Modified cytidine deaminases and methods of use

Abstract

The present disclosure relates to proteins, methods, compositions, and kits for mapping the methylation state of nucleic acids, including 5-methylcytosine and 5-hydroxymethylcytosine (5hmC). In one embodiment, proteins are provided that selectively act on specific modified cytosines in a target nucleic acid, converting them to thymidine or modified thymidine analogs. In another embodiment, proteins are provided that selectively act on specific modified cytosines in a target nucleic acid, converting them to uracil or thymidine, and not on other specific modified cytosines in the target nucleic acid. Compositions and kits comprising one or more of the proteins, as well as methods of using one or more of the proteins, are also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 328,444, filed April 7, 2022, and U.S. Provisional Application No. 63 / 350,068, filed June 8, 2022, each of which is incorporated by reference in its entirety herein.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically to the U.S. Patent and Trademark Office via EFS-Web as an XML file entitled "0531.002278WO01.xml" which is 126 kilobytes in size and was created on April 7, 2023. The information contained in the Sequence Listing is incorporated herein by reference.

[0003] FIELD OF THEINVENTION Embodiments of the present disclosure relate to preparing nucleic acids for sequencing or other uses. In particular, embodiments of the proteins, methods, compositions, and kits provided herein relate to mapping methylation states through the use of sequencing libraries and other methods. [Background technology]

[0004] Modified DNA cytosines, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), are well-studied epigenetic modifications that play fundamental roles in human development and disease. Their genome-wide distribution differs between tissue types and between healthy and diseased states. In recent years, 5mC has also attracted attention as a tool for clinical diagnostics: its distribution in cell-free DNA (cfDNA) obtained from liquid biopsies can be used for tissue-specific prediction of early cancer or monitoring cancer recurrence or remission after treatment. As a result, there has been a strong focus on developing methods to map modified DNA cytosines at single-base resolution while minimizing the loss in quantity, quality, and complexity of sample DNA. However, current methods for mapping modified DNA cytosines exhibit limitations including: (i) degradation of sample DNA due to prolonged chemical treatment at non-neutral pH and high temperature; (ii) loss of complexity of sample DNA due to conversion of unmethylated DNA bases to uracil, resulting in low complexity genome mapping; (iii) multi-step conversion requiring both enzymatic and chemical treatment; and (iv) antibody-based 5mC detection limits the resolution of detection to approximately 150 bp, preventing identification of its exact location in the genome.

[0005] 5-Hydroxymethylcytosine (5hmC) is an oxidized derivative of the widely studied epigenetic modification 5-methylcytosine (5mC). Increasing evidence supports the biological importance of 5hmC in diverse developmental processes, such as neurogenesis in mammals. Thus, there is widespread interest in determining the localization of 5hmC in DNA from healthy and diseased patients. The majority of methods for mapping 5-hydroxymethylcytosine (5hmC) require bisulfite treatment, which results in significant DNA loss and damage. Recent methods for mapping 5hmC have been developed, such as oxBS-seq, TAB-seq, and ACE-seq, but some involve bisulfite treatment and all involve multiple steps using different enzymes. Summary of the Invention

[0006] The present disclosure provides proteins, methods, compositions, and kits for determining the methylation status of DNA and RNA. Different from current methods for mapping the methylation status of cytosine (C) nucleotides, the present disclosure presents a one-step, fully enzymatic method using modified cytidine deaminases that selectively act on specific modified cytosines of target nucleic acids and convert them to thymidine (T) or modified thymidine analogs. The modified cytidine deaminases described herein circumvent the limitations of currently available methods for mapping methylated cytosine nucleotides because (i) they are active at near-neutral pH and physiological temperature, (ii) unmethylated cytosines react at a reduced rate, thus preserving the DNA complexity of the sample, (iii) the conversion of methylated C to T is a single-step enzymatic reaction, (iv) this process detects methylated C with single-base resolution, and (v) this process preserves DNA complexity and simplifies analysis by next-generation sequencing.

[0007] The present disclosure also provides proteins, methods, compositions, and kits for mapping 5-hydroxymethylcytosine (5hmC) nucleotides present in DNA and RNA. Current methods for mapping the methylation status of 5hmC nucleotides include modifying or blocking 5hmC nucleotides. For example, the ACE-seq method (Schutsky et al., Nature biotechnology, 10.1038 / nbt.4204.8 October 2018, doi:10.1038 / nbt.4204) blocks 5hmC by conversion to 5ghmC using the enzyme β-glucosyltransferase (βGT). Unlike current methods for mapping the methylation status of 5hmC nucleotides, the methods presented herein do not require modifying or blocking 5hmC nucleotides. Instead, the present disclosure presents a one-step, fully enzymatic method that uses modified cytidine deaminases that selectively act on specific modified cytosines in target nucleic acids, converting them to uracil (U) or thymidine (T), but not on 5hmC, 5-formylcytosine (5fC), or 5-carboxycytosine (5-caC). The modified cytidine deaminases described herein circumvent the limitations of currently available methods for mapping 5hmC nucleotides because (i) no harsh chemical treatments that cause significant loss of DNA and RNA are required, (ii) the conversion is a single-step enzymatic reaction, and (iii) the process results in the detection of 5hmC with single-base resolution.

[0008] The present disclosure includes modified cytidine deaminase. In one embodiment, the modified cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 and Tyr132 in a wild-type APOBEC3A protein. In another embodiment, the modified cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 in a wild-type APOBEC3A protein, the substitution mutation being (Tyr / Phe)130Trp. The (Tyr / Phe)130 of the modified cytidine deaminase can be Tyr130, and the wild-type APOBEC3A protein is SEQ ID NO:3. The present disclosure also includes a polynucleotide encoding the modified cytidine deaminase.

[0009] The present disclosure also provides a composition comprising the modified cytidine deaminase described herein. In one embodiment, the composition can further comprise at least one of: (i) a sample comprising DNA comprising at least one modified cytosine, the modified cytosine being 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), or a combination thereof; or (ii) a buffer having a pH of less than 7; or (iii) a combination thereof.

[0010] The present disclosure also provides a method. In one embodiment, the method includes providing a sample of DNA suspected of containing single-stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5CaC), or combinations thereof; contacting the single-stranded DNA with a modified cytidine deaminase under conditions suitable for (i) conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than conversion of cytosine (C) to uracil (U) by deamination, or (ii) conversion of C to U and 5mC to T by deamination at a rate greater than conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination to produce converted single-stranded DNA; and processing the converted single-stranded DNA to produce a sequencing library.

[0011] In another embodiment, the method includes providing a sample of DNA suspected of containing double-stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5caC), or combinations thereof; treating the double-stranded DNA to generate a sequencing library; denaturing the sequencing library to produce single-stranded DNA; (i) converting cytosine (C) to uracil (U) by deamination; or (ii) conversion of C to U and conversion of 5mC to T by deamination at a rate greater than conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination to produce a converted single-stranded DNA, and converting the converted single-stranded DNA into a converted double-stranded DNA sequencing library.

[0012] In one embodiment, a method can include detecting the location of a modified cytosine in a target nucleic acid. The method can include (a) contacting a target nucleic acid suspected of containing at least one modified cytosine with a modified cytidine deaminase according to claim 1 or 2 to generate a converted nucleic acid containing at least one converted cytosine; and (b) detecting the at least one converted cytosine in the converted nucleic acid of (a). The detection can include sequencing the converted nucleic acid or hybridizing a nucleic acid probe to the converted nucleic acid.

[0013] In embodiments in which detection involves sequencing the converted nucleic acid, the method may further include (c) comparing the sequence of the converted nucleic acid to an untreated reference sequence to determine which cytosines in the target nucleic acid have been modified.

[0014] In an embodiment in which the detection comprises hybridizing the converted nucleic acid to a nucleic acid probe, the method can further comprise that the nucleic acid probe can be present on an analyte array, and the method can further comprise sequencing the hybridized converted nucleic acid. In another embodiment, the method can further comprise amplifying the converted nucleic acid, the nucleic acid probe comprises two primers for amplification of a predetermined sequence, the primers anneal to a region of the converted nucleic acid that contains at least one converted cytosine with greater affinity than to a region of the converted nucleic acid in which the at least one cytosine is not a converted cytosine, and the presence of an amplification product indicates a modified cytosine in the target nucleic acid. In another embodiment, the method can further comprise cleaving a single-stranded DNA (ssDNA) reporter substrate by a CRISPR-based system, the ssDNA reporter substrate comprises a fluorophore and a quencher, and the presence of fluorescence indicates a modified cytosine in the target nucleic acid. In another embodiment, the converted nucleic acid can be present in fixed cells, and the nucleic acid probe comprises a fluorescently labeled probe, which anneals to a predetermined sequence of the converted nucleic acid that contains at least one converted cytosine with greater affinity than to regions of the converted nucleic acid in which the at least one cytosine is not a converted cytosine, and the presence of cell-associated fluorescence indicates a modified cytosine in the target nucleic acid.

[0015] In one embodiment of detecting the location of modified cytosines in a target nucleic acid, the target nucleic acid can be obtained from a subject, and detecting can include obtaining a pattern of cytosine modifications in a converted nucleic acid. In some embodiments, the method can further include comparing the pattern of cytosine modifications in the converted nucleic acid with a pattern of cytosine modifications in a reference nucleic acid. For example, the subject can be a subject having or at risk of having a disease or condition, and the reference nucleic acid can be from a normal subject. In one embodiment, the pattern of cytosine modifications is linked in cis to a coding region that correlates with the disease or condition. For example, the pattern of cytosine modifications is linked in cis to a coding region, and the coding region in the reference nucleic acid is transcriptionally active or transcriptionally inactive. Comparing can further include determining whether the pattern of cytosine modifications in the converted nucleic acid indicates that the coding region is transcriptionally active or transcriptionally inactive in the subject. Transcription of the coding region can be correlated with a disease or condition.

[0016] In any method disclosed herein that includes distinct steps, the steps may be performed in any practicable order, and, suitably, any combination of two or more steps may be performed simultaneously.

[0017] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The following description more particularly illustrates exemplary embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. [Brief description of the drawings]

