component comprising an acid or base solution; and c) optionally instructions for processing a polynucleotide sample.

89. A composition comprising a mixture of a bisulfite solution and a base or acid solution, wherein the concentration of bisulfite in the composition is between about 3 M and 6 M, and the pH of the composition is between about 5.0 and 6.5298000704.1178ARCD.P0841WO / 24-T-190 90. A method for converting Ψ to Ψ-BS and C to U in an RNA polynucleotide, the method comprising incubating a composition comprising an RNA polynucleotide, a sulfite solution, and a base or acid solution, wherein a) the bisulfite concentration is about 4 M to 6 M, b) the pH is about 5-6.1, c) the temperature is between about 50 to 98 °C, and d) the incubation is between about 1-180 minutes.

91. A composition comprising a mixture of a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and DNA.

92. A composition comprising a mixture of a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, a bisulfite stabilizer, a single stranded nucleic acid stabilizer, an antioxidant, and RNA.

93. A kit comprising a 60-72% ammonium bisulfite solution, an about 20 M KOH solution, an about 3 M KOH solution, a bisulfite stabilizer solution, a single stranded nucleic acid stabilizer solution, a desulphonation solution, an antioxidant solution, and / or desulphonation beads or columns.

94. A composition comprising ammonium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the concentration of Trolox is between 0.1% and 4% w / v.

95. A composition comprising ammonium bisulfite, sodium bisulfite, KOH, tetrahydrofurfuryl alcohol, and Trolox, wherein the concentration of Trolox is between 0.1% and 4% w / v.

96. Use of the composition, kit, or method of any one of the preceding claims in diagnosing, identifying, and / or treating a disease and / or disorder in an individual.298000704.1179