[0018] The following detailed description of the exemplary embodiments of the present disclosure can be best understood when read in conjunction with the following drawings. [Figure 1A]FIG. 1 shows the deamination scheme of APOBEC3A. Cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) nucleobases in single-stranded DNA are well-characterized substrates for APOBEC3A. FIG. 1A shows the conversion of C to uracil (U) by APOBEC3A. [ka] refers to the attachment of a nucleobase to a DNA molecule. [Figure 1B] FIG. 1B shows the deamination scheme of APOBEC3A. Cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) nucleobases in single-stranded DNA are well-characterized substrates of APOBEC3A. FIG. 1B shows the conversion of 5mC to thymidine (T) by APOBEC3A. [ka] refers to the attachment of a nucleobase to a DNA molecule. [Figure 1C] FIG. 1C shows the deamination scheme of APOBEC3A. Cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) nucleobases in single-stranded DNA are well-characterized substrates of APOBEC3A. FIG. 1C shows the conversion of 5hmC to 5-hydroxymethyluracil (5hmU) by APOBEC3A. [ka] refers to the attachment of a nucleobase to a DNA molecule. [Figure 1D] The results of treating DNA samples with wild-type APOBEC3A enzyme are shown (FIG. 1D). The top strands of FIG. 1D-F show C, 5mC, and / or 5hmC bases, and the bottom strands of FIG. 1D-F show the altered bases underlined. In FIG. 1D, 5mC nucleobases are marked with CH3, 5hmC nucleobases are marked with CH2-OH, 5-hydroxymethyluracil nucleobases are indicated with a lower case "u" and uracil nucleobases are indicated with an upper case "U". [Figure 1E]An example of one-step detection of 5mC using modified cytidine deaminases described herein (FIG. 1E) is shown. The top strands of FIG. 1D-F show C, 5mC, and / or 5hmC bases, and the bottom strands of FIG. 1D-F show the altered bases underlined. In FIG. 1D, 5mC nucleobases are marked with CH3, 5hmC nucleobases are marked with CH2-OH, 5-hydroxymethyluracil nucleobases are indicated with lowercase "u" and uracil nucleobases are indicated with uppercase "U". [Figure 1F] An example of one-step detection of 5hmC using different modified cytidine deaminases described herein (FIG. 1F) is shown. The top strands of FIG. 1D-F show C, 5mC, and / or 5hmC bases, and the bottom strands of FIG. 1D-F show the altered bases underlined. In FIG. 1D, 5mC nucleobases are marked with CH3, 5hmC nucleobases are marked with CH2-OH, 5-hydroxymethyluracil nucleobases are indicated with lower case "u" and uracil nucleobases are indicated with upper case "U". [Diagram 2] 1 shows examples of cytosine and modified cytosine nucleobases in DNA. [ka] represents the attachment of a nucleobase to a DNA molecule. [Diagram 3]Schematic diagram showing alignment of cytidine deaminase amino acid sequences using the Clustal O algorithm. "*" (asterisk) indicates positions with a single perfectly conserved residue among all cytidine deaminases. ":" (colon) indicates conservation between groups of very high similarity - approximately equivalent to a scoring of >0.5 in the Gonnet PAM 250 matrix. "." (period) indicates conservation between groups of weak similarity - approximately equivalent to a scoring of =<0.5 and >0 in the Gonnet PAM 250 matrix. Amino acids marked with "^" indicate ZDD motif SEQ ID NO: 12 (e.g., on amino acids 70-106 of sp|P31941|1-199). Amino acids marked with "^" and "#" indicate ZDD motif SEQ ID NO: 13 (e.g., on amino acids 70-153 of sp|P31941|1-199). sp|P31941|1-199 is human APOBEC3A, sequence number 3; XP_045219544.1 is APOBEC3A from Macaca fascicularis, sequence number 19; AER45717.1 is APOBEC3A from Pongo pygmaeus, sequence number 20; XP_003264816.1 is APOBEC3A from Nomascus leucogenys, sequence number 21; PNI48846.1 is APOBEC3A from Pan troglodytes, sequence number 22; and ADO85886.1 is APOBEC3A from Gorilla gorilla, sequence number 23. [Figure 4] Schematic diagram of a restriction enzyme (SwaI)-based assay for deamination by cytidine deaminase. X is H in the context of C, X is methyl in the context of 5mC, and X is hydroxymethyl in the context of 5hmC. "Match oligo" refers to perfect complementarity between the two oligonucleotides. "Mismatch oligo" refers to incomplete complementarity between the two oligonucleotides. Mismatches result in cleavage of the double-stranded oligonucleotide by SwaI. [Figure 5A]Positive control experiment of SwaI-based assay using synthetic oligonucleotides. Figure 5A shows the sequences of the synthesized oligonucleotides: oLB1609, SEQ ID NO:24; oLB1610, SEQ ID NO:25; oLB1611, SEQ ID NO:26; oLB1612, SEQ ID NO:27; oLB1679, SEQ ID NO:28; oJT1910, SEQ ID NO:57; and oJT1911, SEQ ID NO:58. [Figure 5B] A positive control experiment of the SwaI-based assay using synthetic oligonucleotides is shown. Figure 5B shows the visualization of the results of SwaI digestion. "Matched" refers to perfect complementarity between the two oligonucleotides. "Mismatched" refers to incomplete complementarity between the two oligonucleotides. Mismatching results in cleavage of the double-stranded oligonucleotide by SwaI. [Figure 6] A positive control deamination experiment using a commercially available APOBEC3A enzyme is shown. [Figure 7A] 7A shows an SDS-PAGE panel of APOBEC3A(Y130X) protein. FIG 7A shows an SDS-PAGE analysis of purified APOBEC3A(Y130X) mutant protein. [Figure 7B] Figure 7B shows an SDS-PAGE panel of APOBEC3A(Y130X) protein.Figure 7B shows an SDS-PAGE analysis of purified APOBEC3A(Y130A_Y132H) mutant protein. [Figure 8] 1 shows the results of an APOBEC3A(Y130X) deamination endpoint assay panel using a SwaI assay readout. [Figure 9] 1 shows a bar graph representation of APOBEC3A(Y130X) deaminase activity. [Figure 10] Deaminase activity of wild-type (NEB APOBEC) and mutant APOBEC variants on C, 5mC, and 5hmC substrates is shown. Percent deamination values ​​were determined from SwaI restriction enzyme assays and quantified as in Figure 4. C deamination activity was measured in two independent experiments corresponding to the left and right panels. [Figure 11A]Time course C and 5mC deamination reactions of APOBEC3A (Y130A). Figures 11A-C show reactions at 37°C. Figures 11A and 11D) Y130A deamination SwaI-based assays at 37°C and 22°C, respectively, visualized by 15% urea-PAGE using FAM filters. [Figure 11B] Figure 11A-C show reactions at 37°C. Figure 11B and Figure 11E) Graphical representation of Y130A time course C and 5mC deamination reactions at 37°C and 22°C, respectively. [Figure 11C] Time course C and 5mC deamination reactions of APOBEC3A(Y130A). Figures 11A-C show reactions at 37°C. Figures 11C and 11F) Tables showing deamination rates at various time points at 37°C and 22°C, respectively. [Figure 11D] Time course C and 5mC deamination reactions of APOBEC3A (Y130A). Figures 11D-F show reactions at 22°C. Figures 11A and 11D) Y130A deamination SwaI-based assays at 37°C and 22°C, respectively, visualized by 15% urea-PAGE using FAM filters. [Figure 11E] Time course C and 5mC deamination reactions of APOBEC3A (Y130A). Figures 11D-F show reactions at 22°C. Figures 11B and 11E) Graphical representation of Y130A time course C and 5mC deamination reactions at 37°C and 22°C, respectively. [Figure 11F] Time course C and 5mC deamination reactions of APOBEC3A(Y130A). Figures 11D-F show reactions at 22°C. Figures 11C and 11F) Tables showing deamination rates at various time points at 37°C and 22°C, respectively. [Figure 12] 1 shows preliminary Michaelis-Menten kinetics of Y130A toward C and 5mC oligonucleotide substrates. [Figure 13]1 shows DNA oligonucleotide substrates for assessing the deaminase activity of double mutant cytidine deaminases: Set (A), SEQ ID NO: 29; Set (B), SEQ ID NOs: 30, 31, 32, and 36, respectively. [Figure 14] Deamination rates at each NCN motif in DNA oligo substrate (A) after incubation with APOBEC3A mutants (37°C, 6 hour reaction). This metric is calculated as the percentage of C>T (cytosine to thymidine) mutations at each position as determined by DNA sequencing. [Figure 15] The deamination rate at each NCN motif in the DNA oligo substrate (A) after incubation with APOBEC3A mutants (37° C., 1 hour reaction) is shown. This metric is calculated as the percentage of C>T mutations at each position. [Figure 16] Deamination rates at each NCpGN motif in the oligo substrate (B) after incubation with APOBEC3A mutants are shown. Four different DNA oligos were mixed together as substrates for APOBEC3A deamination. Percentage deamination was calculated as the percentage of C>T mutations in each NCpGN motif. Methylated and unmethylated forms of each NCpGN motif were assayed (32 sites in total). [Figure 17A] Figure 17A shows the time course of APOBEC3A(Y130W) deamination for C, 5mC, and 5hmC-containing substrates. A SwaI restriction enzyme assay was performed to measure the deaminase activity of APOBEC3A(Y130W). The deamination rate is calculated as the ratio of the cleaved band intensity to the (uncleaved + cleaved) band intensity. Figure 17A, gel image. [Figure 17B] FIG. 17B shows the time course of APOBEC3A(Y130W) deamination for C, 5mC, and 5hmC-containing substrates. A SwaI restriction enzyme assay was performed to measure the deaminase activity of APOBEC3A(Y130W). The deamination rate is calculated as the ratio of the cleaved band intensity to the (uncleaved + cleaved) band intensity. FIG. 17B, Quantification of band intensity. [Figure 18]Comparison of APOBEC3A deaminase activity for C, 5mC, and oxidized derivatives is shown. SwaI restriction enzyme assays were performed with a 90 min reaction time to measure deaminase activity of APOBEC3A wild type and Y130W mutant enzymes. A no protein control was included to account for potential degradation of the oligonucleotide substrate and to account for non-specific activity of SwaI during the course of the assay. Deamination rates were calculated as the ratio of cleaved band intensity to (uncleaved + cleaved) band intensity. Figure 18A, gel image, and Figure 18B, quantification of band intensity subtracted from the corresponding no protein control lane. [Figure 19] 1 shows a method for detecting 5hmC using deaminase-based sequencing. [Figure 20A] Figure 20 shows a comparison of different reaction conditions and methylation reports on pUC19 (CG methylated) and Lambda (fully unmethylated) obtained by modified cytidine deaminase with Y130A and Y132H. Figure 20A shows the methylation levels of methylated pUC19 and unmethylated Lambda DNA using modified cytidine deaminase under different concentrations. [Figure 20B] Figure 20B shows a comparison of different reaction conditions and methylation reports on pUC19 (CG methylated) and Lambda (fully unmethylated) obtained by modified cytidine deaminase with Y130A and Y132H. Figure 20B shows the methylation levels of methylated pUC19 and unmethylated Lambda DNA using modified cytidine deaminase under different buffers. [Figure 21] 1 shows the effect of RNAse A on the deamination of methylated pUC19 and unmethylated lambda as determined by sequencing. [Figure 22A] Activity of APOBEC Y130A-Y132H against 5hmC is shown. (FIG. 22A) Construction strategy of control oligos. [Figure 22B] Activity of APOBEC Y130A-Y132H against 5hmC is shown. (FIG. 22B) Methylation levels observed with control oligo substrates. mC, 5mC; hmC, 5hmC. [Diagram 23]We show that analysis of regional methylation in CpG islands indicates that deaminase-based assays yield the expected methylation profiles. mC-deaminase-seq, APOBEC3A Y130A-Y132H. [Figure 24] Shows APOBEC3A Y130A-Y132H, mC-deaminase-seq, SNV and indel calling performance with and without methylation conversion by APOBEC3A Y130A-Y132H. [Diagram 25] Figures 25A-F show visualization of DMRs identified by EM-Seq™ and modified cytidine deaminase assay in the ZNF154 gene. Representation of methylation levels across this region is shown in (Figure 25A) HCC2218-normal, EM-Seq™ conversion, (Figure 25B) HCC2218-tumor, EM-Seq™ conversion, (Figure 25C) HCC2218-normal, modified cytidine deaminase assay, (Figure 25D) HCC2218-tumor, modified cytidine deaminase assay, (Figure 25E) variably methylated regions (DMRs) called between tumor / normal samples using EM-Seq™ data, and (Figure 25F) variably methylated regions (DMRs) called between tumor / normal samples using modified cytidine deaminase data. [Figure 26] The recall and precision of DMRs in HCC1187 tumor / normal and HCC2218 tumor / normal paired genomes are shown. The DMRs from each workflow were compared to the DMRs called by EM-Seq™, which was used as the truth set. Bisulfite identifies the majority of the DMRs identified by EM-Seq™. Furthermore, the mC selective deamination protocols described herein in Method A, Method B, and Method C can identify the majority of the DMRs identified by EM-Seq™, mC-deaminase-seq, and APOBEC3A Y130A-Y132H. [Figure 27A]Methylation levels detected in promoter regions using Method A and EM-Seq™ are shown. (FIG. 27A) Methylation levels at H3K36me3 regions that are expected to be highly methylated. The dotted line indicates the methylation level detected in promoter regions using Method A, and the solid line indicates the methylation level detected in promoter regions using EM-Seq™. [Figure 27B] Methylation levels detected in promoter regions using Method A and EM-Seq™ are shown. (Figure 28B) Methylation levels at H3K27ac regions that are expected to be hypomethylated. The dotted line indicates the methylation level detected in promoter regions using Method A, and the solid line indicates the methylation level detected in promoter regions using EM-Seq™. [Figure 28] Tumor signal is shown for 0% spike-in of tumor DNA to normal DNA vs. 10% spike-in of tumor DNA to normal DNA. Methylation levels of HCC2218 normal DNA at individual CpG sites in the PanSeer cancer panel were evaluated to create a baseline. Methylation levels at individual CpG sites were then evaluated in separate replicates of HCC2218 normal DNA and in 10% spike-in of HCC2218 tumor DNA to HCC2218 normal DNA. Tumor signal shows the percentage of CpGs with significantly different methylation levels compared to background. [Figure 29] A diagram showing the advantage of 5mC>T conversion for enrichment is shown. [Figure 30A] Different workflows for enrichment of methyl-converted libraries are shown. (FIG. 30A) Hybridization after conversion and amplification, where the specialized probe design shown in FIG. 29 is typically used. [Figure 30B] Different workflows for enrichment of methyl-converted libraries are shown (FIG. 30B). The Illumina Methyl Capture EPIC workflow, in which standard probe design is used prior to bisulfite conversion, requires higher DNA input. [Figure 30C]Different workflows for enrichment of methyl-converted libraries are shown. (FIG. 30C) Data workflow presented for use with modified cytidine deaminase (mC-deaminase) with enrichment. [Figure 31A] Enrichment performance of engineered cytidine deaminase (mC-deaminase-seq) libraries is shown. (Figure 31A) Lead enrichment performance of engineered cytidine deaminase libraries compared to libraries without methylation conversion. [Figure 31B] Enrichment performance of modified cytidine deaminase (mC-deaminase-seq) libraries. (FIG. 31B) Correlation of local methylation levels in CpG islands measured in non-enriched (WGS) samples compared to enriched samples. [Diagram 32] A schematic diagram of qPCR-based detection of 5mC at a genomic locus of interest is shown. Selective 5mC deamination by APOBEC3A(Y130A / Y132H) results in a DNA template that is perfectly complementary to the qPCR primers and can therefore be amplified. No deamination by APOBEC3A(Y130A / Y132H) is observed in unmethylated substrates, which would result in the qPCR primers mismatching to the target site and therefore impaired amplification. "Selective 5mC→T deamination" refers to the result of incubating ssDNA substrates with the modified cytidine deaminase APOBEC3A Y130A / Y132H, and the underlined thymidine nucleotides are the result of selective deamination of 5mC. "Non-selective 5mC→T and C→U deamination" refers to the result of incubating the ssDNA substrate with wild-type APOBEC3A; the uracil and underlined thymidine nucleotides are the result of non-selective deamination by wild-type APOBEC3A. [Diagram 33]We demonstrate that qPCR assays using purified APOBEC proteins show a decrease in Cq values ​​after treatment of methylated ssDNA substrates with Y130A_Y132H. NEB APOBEC, APOBEC proteins from New England Biolabs; Y130A, Y130A / Y132H, Y130A / E72A, and Y130A / Y132 / E72A are substitution mutations present in four APOBEC3A proteins. The E72A mutation abolishes APOBEC activity and is used as a negative control to show that the observed difference in Cq values ​​is due to the mutant APOBEC enzyme. [Diagram 34] CRISPR-Cas12-mediated detection of 5mC. Modified cytidine deaminase-mediated conversion of 5mC to T restores perfect complementarity of the Cas12 guide RNA to its DNA target, allowing the Cas12-guide RNA protein complex to engage the converted substrate. This activates the concomitant cleavage activity of Cas12, cleaving the reporter ssDNA containing a fluorophore and a quencher. Release of the fluorophore results in an increase in fluorescence, which is measured in a standard fluorometer. F, fluorophore; q, quencher. [Figure 35A]Figure 35A-1 to Figure 35A-3 show that incubation of substrate DNA with APOBEC3A(Y130A / Y132H) results in high 5mC deamination and low but detectable C deamination. The ssDNA oligo substrate detailed in Figure 32 was treated with the enzyme and subsequently analyzed by Illumina sequencing. The % methylation at each C or 5mC site in the oligo is calculated as the % conversion to T. In this experiment, various concentrations of APOBEC3A(Y130A / Y132H) were tested at different reaction temperatures and times. Elevated levels of C deamination were observed at 25°C with increasing enzyme concentration and reaction time. 1 h, 3 h, and 6 h refer to the hours of incubation time, 0.75 uM, 1.5 uM, and 4 uM refer to the micromolar amount of enzyme used in each reaction, and 25C, 30C, and 37C refer to the reaction temperatures. In each histogram, the 17 bars on the left are C deaminations and the 16 bars on the right are 5mC deaminations. The nucleotide triplets on the x-axis of the histograms for each micromolar amount of enzyme are as follows: ACT, ACT, CCT, TCT, ACA, GCA, CCA, GCT, GCC, ACG, ACG, ACT, TCG, CCT, TCG, GCA, ACG, TCA, TCC, ACG, ACA, ACG, GCC, ACG, TCG, CCA, GCA, GCG, TCG, GCC, ACA, GCG, TCA. [Figure 35B]Figure 35B-1 to Figure 35B-3 show that incubation of substrate DNA with APOBEC3A(Y130A / Y132H) results in high 5mC deamination and low but detectable C deamination. The ssDNA oligo substrate detailed in Figure 32 was treated with the enzyme and subsequently analyzed by Illumina sequencing. The % methylation at each C or 5mC site in the oligo is calculated as the % conversion to T. In this experiment, various concentrations of APOBEC3A(Y130A / Y132H) were tested at different reaction temperatures and times. Elevated levels of C deamination were observed at 25°C with increasing enzyme concentration and reaction time. 1 h, 3 h, and 6 h refer to the hours of incubation time, 0.75 uM, 1.5 uM, and 4 uM refer to the micromolar amount of enzyme used in each reaction, and 25C, 30C, and 37C refer to the reaction temperatures. In each histogram, the 17 bars on the left are C deaminations and the 16 bars on the right are 5mC deaminations. The nucleotide triplets on the x-axis of the histograms for each micromolar amount of enzyme are as follows: ACT, ACT, CCT, TCT, ACA, GCA, CCA, GCT, GCC, ACG, ACG, ACT, TCG, CCT, TCG, GCA, ACG, TCA, TCC, ACG, ACA, ACG, GCC, ACG, TCG, CCA, GCA, GCG, TCG, GCC, ACA, GCG, TCA. [Figure 35C]Figure 35C-1 to Figure 35C-3 show that incubation of substrate DNA with APOBEC3A(Y130A / Y132H) results in high 5mC deamination and low but detectable C deamination. The ssDNA oligo substrate detailed in Figure 32 was treated with the enzyme and subsequently analyzed by Illumina sequencing. The % methylation at each C or 5mC site in the oligo is calculated as the % conversion to T. In this experiment, various concentrations of APOBEC3A(Y130A / Y132H) were tested at different reaction temperatures and times. Elevated levels of C deamination were observed at 25°C with increasing enzyme concentration and reaction time. 1 h, 3 h, and 6 h refer to the hours of incubation time, 0.75 uM, 1.5 uM, and 4 uM refer to the micromolar amount of enzyme used in each reaction, and 25C, 30C, and 37C refer to the reaction temperatures. In each histogram, the 17 bars on the left are C deaminations and the 16 bars on the right are 5mC deaminations. The nucleotide triplets on the x-axis of the histograms for each micromolar amount of enzyme are as follows: ACT, ACT, CCT, TCT, ACA, GCA, CCA, GCT, GCC, ACG, ACG, ACT, TCG, CCT, TCG, GCA, ACG, TCA, TCC, ACG, ACA, ACG, GCC, ACG, TCG, CCA, GCA, GCG, TCG, GCC, ACA, GCG, TCA. [Diagram 36] Detection of 5mC in fixed cell or tissue preparations using FISH. Fixed biological samples are permeabilized and denatured to make the DNA accessible to modified cytidine deaminase. Enzymatic deamination selectively converts 5mC to T. Methylation events are detected using a fluorescent probe specific for the converted DNA sequence. [Figure 37A] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37B] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37C] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37D]The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37E] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37F] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown. [Figure 37G] The amino acid sequences of SEQ ID NOs: 16, 17, 37 to 56, and 61 to 63 are shown.

[0019] The schematic diagrams are not necessarily to scale. Like numbers used in the figures refer to like components, steps, etc. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. Moreover, the use of different numbers to refer to a component is not intended to indicate that the differently numbered component may not be the same or similar to the other numbered components. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Terms used herein shall be understood to have their ordinary meaning in the relevant art unless otherwise specified. Some terms used herein and their meanings are described below.

[0021] As used herein, the terms "organism" and "subject" are used interchangeably and refer to microorganisms (e.g., prokaryotes or eukaryotes), animals, and plants. An example of an animal is a mammal, such as a human.

[0022] As used herein, the term "target nucleic acid," when used in reference to a nucleic acid, is intended as a semantic identifier of the nucleic acid in the context of the methods or compositions or kits described herein and does not necessarily limit the structure or function of the nucleic acid other than as otherwise expressly indicated. Reference to a nucleic acid, such as a target nucleic acid, includes both single-stranded and double-stranded nucleic acids, and both DNA and RNA, unless otherwise indicated. The term library refers to a collection of target nucleic acids that contain known common sequences, such as universal sequences or adapters, at the 3' and 5' ends.

[0023] As used herein, the term "adapter" and its derivatives, such as universal adaptor, generally refers to any linear oligonucleotide that can be added to a target nucleic acid. An adaptor can be single-stranded or double-stranded DNA, or can include both double-stranded and single-stranded regions. An adaptor can include a sequence that is substantially identical to or substantially complementary to at least a portion of a primer, such as a universal sequence; an index (also referred to herein as a barcode or tag) to aid in downstream error correction, identification, or sequencing, and / or a unique molecular identifier. In some embodiments, an adaptor is substantially non-complementary to the 3' or 5' end of any target sequence present in a sample. In some embodiments, suitable adaptor lengths range from about 6-100 nucleotides, about 12-60 nucleotides, or about 15-50 nucleotides in length. For example, the terms "adaptor" and "adapter" are used interchangeably.

[0024] As used herein, the term "universal" when used to describe a nucleotide sequence refers to a region of sequence common to two or more nucleic acid molecules, where the molecules also have regions of sequence that differ from one another. Universal sequences present in different members of a nucleic acid collection can be used as "landing pads" in a subsequent step of annealing nucleotide sequences that can be used as primers to add another nucleotide sequence, such as an index, to a target nucleic acid. Universal sequences present in different members of a nucleic acid collection can capture multiple different nucleic acids using a population of universal capture nucleic acids, e.g., capture oligonucleotides that are complementary to a portion of the universal sequence, e.g., universal capture sequences. Non-limiting examples of universal capture sequences include sequences identical to or complementary to P5 and P7 primers. Similarly, universal sequences present in different members of a collection of molecules can replicate (e.g., sequence) or amplify multiple different nucleic acids using a population of universal primers, e.g., universal anchor sequences, that are complementary to a portion of the universal sequence. In one embodiment, the universal anchor sequence is used as a site to which a universal primer (e.g., sequencing primer for read 1 or read 2) anneals for sequencing. Thus, the capture oligonucleotide or universal primer comprises a sequence that can specifically hybridize to a universal sequence.

[0025] The terms "P5" and "P7" may be used to refer to a universal capture sequence or capture oligonucleotide. The terms "P5'" (P5 prime) and "P7'" (P7 prime) refer to the complements of P5 and P7, respectively. It will be understood that any suitable universal capture sequence or capture nucleotide may be used in the methods presented herein, and the use of P5 and P7 is only an exemplary embodiment. The use of capture nucleotides such as P5 and P7 or their complements on a flow cell is known in the art, as exemplified by the disclosures of WO 2007 / 010251, WO 2006 / 064199, WO 2005 / 065814, WO 2015 / 106941, WO 1998 / 044151, and WO 2000 / 018957, which are incorporated by reference with respect to P5 and P7 and their uses. For example, any suitable forward amplification primer can be useful in the methods presented herein for amplifying complementary sequences and sequences, whether immobilized or in solution. Similarly, any suitable reverse amplification primer can be useful in the methods presented herein for amplifying complementary sequences and sequences, whether immobilized or in solution. Those skilled in the art will understand how to design and use suitable primer sequences for capturing and / or amplifying nucleic acids as presented herein.

[0026] As used herein, the term "primer" and its derivatives generally refer to any nucleic acid that can hybridize to a target sequence of interest. Typically, a primer serves as a substrate to which nucleotides can be polymerized by a polymerase or to which a polynucleotide can be ligated, but in some embodiments, a primer can be incorporated into a synthesized nucleic acid strand to provide a site to which another primer can hybridize to prime the synthesis of a new strand complementary to the synthesized nucleic acid molecule. In some embodiments, a primer can be used for hybridization to a predetermined sequence, for example, a predetermined sequence that includes one or more nucleotides that specify the position of a modified cytosine. In one embodiment, a "primer" includes a sequence present in a guide RNA that is used with a CRISPR-based system to hybridize to a predetermined sequence. A primer can include any combination of nucleotides or analogs thereof. In some embodiments, a primer is a single-stranded oligonucleotide or polynucleotide.

[0027] The terms "polynucleotide" and "oligonucleotide" and "nucleic acid" are used interchangeably herein to refer to polymeric forms of nucleotides of any length and may include ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. It is understood that these terms include, as equivalents, any analog of DNA, RNA, cDNA, or antibody-oligoconjugates made from nucleotide analogs, and are applicable to single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term also encompasses cDNA, which is the complementary or copy DNA generated from an RNA template, for example, by the action of reverse transcriptase.

[0028] As used herein, "index" (also referred to as "index region", "index adapter", "tag", or "barcode") refers to a unique nucleic acid tag that can be used to identify a sample or source of nucleic acid material, or a compartment in which a target nucleic acid is present. The index can be in solution or on a solid support, or can be attached or bound to a solid support and released into a solution or compartment. When nucleic acid samples are from multiple sources, the nucleic acids in each nucleic acid sample can be tagged with a different nucleic acid tag so that the source of the sample can be identified. Any suitable index or set of indexes can be used, as known in the art and as exemplified by the disclosures of U.S. Pat. No. 8,053,192, WO 05 / 068656, and U.S. Patent Application Publication No. 2013 / 0274117. In some embodiments, the index may include a 6-base index 1 (i7) sequence, an 8-base index 1 (i7) sequence, an 8-base index 2 (i5e) sequence, a 10-base index 1 (i7) sequence, or a 10-base index 2 (i5) sequence from Illumina, Inc. (San Diego, Calif.).

[0029] As used herein, the term "amplicon" when used in reference to a nucleic acid refers to a product of copying a nucleic acid, which product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be generated by any of a variety of amplification methods using a nucleic acid or its amplicon as a template, including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule that has a single copy of a particular nucleotide sequence (e.g., a PCR product) or multiple copies of a nucleotide sequence (e.g., a concatemeric product of RCA). A first amplicon of a target nucleic acid is typically a complementary copy. Subsequent amplicons are copies made from the target nucleic acid or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to the target nucleic acid or substantially identical to the target nucleic acid.

[0030] As used herein, "amplify", "amplification" or "amplification reaction" and their derivatives generally refer to any act or process in which at least a portion of a nucleic acid molecule is replicated or copied to at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally comprises a sequence that is substantially identical or substantially complementary to at least a portion of the template nucleic acid molecule. The template nucleic acid molecule may be single-stranded or double-stranded, and the additional nucleic acid molecule may be independently single-stranded or double-stranded. Amplification typically involves exponential replication of the nucleic acid molecule. In some embodiments, such amplification may be performed using isothermal conditions, and in other embodiments, such amplification may involve thermal cycling. In some embodiments, amplification is a multiplex amplification that involves simultaneous amplification of multiple target sequences in a single amplification reaction. In some embodiments, "amplification" involves amplifying at least a portion of DNA and RNA-based nucleic acids, alone or in combination. The amplification reaction may involve any of the amplification processes known to those of skill in the art. In some embodiments, the amplification reaction involves polymerase chain reaction (PCR).

[0031] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202), which describes a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying a polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to a DNA mixture containing the desired polynucleotide of interest, followed by a series of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to each strand of the double-stranded polynucleotide of interest. The mixture is first denatured at a higher temperature, and then the primers are annealed to complementary sequences within the polynucleotide of the molecule of interest. After annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension can be repeated many times (called thermal cycling) to obtain a high concentration of the amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired target polynucleotide (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore this length is a controllable parameter. By repeating this process, the method is called PCR. The desired amplified segments of the target polynucleotide become the predominant nucleic acid sequences (in terms of concentration) in the mixture, and therefore are said to be "PCR amplified". In a modification of the above method, the target nucleic acid molecule can be PCR amplified using multiple different primer pairs, and in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction.

[0032] As used herein, "amplification conditions" and its derivatives generally refer to conditions suitable for amplifying one or more nucleic acid sequences. In some embodiments, amplification conditions can include isothermal conditions, or can include thermal cycling conditions, or a combination of isothermal and thermal cycling conditions. In some embodiments, conditions suitable for amplifying one or more nucleic acid sequences include polymerase chain reaction (PCR) conditions. Typically, amplification conditions refer to a reaction mixture sufficient to amplify a nucleic acid, such as a universal sequence, or one or more target sequences adjacent to a target-specific primer, or amplify an amplified target sequence adjacent to one or more adapters. Generally, amplification conditions include a catalyst for amplification, or nucleic acid synthesis, such as a polymerase, a primer having a degree of complementarity to the nucleic acid to be amplified, and a nucleotide, such as deoxyribonucleotide triphosphate (dNTP), to facilitate extension of the primer when hybridized to the nucleic acid. Amplification conditions may require steps of hybridization or annealing of the primer to the nucleic acid, extension of the primer, and denaturation, in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, but not necessarily, amplification conditions include thermal cycling, but in some embodiments, amplification conditions include multiple cycles in which the steps of annealing, extension, and separation are repeated. Typically, amplification conditions include, but are not limited to, Mg 2+ or Mn 2+ and may also include various modifiers of ionic strength.

[0033] As defined herein, "multiplex amplification" refers to the selective and non-random amplification of two or more target sequences in a sample using at least one target-specific primer. In some embodiments, multiplex amplification is performed such that some or all of the target sequences are amplified in a single reaction vessel. The "plex" of a given multiplex amplification generally refers to the number of different target-specific sequences that are amplified during that single multiplex amplification. In some embodiments, the plex can be about 12-plex, 24-plex, 48-plex, 96-plex, 192-plex, 384-plex, 768-plex, 1536-plex, 3072-plex, 6144-plex, or more. The amplified target sequences can be analyzed by a number of different methodologies (e.g., gel electrophoresis followed by densitometry, quantification by bioanalyzer or quantitative PCR, hybridization with labeled probes, incorporation of biotinylated primers followed by detection of avidin-enzyme conjugates, detection of the amplified target sequences, and detection of the amplified target sequences by a number of different methods (e.g., quantification by gel electrophoresis followed by densitometry, bioanalyzer or quantitative PCR, hybridization with labeled probes, incorporation of biotinylated primers followed by detection of avidin-enzyme conjugates, detection of the amplified target sequences, detection of the amplified target sequences by a number of different methods (e.g., quantification by gel electrophoresis followed by densitometry, bioanalyzer or quantitative PCR, detection of the avidin-enzyme conjugates, detection of the amplified target sequences, detection of the avidin-enzyme conjugates ... 32 It is also possible to detect the presence of .P-labeled deoxynucleotide triphosphates.

[0034] As used herein, the term "amplification site" refers to a site within or on an array where one or more amplicons can be generated. An amplification site can be further configured to contain, hold, or attach at least one amplicon generated at the site.

[0035] As used herein, the terms "array", "analyte array", and "microarray" are used interchangeably and refer to a collection of sites that can be distinguished from one another according to their relative positions. Different molecules at different sites of an array can be distinguished from one another according to the site's position within the array. An individual site of an array can contain one or more molecules of a particular type. For example, a site can contain a single target nucleic acid molecule with a particular sequence, or a site can contain several nucleic acid molecules with the same sequence (and / or its complementary sequence). The sites of an array can be different features located on the same substrate. Exemplary features include, but are not limited to, droplets, wells in a substrate, beads (or other particles) in or on a substrate, protrusions from a substrate, bumps on a substrate, or channels within a substrate. The sites of an array can be separate substrates, each with different molecules. The different molecules attached to the separate substrates can be identified according to the substrate's position on a surface to which the substrates are associated, or according to the substrate's position within a liquid or gel. Exemplary arrays in which separate substrates are located on a surface include, but are not limited to, those with beads in wells.

[0036] As used herein, the term "compartment" is intended to mean an area or volume that separates or isolates something from another. Exemplary compartments include, but are not limited to, vials, tubes, wells, droplets, boluses, beads, containers, surface features, flow cells, or areas or volumes separated by physical forces such as fluid flow, magnetism, electric current, etc. In one embodiment, the compartment is a well of a multi-well plate, such as a 96 or 384 well plate. As used herein, a droplet may include hydrogel beads, which are beads for encapsulating one or more nuclei or cells, including hydrogel compositions. In some embodiments, the droplet is a homogenous droplet of hydrogel material or is a hollow droplet with a polymer hydrogel shell. Whether homogenous or hollow, the droplet may be capable of encapsulating one or more nuclei or cells. In some embodiments, the droplet is a surfactant-stabilized droplet. In some embodiments, a single cell or nucleus is present per compartment. In some embodiments, there are two or more cells or nuclei per compartment. In some embodiments, each compartment includes a compartment-specific index. In some embodiments, the index is in solution or is attached or bound to a solid phase within each compartment.

[0037] As used herein, the term "flow cell" refers to a chamber that includes a solid surface through which one or more fluidic reagents can flow. Examples of flow cells and related fluidic systems and detection platforms that can be easily used in the methods of the present disclosure are described in, for example, Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Patent No. 7,057,026, WO 91 / 06678, U.S. Patent No. 07 / 123744, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211,414, U.S. Patent No. 7,315,019, U.S. Patent No. 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082.

[0038] As used herein, the term "clonal population" refers to a population of nucleic acids that are homogeneous for a particular nucleotide sequence. A homogeneous sequence is typically at least 10 nucleotides in length, but may be longer, for example, at least 50, 100, 250, 500, or 1000 nucleotides in length. A clonal population may be derived from a single target or template nucleic acid. Typically, all nucleic acids in a clonal population have the same nucleotide sequence. It will be understood that a small number of mutations (e.g., due to amplification artifacts) may occur in a clonal population without departing from clonality.

[0039] As used herein, a "pattern of cytosine modification", also referred to as a "methylation profile", refers to the pattern in which both methylated and unmethylated cysteines are distributed in the genome of a cell or organism. The "pattern" includes both modified and unmodified cytosines. This pattern can be defined in several distribution dimensions: by organ, by tissue, by disease or pathological state (e.g., cancer, neurophysiological), by genome segment (e.g., chromosome or genetic coordinates on a chromosome), by gene, by CpG island, by group of cytosines, or by site of modified cytosine. The pattern of cytosine modification can have a known correlation with a disease or pathological state, or the correlation between the pattern of cytosine modification and a disease or pathological state can be identified using the methods described herein. The pattern of cytosine modification can be present at a specific locus (e.g., position) in the genome, which can be a single modified cytosine or a set of modified cytosines, e.g., a CpG island. The pattern of cytosine modification can be identified by using a predetermined sequence, for example, a method using a modified cytidine deaminase can be designed and implemented with the intent of determining the pattern of cytosine modification, for example, the methylation state of one or more specific cytosines, the methylation state of one or more specific cytosines present at a particular location in the genome, or a combination thereof.

[0040] As used herein, the term "each," when used in reference to a collection of items, is intended to identify each individual item in the set, but does not necessarily refer to every item in the set, unless the context clearly dictates otherwise.

[0041] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" cannot mean "and / or" in other instances.

[0042] Unless otherwise stated, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0043] As used in this specification and the appended claims, the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" cannot mean "and / or" in other instances.

[0044] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may provide certain benefits, under particular circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0045] As used in this specification, the words "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising," and the like are used in their inclusive, open-ended sense and generally mean "including, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0046] Wherever words such as "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising," and the like are used herein, it is understood that similar embodiments are also provided that are otherwise described with the terms "consisting of" and / or "consisting essentially of." The term "consisting of" is meant to include what follows the phrase "consisting of." That is, "consisting" indicates that the recited elements are required or essential, and that no other elements may be present. The term "consisting essentially of" indicates that any elements recited after the phrase are included, and that other elements other than those recited may be included, so long as those elements do not interfere with or contribute to the activity or function specified in the disclosure of the recited elements.

[0047] Conditions that are "favorable" or "suitable" for an event to occur, such as converting 5-methylcytosine to thymidine by deamination, are conditions that do not prevent such an event from occurring. These conditions thus enable, enhance, facilitate, and / or favor the event.

[0048] As used herein, "providing" in the context of a protein, DNA or RNA sample, or composition means making the protein, DNA or RNA sample, or composition; purchasing the protein, DNA or RNA sample, or composition; or obtaining the protein, DNA or RNA sample, or composition.

[0049] References to "one embodiment," "an embodiment," "particular embodiment," or "some embodiments" or the like mean that the particular feature, configuration, composition, or characteristic described in connection with this embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily refer to the same embodiment of the present disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0050] Although the polynucleotide sequences encoding modified cytidine deaminases are described herein as DNA sequences, it is understood that the complement, reverse sequence, and reverse complement of the DNA sequences can be readily determined by one of ordinary skill in the art. It is also understood that the sequences described herein as DNA sequences can be converted from DNA sequences to RNA sequences by replacing each thymidine nucleotide with a uracil nucleotide.

[0051] Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 4.5, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0052] In the description herein, a particular embodiment may be described in isolation for clarity. A particular embodiment may include any combination of compatible features described herein in connection with one or more embodiments, unless expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0053] Described herein is a one-step enzymatic method for mapping modified cytosines such as 5mC and 5hmC at single base resolution using engineered cytidine deaminases. The examples provided herein describe engineered cytidine deaminases based on APOBEC3A, and it is anticipated that other APOBEC proteins engineered as described herein can be used.

[0054] Wild-type APOBEC3A efficiently deaminates cytosine (C), 5-methylcytosine (5mC), and 5-hydroxymethylcytosine (5hmC) in single-stranded DNA (Figure 1A-C). Treatment of DNA, such as genomic DNA, with wild-type APOBEC3A converts C to uracil (U), 5mC to thymidine (T), and 5hmC to 5-hydroxyuracilcytosine (5hmC), reducing the complexity of DNA for sequencing to three bases (Figure 1D). Point mutations in the human APOBEC3A protein were generated in a previous analysis to determine the ability of the mutant APOBEC3A proteins to convert cytosine to uracil. Altering the tyrosine residue at position 130 to alanine (Y130A) consistently resulted in APOBEC proteins that were inactive (see Figure 6c in Bulliard et al., 2011, J Virol., 85(4):1765-1776, and Figure 5a in Shi et al., 2017, Nat Struct Mol Biol., 24(2):131-139). Going against Bulliard and Shi, we made the surprising and unexpected discovery that specific mutations at position 130 in APOBEC3A alter the enzyme's deamination rate for 5mC relative to C substrates.

[0055] As described herein, the cognate tyrosine (Y) at position 130 was individually mutated to all possible standard amino acid substitutions, including A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, and W, and assessed for activity against C, 5mC, and 5hmC substrates. For example, an APOBEC3A mutant containing a tyrosine to alanine point mutation at position 130 (Y130A) was found to preferentially deaminate 5mC instead of C (5mC is converted to T at a faster rate than C is converted to U), and an APOBEC3A mutant containing a tyrosine to leucine point mutation at position 130 (Y130L) was found to preferentially deaminate C instead of 5mC (C is converted to U at a faster rate than 5mC is converted to T). Deamination of 5mC to T results in a C to T mutation that can be identified by standard sequencing methods. As a result, in one embodiment, treatment of DNA with a modified cytidine deaminase of the present disclosure preferentially converts 5mC to thymidine (FIG. 1E). Analysis of sample DNA after treatment with a modified cytidine deaminase described herein, for example by sequencing the sample DNA and optionally comparing to a reference (e.g., a reference sequence), allows for the ready identification of C to T point mutations, which are inferred as 5mC positions.

[0056] In another example, an APOBEC3A mutant containing a tyrosine to tryptophan point mutation at position 130 (Y130W) maintained the ability to deaminate C and 5mC to U and T, respectively, but lost the ability to deaminate 5hmC, 5fC, and 5caC. When nucleic acids exposed to this APOBEC3A mutant are sequenced, C and 5mC are deaminated to U and T, respectively, and are read as T by the sequencer. On the other hand, 5hmC is not deaminated by the APOBEC3A mutant and is read as C. (Figure 1F). 5fC and 5caC are also not deaminated by this APOBEC3A mutant and therefore cannot be distinguished from 5hmC. However, the abundance of 5fC and 5caC is several orders of magnitude lower than 5hmC in human genomic DNA, approaching the detection limit of the mass spectrometers used for such measurements (Ito et al., 2011, Science 333, 1300-1303 (2011); Wagner et al., 2015, Angew. Chem. Int. Edn Engl. 54, 12511-12514 (2015); Bachman et al., 2015, Nat. Chem. Biol. 11, 555-557 (2015)). Therefore, the signal from 5fC and 5caC should be insignificant compared to 5hmC.

[0057] Modified cytidine deaminase Provided herein are modified cytidine deaminases, compositions comprising modified cytidine deaminases, methods of using modified cytidine deaminases, and kits comprising modified cytidine deaminases. The present disclosure provides three types of modified cytidine deaminases. One type of modified cytidine deaminase preferentially deaminates 5mC instead of C (i.e., converts 5mC to T at a faster rate than it converts C to U), and is referred to herein as having "cytosine-deficient deaminase activity". A second type of modified cytidine deaminase preferentially deaminates C instead of 5mC (i.e., converts C to U at a faster rate than it converts 5mC to T), and is referred to herein as having "5mC-deficient deaminase activity". A third type of modified cytidine deaminase preferentially deaminates C and 5mC to U and T, respectively, and has significantly reduced deamination of 5hmC, 5fC, and 5caC. The third type is referred to herein as having "5hmC-deficient deaminase activity."Unless the context indicates otherwise, reference to modified cytidine deaminases includes modified cytidine deaminases with cytosine-deficient deaminase activity, modified cytidine deaminases with 5mC-deficient deaminase activity, and modified cytidine deaminases with 5mC-deficient deaminase activity.

[0058] The modified cytidine deaminases include apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) and activation-induced cytidine deaminase (AID). Wild-type APOBEC and AID cytidine deaminases have the activity of deaminating cytidine (C) in DNA and / or RNA to form uridine (U). The modified cytidine deaminases of the present disclosure have altered rates of deamination of C, 5mC, and / or 5hmC when compared to the wild-type enzyme. The cytidine deaminases of the disclosure may be referred to herein as "altered cytidine deaminases," "recombinant cytidine deaminases," "mutated cytosine deaminases," or "modified cytidine deaminases," and refer to any of the altered cytosine deaminases described herein that contain one or more changes from a reference (i.e., wild-type) amino acid sequence that provide the unexpected property of an altered deamination profile, e.g., that alters its ability to preferentially deaminate one form of cytosine over other forms.

[0059] Whether a protein has cytidine deaminase activity may be determined by an in vitro assay. One example of an in vitro assay is based on digestion with the restriction enzyme SwaI (see Example 1). A protein that can deaminate 5mC to thymidine has cytidine deaminase activity.

[0060] An engineered cytidine deaminase that preferentially deaminates 5mC instead of C (i.e., has cytosine-deficient deaminase activity) can have at least 10-fold, at least 50-fold, or at least 100-fold greater catalytic efficiency for 5mC than a C substrate. In one embodiment, an engineered cytidine deaminase that preferentially deaminates 5mC instead of C can have up to 1500-fold greater catalytic efficiency for a 5mC substrate than a C substrate.

[0061] An engineered cytidine deaminase that preferentially deaminates C instead of 5mC (i.e., has 5mC deficient deaminase activity) can have at least 10-fold, at least 50-fold, or at least 100-fold greater catalytic efficiency for C over a 5mC substrate. In one embodiment, an engineered cytidine deaminase that preferentially deaminates C instead of 5mC can have up to 1500-fold greater catalytic efficiency for a C substrate over a 5mC substrate.

[0062] When compared to wild-type cytidine deaminase, an engineered cytidine deaminase that deaminates C and 5mC to U and T, respectively, and has significantly reduced deamination of 5hmC (i.e., has 5hmC-deficient deaminase activity), deamination of 5hmC by the engineered cytidine deaminase disclosed herein is reduced by at least 80%, at least 90%, or at least 99% compared to the wild-type cytidine deaminase. In one embodiment, deamination of 5hmC by the engineered cytidine deaminase disclosed herein is undetectable using an assay such as the SWaI-based assay described herein.

[0063] In certain embodiments, the modified cytidine deaminase of the present disclosure is based on a member of the APOBEC protein family. By modified cytidine deaminase of the present disclosure "based on" a member of the APOBEC protein family, we mean that the modified cytidine deaminase is an APOBEC protein that comprises one or more substitution mutations as described herein compared to a reference APOBEC sequence. The modified cytidine deaminase of the present disclosure "based on" a member of the APOBEC protein family can also include conservative and / or non-conservative mutations as described herein.

[0064] The APOBEC protein family includes subfamilies AID, APOBEC1, APOBEC2, APOBEC3 (including 3A, 3B, 3C, 3D, 3F, 3G, 3H) and APOBEC4. The modified cytidine deaminase of the present disclosure can be based on a member of the AID subfamily, the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3 subfamily (e.g., the 3A subfamily, the 3B subfamily, the 3C subfamily, the 3D subfamily, the 3F subfamily, the 3G subfamily, or the 3H subfamily), or the APOBEC4 subfamily. The modified cytidine deaminase of the present disclosure can be based on a member of the APOBEC protein family from a vertebrate, such as a mammal. Examples of mammals include, but are not limited to, rodents, primates, rabbits, bovines (e.g., cows), porcines (e.g., pigs), and equines (e.g., horses). Examples of primates are humans and chimpanzees.

[0065] The APOBEC protein family is a member of the large cytidine deaminase superfamily that contains a canonical zinc-dependent deaminase (ZDD) signature motif embedded within a core cytidine deaminase fold that includes a five-stranded mixed beta (b)-sheet surrounded by six alpha (a)-helices with the order a1-b1-b2-a2-b3-a3-b4-a4-b5-a5-a6 (Salter et al., Trends Biochem Sci. 2016 41(7):578-594. doi:10.1016 / j.tibs.2016.05.001; Salter et al., Trends Biochem. Sci. 2018, 43(8):606-622 doi.org / 10.1016 / j.tibs.2018.04.013). The core structure of the cytidine deaminase domain of each APOBEC protein contains the zinc-binding motif H-[P / A / V]-EX [23-28] -PCX [2-4]-C (SEQ ID NO: 12) (herein referred to as the ZDD motif, where X is any amino acid and the subscript range of the number after X refers to the number of amino acids) containing a highly conserved spatial arrangement of catalytic core residues. Salter et al., Trends Biochem Sci. 2016 41(7):578-594. doi:10.1016 / j.tibs.2016.05.001). Without intending to be limited by theory, the H and two C residues coordinate to the Zn atom, and the E residue polarizes a water molecule near the Zn atom for catalysis (Chen et al., 2021, Viruses, 13:497, doi.org / 10.3390 / v13030497).

[0066] Some members of the APOBEC protein family, such as the AID subfamily, APOBEC1 subfamily, APOBEC2 subfamily, APOBEC3A subfamily, APOBEC3C subfamily, APOBEC3H subfamily, and APOBEC4 subfamily, contain one copy of the ZDD motif. Other members of the APOBEC protein family, such as the APOBEC3B subfamily, APOBEC3D subfamily, APOBEC3F subfamily, and APOBEC3G subfamily, contain two copies of the ZDD motif, but often only the C-terminal copy is active (Salter et al., Trends Biochem Sci. 2016 41(7):578-594. doi:10.1016 / j.tibs.2016.05.001). Thus, the modified cytidine deaminases disclosed herein contain one or two ZDD motifs. In one embodiment, the modified cytidine deaminase based on a member of the APOBEC3A subfamily contains the following ZDD motif: HXEX 24 SW(S / T)PCX [2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] M (SEQ ID NO: 13), where X is any amino acid and the subscript or number range after X refers to the number of amino acids (Salter et al., Trends Biochem Sci. 2016 41(7):578-594. doi:10.1016 / j.tibs.2016.05.001).

[0067] In one embodiment, the modified cytidine deaminase disclosed herein is a member of the following subfamilies, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, and APOBEC3G, and can include one or more highly conserved sites that are part of the active site and are within the ZDD motif of SEQ ID NO: 12. The sites include tryptophan at position 98 and serine or threonine at position 99 (Kouno et al., 2017, Nat. Comm, 8:15024, DOI:10.1038 / ncomms15024).

[0068] In addition to the ZDD motif, members of the APOBEC protein family also contain other highly conserved residues that are part of the active site but are not present as part of the ZDD motif SEQ ID NO:12. Members of the APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, and APOBEC3G subfamilies typically contain one or more of the following highly conserved sites that are part of the active site: arginine at position 28; histidine, asparagine, or arginine at position 29; serine or threonine, preferably threonine, at position 31; asparagine or aspartic acid at position 57; tyrosine or phenylalanine at position 130; asparagine or tyrosine at position 131; asparagine, tyrosine, or phenylalanine, preferably tyrosine, at position 132; and arginine or lysine at position 189 (Kouno et al., 2017, Nat. Comm, 8:15024, DOI:10.1038 / ncomms15024).

[0069] The modified cytidine deaminase of the present disclosure comprises substitution mutations at one or more residues when compared with reference cytidine deaminase.The substitution mutations can be at the same position or functionally equivalent positions compared with reference cytidine deaminase.The reference cytidine deaminase and functionally equivalent positions are described in detail herein.Those skilled in the art will easily understand that the modified cytidine deaminase described herein does not exist in nature.

[0070] The reference cytidine deaminase can be a member of the APOBEC protein family. Essentially, any known member of the APOBEC protein family can be the reference cytidine deaminase. Those skilled in the art can easily identify members of each subfamily by using publicly available databases, such as the protein database available at the National Center for Biotechnology Information (ncbi.nlm.nih.gov / protein), and searching for APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, or activation-induced cytidine deaminase when identifying a member of the AID family. The wild-type reference cytidine deaminase has the activity of binding to single-stranded DNA (ssDNA) and deaminating cytosine present on ssDNA to convert it to uracil. In one embodiment, the wild-type reference cytidine deaminase has the activity of binding to single-stranded RNA (ssRNA) and deaminating cytosines present on the ssRNA to convert them to uracil. Methods for determining whether a protein binds to ssDNA or ssRNA and deaminates cytosines present therein are known to those of skill in the art.

[0071] In one embodiment, the modified cytidine deaminase has an amino acid sequence based on a reference sequence that is a member of the APOBEC protein family and has a ZDD motif H-[P / A / V]-EX [23-28] -PCX[2-4] -C (SEQ ID NO: 12) and at least one substitution mutation disclosed herein. Optionally, the modified cytidine deaminase comprises other active site residues disclosed herein. Non-limiting examples of reference cytidine deaminase proteins are shown in the table below.

[0072] [Table 1]

[0073] In one embodiment, the modified cytidine deaminase has amino acids based on a reference sequence that is a member of the APOBEC3A subfamily and has a ZDD motif

[0074] [Table 2]

[0075] In one embodiment, the amino acid sequence of the modified cytidine deaminase is the amino acid sequence of a member of the APOBEC3A subfamily:

[0076] [Table 3]

[0077] In one embodiment, the amino acid sequence of the modified cytidine deaminase is the amino acid sequence of a member of the APOBEC3A subfamily:

[0078] [Table 4]

[0079] The substitution mutation can be at the same position or at a functionally equivalent position compared to the reference cytidine deaminase. By "functionally equivalent" it is meant that the modified cytidine deaminase has an amino acid substitution at an amino acid position of the reference cytidine deaminase that has the same functional role in both the reference cytidine deaminase and the modified cytidine deaminase.

[0080] In general, functionally equivalent substitution mutations in two or more different cytidine deaminases occur at homologous amino acid positions in the amino acid sequence of cytidine deaminases. Thus, the use of the term "functionally equivalent" herein also encompasses mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutated amino acid is known. Based on sequence alignment and / or molecular modeling, it is possible to identify functionally equivalent and positionally equivalent amino acid residues in the amino acid sequence of two or more different cytidine deaminases. An example of sequence alignment for identifying positionally equivalent and / or functionally equivalent residues is shown in Figure 3. For example, the residues in the members of the APOBEC3A subfamily in Figure 3, which are vertically aligned, are considered to be positionally equivalent and functionally equivalent to the corresponding residues in the human APOBEC3A amino acid sequence. Thus, for example, the tyrosine at residue 130 of the APOBEC3A proteins of humans (Homo sapiens), Bornean orangutans (Pongo pygmaeus), Nomascus leucogenys, chimpanzees (Pan troglodytes), and gorillas (Gorilla gorilla), and the tyrosine at residue 133 of the APOBEC3A protein from cynomolgus monkeys (Macaca fascicularis), are functionally equivalent and positionally equivalent, as shown in Figure 3. One of skill in the art can readily identify functionally equivalent residues in cytidine deaminases.

[0081] In one embodiment, the modified cytidine deaminase has an amino acid sequence structurally similar to a reference cytidine deaminase disclosed herein, hi one embodiment, the reference cytidine deaminase comprises the amino acid sequence of a sequence listed in Table 1, SEQ ID NO: 14, or SEQ ID NO: 15.

[0082] As used herein, a modified cytidine deaminase may be "structurally similar" to a reference cytidine deaminase if the amino acid sequence of the modified cytidine deaminase has a particular amount of sequence similarity and / or sequence identity compared to the reference cytidine deaminase.

[0083] The structural similarity of two amino acid sequences can be determined by aligning the residues of the two sequences (e.g., a candidate modified cytidine deaminase and a reference cytidine deaminase described herein) to optimize the number of identical amino acids along the length of the sequences, and gaps in either or both sequences are allowed in the alignment to optimize the number of identical amino acids, but the amino acids in each sequence must nevertheless remain in their proper order. A candidate modified cytidine deaminase is a cytidine deaminase that is compared to a reference cytidine deaminase. A candidate modified cytidine deaminase that has structural similarity and cytidine deaminase activity with a reference cytidine deaminase is a modified cytidine deaminase.

[0084] Unless otherwise modified as described herein, pairwise comparison analysis of amino acid sequences may be performed, for example, by the homology alignment algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity method study of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally, Current Protocols in Molecular Biology, Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., supplemented through 2004). One example of an algorithm that is suitable for determining structural similarity is the BLAST® algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). The BLAST® algorithm can be used to calculate percent sequence identity and percent sequence similarity between two sequences. Software for performing BLAST® analyses is publicly available through the National Center for Biotechnology Information.

[0085] In comparing two amino acid sequences, structural similarity may be referred to by the percentage "identity" or by the percentage "similarity". "Identity" refers to the presence of identical amino acids. "Similarity" refers not only to the presence of identical amino acids, but also to the presence of conservative substitutions. Thus, in one embodiment, the amino acid sequence of a cytidine deaminase protein having sequence similarity to a reference sequence may include conservative substitutions of amino acids present in the reference sequence.

[0086] Conservative substitutions for an amino acid in a protein may be selected from other members of the class to which the amino acid belongs. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a group of amino acids having a particular size or characteristic (e.g., charge, hydrophobicity, or hydrophilicity) can be substituted for another amino acid without altering the activity of the protein, especially in regions of the protein not directly related to biological activity. For example, amino acids having nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine, amino acids having hydrophobic side chains include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, amino acids having polar side chains include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, cysteine, tyrosine, and threonine; amino acids having uncharged side chains include glycine, serine, cysteine, asparagine, glutamine, tyrosine, and threonine.

[0087] Thus, as used herein, a reference to a cytidine deaminase as described herein, for example, a reference to an amino acid sequence of one or more SEQ ID NOs as described herein, can include a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence similarity to the reference cytidine deaminase. Examples of modified cytidine deaminases having similarity to a reference amino acid sequence include, for example, those having at least 80%, at least 85%, at least 90%, or at least 95% similarity to SEQ ID NO: 16 and having an alanine at amino acid 130. Other examples of modified cytidine deaminases having similarity to a reference amino acid sequence include, for example, those having at least 80%, at least 85%, at least 90%, or at least 95% similarity or identity to SEQ ID NO:17 and having an alanine at amino acid 130 and a histidine at amino acid 132.

[0088] Alternatively, as used herein, a reference to a cytidine deaminase described herein, for example, a reference to an amino acid sequence of one or more SEQ ID NOs described herein, can include a protein having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the reference cytidine deaminase. Examples of modified cytidine deaminases having identity to a reference amino acid sequence include, for example, those having at least 80%, at least 85%, at least 90%, or at least 95% similarity to SEQ ID NO: 16 and having an alanine (A) at amino acid 130. Other examples of modified cytidine deaminases having identity to a reference amino acid sequence include, for example, those having at least 80%, at least 85%, at least 90%, or at least 95% similarity or identity to SEQ ID NO:17 and having an alanine (A) at amino acid 130 and a histidine (H) at amino acid 132.

[0089] Substitution Mutation The modified cytidine deaminase of the present disclosure comprises a substitution mutation at a position functionally equivalent to tyrosine at position 130 (Y130) in a member of the APOBEC3A subfamily (e.g., SEQ ID NO: 3). Thus, an alignment can be made using a member of the APOBEC3A subfamily (e.g., SEQ ID NO: 3) and another candidate modified cytidine deaminase from the APOBEC3A subfamily or a different APOBEC subfamily. In one embodiment, the candidate is selected from the APOPEC subfamily APOBEC1 or AID. An example of an algorithm that can be used to generate the alignment is Clustal O. In some APOBEC family proteins, the wild type residue at the position functionally equivalent to Y130 is phenylalanine (F).

[0090] In another embodiment, the engineered cytidine deaminase of the disclosure recognizes a ZDD motif in a member of the APOBEC family, such as a member of the APOBEC3A subfamily.

[0091] [Table 5]

[0092] The underlined tyrosine (Y) in SEQ ID NO:13 is the functionally equivalent position to the tyrosine amino acid 130 of the APOBEC3A protein in SEQ ID NO:3.

[0093] In one embodiment, a substitution mutation at a position functionally equivalent to Y130 increases cytidine deaminase activity and preferentially acts on 5mC over cytosine (i.e., has cytosine-deficient deaminase activity). The substitution mutation can be a mutation to alanine (A), glycine (G), phenylalanine (F), histidine (H), glutamine (Q), methionine (M), asparagine (N), lysine (K), valine (V), aspartic acid (D), glutamic acid (E), serine (S), cysteine ​​(C), proline (P), or threonine (T). For example, the modified cytidine deaminase can comprise SEQ ID NO: 61, where X is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P, or T (but not Y), or can comprise SEQ ID NO: 62, where Z is selected from A, G, F, H, Q, M, N, K, V, D, E, S, C, P, or T (but not Y), and preferably, in one embodiment, X or Z is A or L. In an exemplary aspect of this embodiment, the substitution mutation at the position functionally equivalent to Y130 is a mutation to alanine (A) (e.g., SEQ ID NO: 16). Specific examples of modified cytidine deaminases with increased activity and preferentially acting on 5mC over cytosine include SEQ ID NO: 16, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO: 16 and comprising Y130A.

[0094] The modified cytidine deaminase of the disclosure that has cytosine-deficient deaminase activity (i.e., converts 5mC to T at a faster rate than it converts C to U) optionally comprises a second substitution mutation at amino acid position 2, 3, 4, or 5 C-terminal to position Y130, or at a position functionally equivalent to position Y130. In one embodiment, the second mutation is a tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), or phenylalanine (F) at amino acid position 2, 3, 4, or 5 C-terminal to position Y130, or at a position functionally equivalent to position Y130. In one embodiment, the second mutation is at a position functionally equivalent to tyrosine at position 132 (Y132) of a member of the APOBEC3A subfamily (e.g., SEQ ID NO: 3). An APOBEC protein, such as an APOBEC3A protein, that contains substitution mutations at both a first site, which is a position functionally equivalent to Y130, and a second site, which is 2, 3, 4, or 5 amino acids C-terminal to the Y130 position, has increased preferential activity acting on 5mC compared to the same APOBEC protein, such as an APOBEC3A protein, that contains a single substitution mutation at Y130. In one embodiment, the substitution mutation at the second position is an amino acid that has a positively charged side chain and is selected from arginine (R), histidine (H), lysine (L), or has a polar side chain selected from glutamine (Q). In one embodiment, the substitution mutation at the second position is a histidine (H), e.g., Y132 to histidine. A double mutant containing both a first and a second mutation, in any combination, can be any substitution mutation at a position functionally equivalent to Y130 as described herein, and any second substitution mutation at amino acid positions 2, 3, 4, or 5 C-terminal to the Y130 position as described herein. For example, the modified cytidine deaminase can be SEQ ID NO: 3, 15, or 16 and can have substitutions at Y130 and Y132, or at positions functionally equivalent to Y130 and Y132 as described herein. One example of a modified cytidine deaminase is SEQ ID NO: 63, which includes Y130X and Y132Z, where X is selected from (A), (L), or (W) (preferably (A)), and Z is selected from (R), (H), (L), or (Q), preferably (H).This includes, but is not limited to, examples such as Y130A and Y132R, Y130A and Y132H, Y130A and Y132L, Y130A and Y132Q, Y130L and Y132R, Y130L and Y132H, Y130L and Y132L, Y130L and Y132Q, Y130W and Y132R, Y130W and Y132H, Y130W and Y132L, Y130W and Y130Q, or any suitable combination thereof. In one embodiment, the double mutant comprises the substitution mutations Y130A and Y132H. Specific examples of modified cytidine deaminases that have both substitution mutations and act preferentially on 5mC compared to APOBEC proteins that have only a single substitution mutation at cytosine include SEQ ID NO:17 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO:17 and including Y130A and Y132H.

[0095] Those skilled in the art can confirm the 5mC preferential deaminase activity of arginine, glutamine, histidine and lysine substitution mutations at the second position of the double mutants described above. For example, double mutants can be constructed to generate modified cytidine deaminases with a first substitution mutation at a position functionally equivalent to Y130 and a second arginine, glutamine, histidine or lysine substitution mutation at the tyrosine position two amino acids C-terminal to the Y130 position, and then evaluated for deamination of C residues in one assay and for deamination of 5mC residues in a second assay. Using assays such as the SwaI-based assay described herein, the ratio of 5mC deamination to C deamination can be compared to identify double mutants that preferentially deaminate 5mC compared to C. One skilled in the art can similarly test double mutants with a tyrosine at position 3, 4 or 5 C-terminal to a position functionally equivalent to Y130, and identify substitution mutations to arginine, glutamine, histidine or lysine at that position in combination with a mutation at a position functionally equivalent to Y130 (such as Y130A) as double mutants that preferentially deaminate 5mC over C.

[0096] Some embodiments presented herein relate to substitution mutations that result in 5mC-deficient deaminase activity (i.e., convert C to U at a faster rate than convert 5mC to T). In one embodiment, the substitution mutation at a position functionally equivalent to Y130 is a mutation to an amino acid that increases cytidine deaminase activity, preferentially acts on cytosine compared to 5mC, and has a non-polar or hydrophobic side chain, such as leucine (L) or tryptophan (W). In an exemplary aspect of this embodiment, the substitution mutation at a position functionally equivalent to Y130 is a mutation to leucine. Other examples of mutations that result in increased preferential deamination activity for cytosine compared to 5mC include single mutants with Y132P, and double mutants with substitution mutations at Y130V and Y132H, or Y130W and Y132H. Specific examples of modified cytidine deaminases having increased cytidine deaminase activity and acting preferentially on cytosine compared to 5mC include SEQ ID NO: 18, or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO: 18 and comprising Y130L.

[0097] In one embodiment, the substitution mutation is at a position functionally equivalent to Y130 that results in 5hmc-deficient deaminase activity (i.e., preferentially deaminating C and 5mC to U and T, respectively, with significantly reduced deamination of 5hmC). In an exemplary aspect of this embodiment, the substitution mutation at a position functionally equivalent to Y130 is a mutation to an amino acid with a non-polar or hydrophobic side chain, such as tryptophan (W). Specific examples of modified cytidine deaminases that have the ability to deaminate C and 5mC to U and T, respectively, but have a reduced ability to deaminate 5hmC, preferably no detectable ability to deaminate 5hmC, include SEQ ID NO:59 or a sequence having at least 90%, at least 95%, at least 98%, at least 99% sequence identity to SEQ ID NO:59 and comprising Y130W.

[0098] The modified cytidine deaminase described herein can include additional mutations. Typically, the additional mutations do not excessively change the activity of the modified cytidine deaminase. One or more additional mutations can be conservative mutations.

[0099] The modified cytidine deaminase described herein can be a truncated protein. A truncated protein is a fragment of the modified cytidine deaminase of the present disclosure that retains the ability to deaminate 5mC to thymidine. A truncated modified cytidine deaminase can include a deletion of 1-13 amino acids on the N-terminus of the protein, a deletion of 1-3 amino acids on the C-terminus of the protein, or a combination thereof.

[0100] Polynucleotides encoding modified cytidine deaminases The modified cytidine deaminases described herein may also be identified in terms of polynucleotides encoding the proteins.Thus, the present disclosure provides polynucleotides that encode the modified cytidine deaminases described herein or that hybridize under standard hybridization conditions to polynucleotides encoding the modified cytidine deaminases described herein, and the complements of such polynucleotide sequences.

[0101] The polynucleotides described herein can include any polynucleotide that encodes the modified cytidine deaminase of the present disclosure. Thus, the nucleotide sequence of the polynucleotide can be deduced from the amino acid sequence that will be encoded by the polynucleotide. The modified cytidine deaminase can be encoded by multiple codons, and a particular translation system (e.g., a prokaryotic or eukaryotic cell) often exhibits codon bias, e.g., different organisms often prefer one of several synonymous codons that code for the same amino acid. Thus, the polynucleotides presented herein are optionally "codon-optimized," and are synthesized such that they contain codons that are preferred by the particular translation system used to express the protein. For example, if it is desired to express the protein in a bacterial cell (or even a particular strain of bacteria), the polynucleotide can be synthesized to contain the codons most frequently found in the genome of that bacterial cell for efficient expression of the modified cytidine deaminase. If it is desired to express the modified cytidine deaminase in a eukaryotic cell, a similar strategy can be used, e.g., the nucleic acid can contain codons that are preferred by the eukaryotic cell.

[0102] The polynucleotides described herein may also be advantageously included in a suitable expression vector to express the modified cytidine deaminase encoded therein in a suitable host. The incorporation of cloned DNA into a suitable expression vector for subsequent transformation of host cells and subsequent selection of transformed cells is well known to those skilled in the art, as provided in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory. Suitable host cells include, but are not limited to, Escherichia coli (E. coli) and S. cerevisiae.

[0103] Such expression vectors include vectors having the polynucleotides described herein operably linked to heterologous regulatory sequences, such as promoter regions, capable of effecting expression of the DNA fragment. The term "operably linked" refers to a juxtaposition of the described components in a relationship that allows them to function in their intended manner. Such vectors may be transformed into suitable host cells to provide expression of modified cytidine deaminase.

[0104] The nucleic acid molecule may encode a protein having a prosequence, including one that encodes a leader sequence on the mature protein or on a preprotein, which is then cleaved by the host cell to form the mature protein. The vector may be, for example, a plasmid, virus or phage vector with an origin of replication, optionally a promoter for expression of the nucleotide, and optionally a regulator of the promoter. The vector may contain one or more selectable markers, such as, for example, an antibiotic resistance gene.

[0105] Regulatory elements required for expression include promoter sequences for binding RNA polymerase and directing appropriate levels of transcription initiation and also translation initiation sequences for ribosome binding. For example, bacterial expression vectors may contain promoters such as lac promoters, and Shine-Dalgarno sequences and start codon AUG for translation initiation. Similarly, eukaryotic expression vectors may contain heterologous or homologous promoters of RNA polymerase II, downstream polyadenylation signals, start codon AUG, and termination codons for detachment of ribosomes. Such vectors may be commercially available or may be assembled from the sequences described by methods well known in the art.

[0106] The transcription of DNA encoding modified cytidine deaminase may be optimized by including an enhancer sequence in the vector. Enhancer is a cis-acting element of DNA that acts on a promoter to increase the transcription level. Vectors also generally include an origin of replication in addition to a selectable marker.

[0107] Creation and isolation of engineered cytidine deaminases Generally, the polynucleotide encoding the modified cytidine deaminase presented herein can be produced by cloning, recombination, in vitro synthesis, in vitro amplification, and / or other available methods.Various recombinant methods can be used to express the expression vector encoding the modified cytidine deaminase presented herein.Methods for producing recombinant polynucleotides, expressing, and isolating expression products are well known and described in the art.

[0108] The polynucleotide encoding wild-type cytidine deaminase can be obtained from a source and can be subjected to mutagenesis to introduce one or more substitution mutations as described herein.Generally, any available mutagenesis procedure can be used to create the modified cytidine deaminase described herein.The procedures that can be used include, but are not limited to, site-directed point mutagenesis, in vitro or in vivo homologous recombination, oligonucleotide-directed mutagenesis, mutagenesis by total gene synthesis, and many others known to those skilled in the art.

[0109] Further useful references on mutations, recombination, and in vitro nucleic acid manipulation methods (including cloning, expression, PCR, etc.) include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition Ceske (ed) CRC Press (Kaufman); The Nucleic Acid Protocols Handbook Ralph Rapley (ed) (2000) Cold Spring Harbor, Humana Press Inc (Rapley); Chen et al. (ed) PCR Cloning Protocols, Second Edition (Methods in Molecular Biology, volume 192) Humana Press; and in Viljoen et al. (2005) Molecular Diagnostic PCR Handbook Springer, ISBN Examples include 1402034032.

[0110] Additionally, many kits are commercially available for purifying plasmids or other related nucleic acids from cells. The isolated polynucleotides can be further manipulated to generate other polynucleotides used to transfect or transform cells, can be incorporated into related vectors and introduced into cells for expression, and / or the like. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating the expression of a particular target nucleic acid. The vectors optionally contain a generic expression cassette containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes or prokaryotes, or both, (e.g., shuttle vectors), and a selection marker for both prokaryotic and eukaryotic systems. The vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both.

[0111] For example, other useful references for cell isolation and culture (e.g., subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and references cited therein, Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. Construction of vectors containing nucleic acids encoding modified cytidine deaminases as described herein uses standard ligation techniques known in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989) or Ausubel, RM, ed. Current Protocols in Molecular Biology (1994).

[0112] A variety of protein isolation and detection methods are known and can be used, for example, to isolate modified cytidine deaminase from recombinant cultures of cells expressing the recombinant modified cytidine deaminase provided herein. For example, R. Scopes, Protein Purification, Springer-Verlag, NY (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. NY (1990); Sandana (1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al. (1996) Protein Methods,2nd Edition Wiley-Liss,NY;Walker(1996)The Protein Protocols Handbook Humana Press,NJ,Harris and Angal(1990)Protein Purification Applications:A Practical Approach IRL Press at Oxford,Oxford,England;Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford,Oxford,England;Scopes(1993)Protein Purification:Principles and Practice 3rd Edition Springer Verlag,NY;Janson A variety of protein isolation and detection methods are well known in the art, including those described in: and Ryden (1998) Protein Purification: Principles, High Resolution Methods and Applications, Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and references cited therein.Further details regarding protein purification and detection methods can be found in Satinder Ahuja ed., Handbook of Bioseparations, Academic Press (2000).

[0113] The modified cytidine deaminase protein or polynucleotide can be isolated. An "isolated" protein or polynucleotide is one that has been removed from a cell. For example, an isolated protein is a polypeptide that has been removed from the cytoplasm or membrane of a cell, and many of the proteins, nucleic acids, and other cellular materials of its natural environment are no longer present. For example, a protein that is produced outside a cell by chemical or recombinant means is considered isolated by definition, since it was never present inside a cell.

[0114] How to use The modified cytidine deaminase provided by the present disclosure can be easily incorporated into essentially any application for identifying modified cytosines. For example, the modified cytidine deaminase can be incorporated into applications including sequencing library preparation. Examples of sequencing library preparation include, but are not limited to, whole genome, accessible (e.g., ATAC), conformational state (e.g., HiC), and reduced bisulfite sequencing (RRBS). It can be particularly useful in essentially any application that uses low input DNA or RNA, including, but not limited to, single-cell combinatorial indexing (sci) methods such as sci-WGS-seq, sci-MET-seq, and sci-ATAC-seq, sci-RNA-seq, and cell-free DNA-based methods. Specific applications include, but are not limited to, identification of one or more patterns of cytosine modifications, such as determining methylation on CpG islands (Example 15) and variant calling, including reduced bisulfite sequencing (RRBS), SNVs / indels, copy number variation (CNV), short tandem repeats (STRs), and structural variants (SVs) (Example 16); detection of variably methylated regions (DMRs) (Example 17); measuring methylation in promoters (Example 18); and detection of tumor DNA (Example 19).

[0115] The modified cytidine deaminase provided by the present disclosure can be easily incorporated into essentially any application, including locus-specific methylation profiling. Typical locus-specific detection of epigenetically methylated cytosines, such as 5mC, requires the use of 5mC-specific antibodies, or multi-step chemical or chemoenzymatic conversion resulting in deamination of C or 5mC to U / T to allow for the discrimination of the two C isoforms. When combined with various in vitro detection methodologies, these approaches can be powerful approaches to detect 5mC at defined loci. However, these methods can be confounded by antibody cross-reactivity and stability, or the toxicity and complex workflow required by chemical and chemoenzymatic approaches. The use of the modified cytidine deaminase described herein in a single enzymatic deamination protocol allows for selective conversion of 5mC to T, compatible with many in vitro diagnostic modalities, and specifically detects loci of 5mC.

[0116] Instead of using destructive methods to identify methylated cytosines, incorporating the cytidine deaminase provided by the present disclosure into methods for identifying modified cytosines, such as sequencing library production and locus-specific methylation profiling, results in more efficient enzyme-catalyzed conversion of modified cytosines during generation of target nucleic acids, thereby allowing better sequencing data and better retention of genetic information, as demonstrated by higher mutation calling performance (see Example 16).Furthermore, the use of modified cytidine deaminase as an enzymatic method for conversion allows for higher coverage uniformity and lower sample damage, resulting in lower nucleic acid input requirements. Numerous sequencing library methods are known to those of skill in the art that can be used to construct whole genome or targeted libraries (see, e.g., "Sequencing Methods Review," available on the World Wide Web at illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / sequencing-methods-review.pdf; the DNA sequencing methods collection available on the World Wide Web at illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / dna-sequencing-methods-review-web.pdf; and the RNA sequencing methods collection available on the World Wide Web at illumina.com / content / dam / illumina-marketing / documents / products / research_reviews / rna-sequencing-methods-review-web.pdf).

[0117] In general, the method of using modified cytidine deaminase of the present disclosure includes contacting a target nucleic acid, e.g., DNA or RNA, with the enzyme under conditions suitable for conversion of modified cytidine. Because DNA amplification does not preserve the modification state of cytidine (e.g., methylation state of 5mC and 5hmC is not preserved), the use of modified cytidine deaminase is typically performed before amplification of the target DNA. The target nucleic acid can be contacted with modified cytidine deaminase essentially at any time in the method before amplification, provided that the DNA is single-stranded. For example, the target nucleic acid can be contacted with modified cytidine deaminase after isolation of genomic or cell-free DNA or mRNA, before or after fragmentation, or before or after tagmentation, while the nucleic acid is in a fixed or unfixed cell or nucleus. Those skilled in the art will recognize that the target nucleic acid can be contacted with modified cytidine deaminase after addition of universal sequence and / or adapter, as long as the universal sequence and / or adapter are not added by amplification.

[0118] The method of using modified cytidine deaminase can include an optional step of comparing the treated target nucleic acid with untreated nucleic acid or comparing the treated target nucleic acid with a nucleic acid treated with wild-type cytidine deaminase. For example, in an embodiment where the treated nucleic acid is sequenced, the sequence can be compared with a reference sequence, which allows easy identification of point mutations and inference of modified cytosines. Thus, in an embodiment where a modified cytidine deaminase with cytosine-deficient deaminase activity is used (i.e., converts 5mC to T at a faster rate than it converts C to U), point mutations from C to T can be easily identified, and these point mutations are inferred as 5mC positions. In an embodiment where a modified cytidine deaminase with 5hmC-deficient deaminase activity is used (i.e., preferentially deaminates C and 5mC to U and T, respectively, and significantly reduces deamination of 5hmC), the absence of point mutations from C to T can be easily identified, and the absence of point mutations is inferred as 5hmC positions. In embodiments where the treated nucleic acid is not sequenced, the nucleic acid can be treated with a modified cytidine deaminase and compared to untreated, i.e., nucleic acid not contacted with the modified cytidine deaminase, where the readout typically depends on the assay method, e.g., when amplification is used (e.g., Example 21), the relative amount of amplification can be readily identified and the presence or absence of a pattern of 5mC or cytosine modifications in a given sequence can be inferred.

[0119] Suitable reaction conditions for the conversion of modified cytidine, such as the conversion of 5mC to thymidine, by the cytidine deaminase described herein include, but are not limited to, the substrate of the target nucleic acid, which is a single-stranded (ss) DNA or RNA suspected to contain at least one modified cytidine, buffer, pH, reaction temperature, reaction time, and the concentration of modified cytidine deaminase and / or ssDNA or RNA substrate.In one embodiment, double-stranded (ds) DNA can be denatured and exposed to modified cytidine deaminase.Methods for denaturing dsDNA are known and routine, and include heat treatment, chemical treatment such as NaOH, formamide, DMSO, or N,N-dimethylformamide (DMF), or combinations thereof.

[0120] Target nucleic acids useful in the disclosed methods are described herein. Modified cytidines present on substrate single-stranded (ss) DNA or RNA include, but are not limited to, 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5CaC) (Figure 2). In one embodiment, the modified cytidine is 5-methylcytosine. In one embodiment, the modified cytidine is 5-hydroxymethylcytosine. Methods using double-stranded target DNA to generate sequencing libraries can be modified to include denaturation to convert the double-stranded target DNA to ssDNA. In some embodiments, dsDNA used in tagmentation reactions or for adapter binding can be denatured and then treated with modified cytidine deaminase. Denaturation conditions are known and routine. In embodiments in which the ssDNA is contacted with a modified cytidine deaminase and subsequently used in a process requiring dsDNA, such as addition of universal adaptors by tagmentation or ligation, the ssDNA can be converted to dsDNA using routine methods.

[0121] In some embodiments, the modified cytidine deaminase provided herein can be used to distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). In such embodiments, a sample of DNA suspected of containing at least one 5-methylcytosine (5mC) or single-stranded DNA containing 5-hydroxymethylcytosine (5hmC) is modified to prevent modified cytidine deaminase from converting 5hmC to thymidine. Methods for blocking deaminase activity are known in the art, and any one of a number of methods can be used to protect 5hmC from deaminase activity. As an example, target DNA can be treated to modify 5hmC rather than 5mC, such that 5hmC is an inappropriate substrate for cytidine deaminase activity. In a particular example, a glucosyltransferase enzyme can be used to glucosylate 5hmC rather than 5mC. Glucosyltransferase enzymes are known to those skilled in the art, for example, β-glucosyltransferase (βGT). As an example, the enzyme T4 β-glucosyltransferase is commercially available (βGT, NEB) and can be used to modify 5hmC. Methods of using βGT to glucosylate 5hmC are known in the art and can be used in conjunction with the use of modified cytidine deaminase enzymes presented herein. For example, a sample of DNA can be treated with βGT to glucosylate 5hmC in the sample DNA, and then the DNA can be treated with modified cytidine deaminase enzyme. By treating the sample DNA with βGT, 5hmC is protected from the deaminase activity of modified cytidine deaminase enzyme. Thus, 5mC is detected as thymidine in downstream readout (e.g., sequencing, PCR, array, etc.). In contrast, any protected 5hmC site is detected as cytosine in the same readout.Enzymes, buffers, and conditions for glycosylation of 5hmC are known in the art, as exemplified by the methods disclosed in Schutsky et al., Nature biotechnology, 10.1038 / nbt.4204.8 Oct. 2018, doi:10.1038 / nbt.4204. Specific examples of modifications of 5hmC to protect it from cytidine deaminase activity are provided in Examples 9, 10, and 11 below.

[0122] In some embodiments, the modified cytidine deaminase presented herein can be used in an in vitro diagnostic (IVD) approach to locus-specifically profile methylation. Current methylation biomarker detection methods typically involve digestion of genomic DNA with methylation-sensitive enzymes, followed by quantitative PCR (qPCR) at the locus of interest to quantify the extent of restriction enzyme digestion and thus the methylation rate at that site. This is followed by mismatch-sensitive qPCR of bisulfite-treated DNA, where 5mC is read as the absence of 5mC>T conversion. However, these methods have drawbacks. The recognition site of the methyl-sensitive restriction enzyme needs to be present in the methylated region of the target locus. Bisulfite treatment requires a large amount of starting DNA and converts to a low-complexity genome (unmethylated cytosines, which represent the majority of cytosines in the genome, are converted to U and read as T). This low complexity of the genomic template constrains the design of qPCR primers that specifically hybridize to the locus of interest. During bisulfite conversion, DNA is essentially damaged or lost, which can impede downstream analysis. DNA damage can reduce genome coverage uniformity and result in biased coverage. Furthermore, incomplete bisulfite conversion can exaggerate DNA methylation levels, which can adversely affect results (Sam et al., PLoS One. 2018; 13(6); Ehrich et al., Nucleic Acids Res. Oxford University Press; 2007; 35: e29).

[0123] The conversion of 5mC to T by modified cytidine deaminase described herein eliminates the need for restriction enzymes or bisulfite treatment and maintains DNA complexity. The resulting modification of one or more cytosines can be detected using established in vitro diagnostic (IVD) approaches for profiling methylation in a locus-specific manner. Exemplary approaches include detection of 5mC loci by amplification, e.g., quantitative PCR (qPCR) (Example 21), detection of 5mC loci using CRISPR-based systems, e.g., CRISPR-Cas12 (Example 22), spatial detection of 5mC using molecular cytogenesis methods, e.g., fluorescent in situ hybridization (FISH) (Example 23), and array-based detection of 5mC (Example 24). In one embodiment, an in vitro diagnostic (IVD) approach for profiling methylation in a locus-specific manner uses one or more primers to anneal to a predetermined sequence that may contain one or more modified cytosines. After treatment of the target nucleic acid with modified cytidine deaminase, modified cytosines present in the target nucleic acid are converted as described herein (e.g., 5mC is converted to T), and primers can be easily designed to anneal with higher affinity to a given sequence if they contain a nucleotide resulting from the deaminase treatment (e.g., a T nucleotide that was present before the 5mC treatment). For example, as described in Example 21, primers used for amplification bind with greater affinity to nucleic acids containing a T nucleotide where a 5mC nucleotide was present before treatment. Annealing of the primer to a given sequence containing the predicted 5mC to T conversion(s) allows the location of the modified cytosine in the untreated target nucleic acid to be inferred.A primer that binds with higher affinity to a nucleic acid containing a T nucleotide where a 5mC nucleotide was present prior to treatment can contain at least one, at least two, at least three, at least four, or at least five nucleotides that base pair with the nucleotide resulting from the 5mC to T conversion, i.e., adenine (A), and, if amplification is used, can include a second primer for the opposite strand having a T in place of guanine (G).

[0124] In some embodiments, a target nucleic acid obtained from a subject can be treated with a modified cytidine deaminase to obtain a converted nucleic acid, and a pattern of cytosine modifications can be identified in the converted nucleic acid. The pattern of cytosine modifications can optionally be compared to the pattern of cytosine modifications in a reference nucleic acid. In embodiments in which the pattern of cytosine modifications correlates with a disease or condition, the method can be used for diagnostic or prognostic applications. For example, the subject can have or be at risk of having a disease or condition, and the reference nucleic acid can be from a normal subject, e.g., a subject that does not have or is not at risk for the disease or condition. The pattern of cytosine modifications can be associated with a disease or condition (e.g., the target nucleic acid can be a predetermined sequence), and identification in a subject of a pattern of cytosine modifications associated with a disease or condition can indicate that the subject has or is at risk of having a disease or condition. For example, the pattern of cytosine modifications can be linked in cis to a coding region that correlates with a disease or condition, and identification of the pattern or absence of the pattern in a subject can be used for diagnosis or prognosis. In one embodiment, the coding region can be one that is transcriptionally active or transcriptionally inactive in the reference nucleic acid. Comparing the converted nucleic acid to the reference nucleic acid can include determining whether the pattern of cytosine modifications in the converted nucleic acid indicates that the coding region is transcriptionally active or transcriptionally inactive in the subject. If the coding region is associated with a disease or condition, the state of transcriptional activity can be used for diagnosis or prognosis.

[0125] Comparison of patterns of cytosine modification in subjects can also be used to identify changes in patterns of cytosine modification in subjects over time. For example, a subject can have a disease or condition and be undergoing treatment, or a subject can have a disease or condition and be cured (e.g., the subject is treated and no symptoms of the disease or condition are present) or in remission (e.g., the subject is treated and symptoms of the disease or condition are reduced). Target nucleic acids from subjects at different times, e.g., before treatment begins, during treatment, and after treatment is stopped, can be compared to compare the patterns of cytosine modification of sequences, e.g., a given sequence, and can be used to determine the progress of treatment or the state of a disease or condition in a subject.

[0126] In some embodiments where detection of 5mC nucleotides uses amplification, the use of a uracil-disfavored polymerase can help reduce amplification of processed target nucleic acids containing spurious C to U conversions that may result from the use of modified cytidine deaminases. Family B polymerases are known to exhibit a "uracil read-ahead" function that causes the polymerase to stall at uracil residues (Greagg et al., 1999, PNAS USA; 96(16):9045-50). Examples of uracil-disfavored Family B polymerases include archaeal Family B polymerases from Pyrococcus furiosus (Pfu), Thermococcus kodakarensis (KOD), Thermococcus litoralis (Tli / Vent), Pyrococcus woesei (Pwo), and Thermococcus fumicolans (Tfu). Other examples of uracil-disfavored polymerases include Phusion™, Q5™, and Kapa HiFi™. In other embodiments where amplification of nucleic acids containing uracil nucleotides is desired, the use of uracil-resistant polymerases can be used. Examples of uracil-resistant polymerases include Phusion UTM, Q5U™, Kapa UTM, Taq, and Dpo4.

[0127] Wild-type cytidine deaminases typically function at near neutral pH, e.g., pH 7. The modified cytidine deaminases described herein can have increased activity below neutral pH. In some embodiments, the pH of the reaction involving the modified cytidine deaminase described herein can be pH 6.9 or less, pH 6.7 or less, pH 6.5 or less, pH 6.3 or less, pH 6.1 or less, pH 6.0 or less, pH 5.9 or less, pH 5.7 or less, pH 5.5 or less, pH 5.3 or less, pH 5.2 or less, or pH 5.1 or less. In some embodiments, the pH of the reaction involving the modified cytidine deaminase described herein can be at least pH 5.1, at least pH 5.3, at least pH 5.5, at least pH 5.7, at least pH 5.9, at least pH 6.1, at least pH 6.3, at least pH 6.5, at least pH 6.7, or at least pH 6.9. In some embodiments, the pH of the reaction involving the modified cytidine deaminase described herein can be pH 7.5 or less, pH 7.3 or less, or pH 7.1 or less. Exemplary pH ranges for the reaction include at least 5.1 to 6.9 or less, at least 5.1 to 6.5 or less, at least 5.1 to 6.3 or less, or at least 5.1 to 6.1 or less. The activity of the modified cytidine deaminase to deaminate a 5mC oligonucleotide substrate can be at least 10-fold greater, at least 50-fold greater, or at least 100-fold greater increased catalytic activity when comparing activities (see Example 3).

[0128] It is expected that the modified cytidine deaminase can function in essentially any buffer. Examples of useful buffers include, but are not limited to, citrate buffers, such as citrate buffers available from Thermo Fisher Scientific (Cat. No. #005000); sodium acetate buffers, Bis Tris-propane HCl; and Tris-HCl Tris. Examples of other buffers include, but are not limited to, bicine, DIPSO, glycylglycine, HEPES, imidazole, malonic acid, MES, MOPS, PB, phosphate, PIPES, SPG, succinic acid, TAPS, TAPSO, trincine. In some embodiments, a reducing agent such as dithiothreitol (DTT) can be present. In some embodiments, no divalent cation is included.

[0129] The deamination reaction can occur at a temperature between 25°C and 37°C, for example 37°C. Some modified cytidine deaminases described herein preferentially deaminate modified cytosines to thymidine at a faster rate than deamination of cytosine to uracil. Thus, in some embodiments, reaction time can be used to maximize the difference between deamination of modified cytosine and deamination of cytosine. In one embodiment, the reaction can proceed for at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, or at least 150 minutes, and for 15 minutes or less, 30 minutes or less, 45 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 150 minutes or less, or 180 minutes or less.

[0130] In one embodiment, the deamination reaction can include a modified cytidine deaminase at a concentration of at least 0.5 micromolar (μM) to no more than 5 μM. For example, the enzyme concentration can be at least 0.5, at least 1 μM, at least 2 μM, at least 3 μM, at least 4 μM, or 5 μM, and / or no more than 5 μM, no more than 4 μM, no more than 3 μM, no more than 2 μM, no more than 1 μM, or no more than 0.5 μM. In one embodiment, the deamination reaction can include a nucleic acid at a concentration of at least 400 nanomolar (nM) to no more than 2 μM. For example, the concentration of nucleic acid can be at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, or 1 μM, and / or 1 μM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, or 400 nM.

[0131] In one embodiment, the deamination reaction can include RNAse. RNase A is involved in increasing the activity of cytidine deaminase (Bransteitter et al., Proceedings of the National Academy of Sciences of the United States of America 100, no. 7 (2003): 4102-7. doi.org / 10.1073 / pnas.0730835100). When the activity of the modified cytidine deaminase of the present disclosure is determined in the presence of RNAse A, the opposite was observed. When RNAse A was included in the reaction, modified cytidine deaminase with cytosine-deficient deaminase activity (i.e., converts 5mC to T at a faster rate than converts C to U) showed reduced activity, and the reduced activity was more pronounced for off-target cytosine deamination. Thus, RNAse A provided greater selectivity for deamination of 5mC compared to C. RNAse A can be included in the deamination reaction at a concentration of at least 1 microgram per milliliter (ug / mL) to 20 μM or less. For example, the concentration of RNAse A can be at least 1 ug / mL, at least 2 ug / mL, at least 3 ug / mL, at least 4 ug / mL, 5 ug / mL, 6 ug / mL, 7 ug / mL, 8 ug / mL, or 9 ug / mL, and / or 50 ug / mL or less, 40 ug / mL or less, 30 ug / mL or less, 20 ug / mL or less, 19 ug / mL or less, 18 ug / mL or less, 17 ug / mL or less, 16 ug / mL or less, 15 ug / mL or less, 14 ug / mL or less, 13 ug / mL or less, 12 ug / mL or less, or 11 ug / mL or less. In one embodiment, the concentration of RNAse A is 2 ug / mL to 13 ug / mL, or 5 ug / mL to 10 ug / mL.

[0132] target nucleic acid The target nucleic acid contacted with the modified cytidine deaminase and used in the methods, compositions, and kits provided herein can be essentially any nucleic acid of known or unknown sequence. Sequencing can result in determining the sequence of the entire or part of the target molecule. In one embodiment, the target nucleic acid can be processed into a template suitable for amplification by placing universal amplification sequences, such as sequences present in universal adaptors, at the ends of each target fragment.

[0133] The target nucleic acid is typically derived from a primary nucleic acid present in a sample, such as a biological sample. The primary nucleic acid may be derived from DNA or RNA. The DNA primary nucleic acid may be derived from a double-stranded DNA (dsDNA) form (e.g., genomic DNA, genomic DNA fragments, cell-free DNA, etc.) from the sample, or may be derived from a single-stranded form from the sample. The RNA primary nucleic acid may be mRNA or non-coding RNA, such as microRNA or small interfering RNA. The exact sequence of the polynucleotide molecule from the primary nucleic acid sample is generally not critical to the present disclosure and may be known or unknown.

[0134] A primary nucleic acid molecule may represent an entire gene complement of an organism, e.g., a genomic DNA molecule that includes both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. A primary nucleic acid molecule may represent an entire gene complement of a particular cell of an organism, e.g., a tumor cell, where the genomic DNA molecule includes both intron and exon sequences, as well as non-coding regulatory sequences such as promoter and enhancer sequences. In one embodiment, a specific subset of genomic DNA may be used, e.g., one or more specific sequences, such as a particular chromosome, DNA associated with open chromatin, DNA associated with closed chromatin, or a region of a particular gene (e.g., targeted sequencing). In one embodiment, a primary nucleic acid molecule may represent a specific subset of DNA, e.g., DNA with a specific sequence that anneals to a primer, such as one used for targeted sequencing or target enrichment. In one embodiment, a specific subset of DNA may be used, such as cell-free DNA, which may include DNA of a subject, including DNA from normal cells, DNA from diseased cells such as tumor cells, and / or DNA from fetal cells.

[0135] The primary nucleic acid molecule may represent the entire transcriptome of a cell of an organism, such as an mRNA molecule. The primary nucleic acid molecule may represent the entire transcriptome from a particular cell of an organism, such as a tumor cell, or a cell of a tissue, for example. In one embodiment, the primary nucleic acid molecule may represent a particular subset of mRNAs, such as an mRNA with a particular sequence that anneals to a primer, such as one used for target sequencing or target enrichment.

[0136] Samples, such as biological samples, can include nucleic acid molecules obtained from biopsies, tumors, scrapings, swabs, blood, mucus, urine, stool, plasma, semen, hair, laser capture microdissections, surgical resections, and other clinically or laboratory obtained samples. In some embodiments, the sample can be an epidemiological, agricultural, forensic, or pathogenic sample. In some embodiments, the sample can include cultured cells. In some embodiments, the sample can include nucleic acid molecules obtained from an animal, such as a human or mammalian source. In another embodiment, the sample can include nucleic acid molecules obtained from a non-mammalian source, such as a plant, bacteria, virus, or fungus. In some embodiments, the source of the nucleic acid molecule can be a preserved or extinct sample or species.

[0137] Further non-limiting examples of sources of biological samples may include whole organisms, as well as samples obtained from patients. Biological samples may be obtained from any biological fluid or tissue and may be in a variety of forms, including fluids such as liquids or blood, tissues, solid tissues, and preserved forms such as dried, frozen, and fixed forms. Samples may be of any biological tissue, cell, or bodily fluid. Such samples include, but are not limited to, sputum, blood, serum, plasma, blood cells (e.g., white blood cells), peritoneal fluid, urine, saliva, tears, sputum, vaginal fluid, saliva, tears, saliva, vaginal fluid, saliva, tears, saliva, vaginal fluid (secretion), washings obtained during medical procedures (e.g., pelvic or other washings obtained during biopsy, endoscopy, or surgery), tissues, nipple aspirates, core or fine needle biopsy samples, cell-containing bodily fluids, peritoneal fluid, and pleural fluid, or cells therefrom, as well as free floating nucleic acids such as cell-free circulating DNA. Biological samples may also include sections of tissues, such as frozen or fixed sections taken for histological purposes or microdissected cells or extracellular portions thereof. In some embodiments, the sample may be a blood sample, such as a whole blood sample. In another example, the sample is an unprocessed dried blood spot (DBS) sample. In yet another example, the sample is a formalin-fixed paraffin-embedded (FFPE) sample. In yet another example, the sample is a saliva sample. In yet another example, the sample is a dried saliva spot (DSS) sample.

[0138] Exemplary biological samples from which target nucleic acids may be derived include, for example, from eukaryotic organisms, e.g., mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates, humans or non-human primates; plants, e.g., Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybeans; algae, e.g., Chlamydomonas reinhardtii; nematodes, e.g., Caenorhabditis elegans; insects, e.g., Drosophila melanogaster, mosquitoes, fruit flies, honeybees, or spiders; fish, e.g., zebrafish; reptiles; amphibians, e.g., frogs or Xenopus laevis; Dictyostelium discoideum, Pneumocystis carinii, Takifugu rubripes, yeast, Sacharamoyces cerevisiae, or Schizosaccharomyces The target nucleic acid may be derived from a fungus, such as Plasmodium pombe, or a protozoan, such as Plasmodium falciparum. The target nucleic acid may also be derived from a bacterium, a prokaryote, such as Escherichia coli, Staphylococcus, or Mycoplasma pneumoniae, an archaea; a virus, such as Hepatitis C virus or human immunodeficiency virus; or a viroid. The target nucleic acid may be derived from a homogenous culture or population of organisms as described herein, or alternatively, may be derived from a collection of several different organisms, for example in a community or ecosystem.

[0139] In some embodiments, the biological sample comprises tissue that is processed to obtain the desired primary nucleic acid. In some embodiments, cells are used to obtain the desired primary nucleic acid. In some embodiments, nuclei are used to obtain the desired primary nucleic acid. The method may further comprise dissociating the cells and / or isolating the nuclei. Methods for isolating cells and nuclei from tissue are available (WO 2019 / 236599).

[0140] In some embodiments, nucleic acids present in tissues, cells, or isolated nuclei can be processed depending on the desired readout. For example, nucleic acids can be fixed during processing, and useful fixation methods are available (WO 2019 / 236599). Fixation can be useful to preserve samples or maintain the continuity of analytes from samples, cells, or nuclei. Fixation methods preserve and stabilize tissue, cell, and nuclear morphology and architecture, inactivate proteolytic enzymes, and strengthen samples, cells, and nuclei so they can withstand further processing and staining and protect against contamination. Examples of methods in which fixation can be useful include, but are not limited to, whole genome sequencing of isolated nuclei and chromosome conformation capture methods such as Hi-C. Common fixation methods include perfusion, immersion, freezing, and drying (Srinivasan et al., Am J Pathol. 2002 Dec;161(6):1961-1971.doi:10.1016 / S0002-9440(10)64472-0). In some embodiments, such as whole genome sequencing, methods are available for processing isolated nuclei to dissociate nucleosomes from DNA while leaving the nuclei intact, generating nucleosome-free nuclei (WO 2018 / 018008).

[0141] In some embodiments, for example, primary nucleic acids in bulk from multiple cells can be used to generate the sequencing libraries described herein, in other embodiments, individual cells or nuclei can be used as a source of primary nucleic acids to obtain sequence information from single cells and nuclei. Many different single cell library preparation methods are known in the art, including but not limited to Drop-seq, Seq-well, and single cell combinatorial indexing ("sci" methods). Companies offering single cell products and related technologies include, but are not limited to, Illumina, 10X Genomics, Takara Biosciences, BD Biosciences, Bio-Rad Laboratories, 1cellbio, Isoplexis, CellSee, NanoSelect, and Dolomite Bio. Sci-seq is a methodological framework that uses split-pool barcoding to uniquely label the nucleic acid content of a large number of single cells or single nuclei. In general, the number of nuclei or cells can be at least two. The upper limit depends on the practical limitations of the equipment used in other steps of the methods described herein (e.g., multi-well plates, number of indexes). The number of nuclei or cells that can be used is not intended to be limiting and can reach billions.

[0142] The target nucleic acid used in the disclosed methods and compositions can be derived by fragmentation. Random fragmentation refers to the fragmentation of polynucleotide molecules from a primary nucleic acid sample in a random manner by enzymatic, chemical, or mechanical methods. Such fragmentation methods are known in the art and use standard methods (Sambrook and Russell, Molecular Cloning, A Laboratory Manual, third edition). Furthermore, random fragmentation is designed to generate fragments without regard to the sequence identity or position of the nucleotides that contain and / or surround the break. In one embodiment, random fragmentation is by mechanical means such as nebulization or sonication to generate fragments about 50 base pairs in length to about 1500 base pairs in length, more specifically 50-700 base pairs in length, even more specifically or 50-400 base pairs in length. Most specifically, this method is used to generate smaller fragments of 50-150 base pairs in length.

[0143] Fragmentation of polynucleotide molecules by mechanical means (e.g., spraying, sonication, and Hydroshear) results in fragments with a heterogeneous mix of blunt ends and 3'- and 5'-overhanging ends. It is therefore desirable to repair the fragment ends using methods or kits known in the art (such as the Lucigen DNA Terminator End Repair Kit) to generate ends that are optimal for insertion into, for example, blunt sites in a cloning vector. In certain embodiments, the fragment ends of the nucleic acid population are blunt. More specifically, the fragment ends are blunt and phosphorylated. Phosphate moieties can be introduced by enzymatic treatment, for example, using polynucleotide kinase.

[0144] In certain embodiments, the target fragment sequence is prepared with a single overhanging nucleotide by the activity of certain types of DNA polymerases, such as, for example, Taq polymerase or Klenow exo minus polymerase with template-independent terminal transferase activity, which adds a single deoxynucleotide, e.g., deoxyadenosine (A), to the 3' end of a DNA molecule, e.g., a PCR product. Such enzymes can be used to add a single nucleotide "A" to the 3' end of the blunt end of each strand of a double-stranded target fragment. Thus, an "A" can be added to the 3' end of each end-repaired strand of a double-stranded target fragment by reaction with Taq or Klenow exo minus polymerase, while the universal adaptor polynucleotide construct can be a T construct with a compatible "T" overhang present at the 3' end of each region of the double-stranded nucleic acid of the universal adaptor. This end modification also prevents self-ligation of both the vector and the target, such that there is a bias to form a target nucleic acid with a universal adaptor at each end.

[0145] In one embodiment, fragmentation can be accomplished using a process often referred to as tagmentation. Tagmentation uses a transposome complex to add universal adaptors in combination with a single step of fragmentation and ligation (WO 2016 / 130704). The transposome complex can be bound to a transposase recognition site and insert the transposase recognition site into the target nucleic acid in a process sometimes referred to as "tagmentation". In some such insertion events, a single strand of the transposase recognition site can be transferred to the target nucleic acid. Such a strand is referred to as the "transfer strand". In one embodiment, the transposome complex comprises a dimeric transposase having two subunits and two non-contiguous transposon sequences. In another embodiment, the transposase comprises a dimeric transposase having two subunits and two non-contiguous transposon sequences.

[0146] Some embodiments may include the use of hyperactive Tn5 transposase and Tn5-type transposase recognition sites (Goryshin and Reznikoff, J. Biol. Chem., 273:7367 (1998)), or MuA transposase and Mu transposase recognition sites that include R1 and R2 end sequences (Mizuuchi, K., Cell, 35:785, 1983; Savilahti, H, et al., EMBO J., 14:4893, 1995). Tn5 mosaic end (ME) sequences may also be used by one of skill in the art.

[0147] Examples of transposon sequences useful in the methods and compositions described herein are provided in U.S. Patent Application Publication Nos. 2012 / 0208705, 2012 / 0208724, and WO 2012 / 061832. In some embodiments, the transposon sequence comprises a first transposase recognition site and a second transposase recognition site.

[0148] Some transposome complexes useful herein comprise a transposase with two transposon sequences.In some such embodiments, the two transposon sequences are not connected to each other, in other words, the transposon sequences are not contiguous to each other.Examples of such transposomes are known in the art (see, for example, US Patent Application Publication No. 2010 / 0120098).

[0149] In one embodiment, tagmentation is used to generate a target nucleic acid that contains a different universal sequence at each end. This can be achieved by using two transposome complexes, each of which contains a different nucleotide sequence that is part of the transport strand.

[0150] The population of target nucleic acids can have an average chain length that is desirable or suitable for a particular application of the methods, compositions, or kits described herein. For example, the average chain length can be less than about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100 nucleotides, or 50 nucleotides. Alternatively, or in addition, the average chain length can be more than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or 100,000 nucleotides. The average chain length of the population of target nucleic acids can be within a range between the maximum and minimum values ​​described herein. It will be understood that the amplicons generated at the amplification site (or otherwise made or used herein) can have an average chain length in a range between the upper and lower limits selected from those exemplified above.

[0151] In some cases, a population of target nucleic acids can be configured under conditions that have a maximum length for its members, or otherwise to have a maximum length for its members. For example, the maximum length of a member used in one or more steps of the methods described herein or present in a particular composition can be less than 100,000 nucleotides, less than 50,000 nucleotides, less than 10,000 nucleotides, less than 5,000 nucleotides, less than 1,000 nucleotides, less than 500 nucleotides, less than 100 nucleotides, or less than 50 nucleotides. Alternatively or in addition, a population of target nucleic acids can be configured under conditions that have a minimum length for its members, or otherwise to have a minimum length for its members. For example, the minimum length of a member used in one or more steps of the methods described herein or present in a particular composition can be more than 10 nucleotides, more than 50 nucleotides, more than 100 nucleotides, more than 500 nucleotides, more than 1,000 nucleotides, more than 5,000 nucleotides, more than 10,000 nucleotides, more than 50,000 nucleotides, or more than 100,000 nucleotides. The maximum and minimum strand lengths of the target nucleic acids in the population can range between the maximum and minimum values ​​listed above. It will be understood that the amplicons produced at the amplification site (or otherwise made or used herein) can have maximum and / or minimum strand lengths that range between the upper and lower limits exemplified above.

[0152] In some embodiments, a sample can be enriched for a sequence of interest, e.g., a predetermined sequence. For example, a subset of genes or regions of the genome are isolated and sequenced, or a subset of genes or regions of the genome are interrogated by other methods, such as locus-specific in vitro diagnostic methods. The predetermined sequence can be, for example, a sequence that can have a pattern of cytosine modifications.

[0153] In some embodiments, target enrichment works by capturing genomic regions of interest by hybridization to target-specific probes, which can be used to physically separate the target DNA hybridized to the bait probe from all other DNA in solution, and then washing away the target.For example, some enrichment methods use biotinylated probes, which are then isolated by magnetic pull-down using streptavidin-coated magnetic particles.In another example, some enrichment methods use analyte arrays, also known as microarrays, which allow hybridization of predetermined sequences.

[0154] Enrichment can be performed, for example, prior to treatment with modified cytidine deaminase. In such embodiments, enriching the nucleic acid of interest or fragments thereof, such as enriching DNA in a sample, may include any suitable enrichment technique. In some embodiments, enrichment of DNA may include molecular inversion probes in solution capture, pull-down probes, bait sets, standard PCR, multiplex PCR, hybrid capture, endonuclease digestion, DNase I hypersensitivity, and enrichment by selective circularization. Enrichment can be achieved by negative selection of nucleic acids by removing undesired material. This type of enrichment includes "footprinting" techniques or "subtractive" hybrid capture. During the former, the target sample is safe from nuclease activity by protein protection or by single- and double-stranded configuration. During the latter, nucleic acids that bind to the "bait" probe are removed.

[0155] In some embodiments, enrichment can include amplification using target-specific primers. In some embodiments, amplification is performed after another form of enrichment. Typically, however, in embodiments using amplification for enrichment, the amplification step is performed after treatment with deaminase to preserve the methylation state of the target DNA. In some such embodiments, amplification can include PCR amplification or genome-wide amplification.

[0156] In some embodiments, enrichment can occur after treatment with modified cytidine deaminase. Typically, in methods used to identify methylated cytosines, the conversion of unmethylated DNA bases to uracil results in loss of DNA complexity, resulting in a three-base genome, limiting the use of sequences that specifically hybridize to a given sequence. Therefore, typical methods for identifying methylated cytosines are more difficult to use in methods that involve enrichment, such as hybrid enrichment sequencing and amplicon-based targeted sequencing, after conversion of methylated cytosines. In contrast, (i) the conversion of 5mC to T by modified cytidine deaminase, and (ii) because only a small percentage of cytosines are expected to be methylated and converted by modified cytidine deaminase. Examples of enrichment-based methods that can be used after treatment of target nucleic acid with modified cytidine deaminase include, but are not limited to, analyte arrays, the use of primers for selective amplification, CRISPR-Cas systems, and molecular cell development techniques such as FISH. Examples of arrays include, for example, methylation arrays (eg, Infinium MethylationEPIC BeadChip, Illumina) for the interrogation of selected methylation sites across the genome.

[0157] Universal Adapter Binding In some embodiments, the target nucleic acid used in the methods, compositions, or kits described herein can include a universal adaptor attached to each end. A target nucleic acid with a universal adaptor at each end can be referred to as a "modified target nucleic acid." Methods for attaching universal adaptors to each end of a target nucleic acid used in the methods described herein are known to those of skill in the art. Attachment can be performed by tagmentation using a transposase complex (WO 2016 / 130704) or standard library preparation techniques using ligation (see US 2018 / 0305753). Attachment of universal adaptors to the ends of the target nucleic acid can occur before or after treatment of the target nucleic acid with a modified cytidine deaminase.

[0158] In one embodiment, double-stranded target nucleic acid from a sample, such as a fragmented sample that has been contacted with modified cytidine deaminase and converted from single-stranded to double-stranded nucleic acid, is first treated by ligating identical universal adapter molecules to the 5' and 3' ends of the double-stranded target nucleic acid. In one embodiment, the universal adapter is a "match" adapter or Y adapter, since the two strands of the adapter are formed by annealing complementary polynucleotide strands. In one embodiment, the universal adapter used in the disclosed method is referred to as a "mismatch" adapter, since the adapter contains a region of sequence mismatch, i.e., is not formed by annealing perfectly complementary polynucleotide strands. General characteristics of mismatch adapters are further described in Gormley et al., U.S. Pat. No. 7,741,463, and Bignell et al., U.S. Pat. No. 8,053,192. Universal adapters typically contain a universal capture binding sequence that serves to immobilize the target nucleic acid on an array for subsequent sequencing, and a universal primer binding site that is useful for sequencing. In another embodiment, a double-stranded target nucleic acid from a sample that has been contacted with a modified cytidine deaminase and converted from single-stranded to double-stranded nucleic acid is subjected to tagmentation by a transposome complex that inserts a universal adaptor or a sequence that can be used to add a universal adaptor into the target nucleic acid.

[0159] The universal adaptor may optionally include at least one index. The index can be used as a marker characteristic of the source of a particular target nucleic acid on a flow cell (US Pat. No. 8,053,192). In general, the index is a synthetic sequence of nucleotides that is part of the universal adaptor that is added to the target nucleic acid as part of the library preparation process. Thus, the index is a nucleic acid sequence that is bound to each of the target molecules of a particular sample, and its presence is used to indicate or identify the sample or source from which the target molecule was isolated.

[0160] Preferably, the index may be up to 20 nucleotides long, more preferably up to 1-10 nucleotides long, most preferably up to 4-6 nucleotides long. A four nucleotide index offers the possibility of multiplexing 256 samples on the same array, whereas a six base index allows the processing of 4096 samples on the same array.

[0161] The exact nucleotide sequence of the universal adaptor is generally not critical to the present disclosure and may be selected by the user such that the desired sequence element is ultimately included in the common sequence of a plurality of different modified target nucleic acids, e.g., to provide a universal capture binding sequence for immobilizing the target nucleic acids on an array for subsequent sequencing, as well as binding sites for a particular set of universal amplification primers and / or sequencing primers. Additional sequence elements may be included, e.g., to provide binding sites for sequencing primers that will ultimately be used in sequencing the target nucleic acids in the library, sequencing the indexes, or products derived from amplification of the target nucleic acids of the library, e.g., on a solid support.

[0162] To prepare a library of deaminase-treated DNA for analysis using a sequencing platform, it may be useful to perform additional modifications on the target DNA, either before or after treatment with modified cytidine deaminase. In some embodiments, the single-stranded deaminase-treated DNA is prepared for sequencing using single-stranded library preparation methods, as known in the art. Such methods include, but are not limited to, template switching-based second strand synthesis, adapters containing single-stranded splint overhangs, and the like. Reagents for performing single-stranded library preparation methods are commercially available. Examples include xGen ssDNA & Low-Input DNA Library Prep Kit (Integrated DNA Technologies catalog number 10009859), previously sold as Accel-NGS (Swift Biosciences), NGS Single Stranded DNA Library Prep Kit (BioDynami catalog number 30082). Another example includes single-reaction single-stranded libraries (SRSLY), as described in Troll et al., BMC Genomics 20, 1023 (2019).

[0163] In some embodiments, library preparation modifications are performed on double-stranded target DNA prior to treatment with modified cytidine deaminase. Methods for library preparation of double-stranded DNA templates are known in the art and include Y-adapter ligation, transposome-based tagmentation, and the like. It will be understood by those skilled in the art that methods for double-stranded library preparation often include one or more amplification steps, for example using PCR. In such methods, the amplification step may be postponed until after modified cytidine deaminase treatment to preserve the methylation state of the template strand. For example, in the Y-adapter ligation method, a Y-adapter can be ligated to the double-stranded template, and then the adapter-ligated template DNA is denatured and treated with modified cytidine deaminase as described elsewhere herein. After treatment with modified cytidine deaminase, the resulting treated single-stranded DNA molecules can be amplified using PCR, bridge amplification, and other methods commonly known in the art.

[0164] Preparation of immobilized samples for sequencing A library of modified target nucleic acids, for example, target nucleic acids with universal adaptors at each end, can be prepared for sequencing. Methods of attaching modified target nucleic acids to substrates are known in the art. In one embodiment, the modified fragments are enriched using multiple capture oligonucleotides with specificity for the modified fragments, and the capture oligonucleotides can be immobilized on the surface of a solid substrate, such as a flow cell or beads. For example, the capture oligonucleotides can include a first member of a universal binding pair, and a second member of the binding pair is immobilized on the surface of the solid substrate. Similarly, methods of amplifying immobilized target nucleic acids include, but are not limited to, bridge amplification and exclusion amplification (also called binding equilibrium exclusion (KEA)). Methods of immobilization and amplification prior to sequencing are described, for example, in Bignell et al. (U.S. Pat. No. 8,053,192), Gunderson et al. (WO 2016 / 130704), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (U.S. Pat. No. 9,309,502).

[0165] Pooled samples can be immobilized in preparation for sequencing. Sequencing can be performed as an array of single molecules or can be amplified prior to sequencing. Amplification can be performed using one or more immobilized primers. The immobilized primers can be, for example, on a flat surface or in a lawn on a pool of beads. The pool of beads can be isolated in an emulsion with a single bead in each "compartment" of the emulsion. At a concentration of only one template per "compartment", only a single template is amplified on each bead.

[0166] As used herein, the term "solid-phase amplification" refers to any nucleic acid amplification reaction carried out on or in association with a solid support such that all or a portion of the amplification product is immobilized on the solid support when formed. Specifically, the term encompasses solid-phase polymerase chain reaction (PCR) and solid-phase isothermal amplification, which are reactions similar to standard solution-phase amplification, except that one or both of the forward and reverse amplification primers are immobilized on a solid support. Solid-phase PCR encompasses systems such as emulsions where one primer is immobilized on a bead and the other is in free solution, and colonization in solid-phase gel matrices where one primer is immobilized on a surface and the other is in free solution.

[0167] In some embodiments, the solid support comprises a patterned surface. "Patterned surface" refers to an arrangement of different regions in or on an exposed layer of a solid support. For example, one or more of the regions can be features in which one or more amplification primers are present. The features can be separated by interstitial regions in which no amplification primers are present. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeating arrangement of features and / or interstitial regions. In some embodiments, the pattern can be a random arrangement of features and / or interstitial regions. Exemplary patterned surfaces that can be used in the methods and compositions described herein are described in U.S. Pat. Nos. 8,778,848, 8,778,849, and 9,079,148, and U.S. Patent Application Publication No. 2014 / 0243224.

[0168] In some embodiments, the solid support comprises an array of wells or depressions on its surface, which can be fabricated as commonly known in the art using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques. As will be appreciated by those skilled in the art, the technique used will depend on the composition and shape of the array substrate.

[0169] The features within the patterned surface can be wells of an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic, or other suitable solid support with a patterned covalently attached gel, such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, see, e.g., U.S. Patent Application Publication Nos. 2013 / 184796, WO 2016 / 066586, and 2015 / 002813). This process creates a gel pad used for sequencing, which can be stable over a large number of cycles of sequencing operations. Covalently attaching the polymer to the wells is useful to maintain the gel in the structured features throughout the life of the structured substrate during various applications. However, in many embodiments, the gel does not need to be covalently attached to the wells. For example, in some conditions, silane free acrylamide (SFA, see, e.g., U.S. Pat. No. 8,563,477), which is not covalently bonded to any part of the structured matrix, can be used as the gel material.

[0170] In certain embodiments, a structured substrate can be created by patterning a solid support material with wells (e.g., microwells or nanowells), coating the patterned support with a gel material (e.g., PAZAM, SFA, or chemically modified variants thereof), such as an azido form of SFA (azido-SFA), and polishing the gel-coated support, for example by chemical or mechanical polishing, thereby retaining the gel in the wells but removing or inactivating substantially all of the gel from the interstitial regions of the surface of the structured substrate between the wells. Primer nucleic acids can be attached to the gel material. A solution of modified target nucleic acids can then be contacted with the polished substrate such that individual modified target nucleic acids are seeded into individual wells via interaction with primers attached to the gel material, however, due to the absence or inactivity of the gel material, the target nucleic acids do not occupy the interstitial regions. Amplification of the modified target nucleic acids will be restricted to the wells because the absence or inactivity of the gel in the interstitial regions prevents migration out of the growing nucleic acid colonies. The process is conveniently manufacturable and scalable, utilizing conventional micro- or nano-fabrication methods.

[0171] Although the present disclosure encompasses "solid phase" amplification methods in which only one amplification primer is immobilized (the other primer is usually in free solution), in one embodiment, a solid support is provided with both immobilized forward and reverse primers. In practice, since the amplification process requires an excess of primers to maintain amplification, there will be "multiple" identical forward primers and / or "multiple" identical reverse primers immobilized on the solid support. References herein to forward and reverse primers should be construed as encompassing a plurality of such primers, unless the context dictates otherwise.

[0172] As will be understood by those skilled in the art, any given amplification reaction requires at least one type of forward primer and at least one type of reverse primer specific to the template to be amplified. However, in certain embodiments, the forward and reverse primers may contain template-specific portions of the same sequence and may have completely identical nucleotide sequences and structures (including any non-nucleotide modifications). In other words, solid-phase amplification can be performed using only one type of primer, and such single primer methods are encompassed within the scope of the present disclosure. Other embodiments may use forward and reverse primers that contain the same template-specific sequence but differ in some other structural features. For example, one type of primer may contain non-nucleotide modifications that are not present in the other.

[0173] Primers for solid phase amplification are preferably immobilized by a single-point covalent bond to the solid support at or near the 5' end of the primer, leaving the template-specific portion of the primer free for annealing to its cognate template and the 3' hydroxyl group free for primer extension. Any suitable covalent attachment means known in the art may be used for this purpose. The attachment chemistry chosen will depend on the nature of the solid support and any derivatization or functionalization applied to it. The primer itself may contain a moiety that may be a non-nucleotidic chemical modification to facilitate attachment. In certain embodiments, the primer may contain a sulfur-containing nucleophile such as a phosphorothioate or thiophosphate at the 5' end. In the case of solid-supported polyacrylamide hydrogels, this nucleophile binds to bromoacetamide groups present in the hydrogel. A more specific means of attaching primers and templates to the solid support is via a 5' phosphorothioate bond to a hydrogel composed of polymerized acrylamide and N-(5-bromoacetamidoylpentyl)acrylamide (BRAPA), as described in WO 05 / 065814.

[0174] Certain embodiments of the present disclosure may utilize solid supports that include an inert substrate or matrix (e.g., glass slides, polymeric beads, etc.) that have been "functionalized" by application of a layer or coating of an intermediate material that includes reactive groups that allow for covalent attachment to a biomolecule, such as a polynucleotide. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass. In such embodiments, the biomolecule (e.g., polynucleotide) may be covalently attached directly to the intermediate material (e.g., hydrogel), although the intermediate material may itself be non-covalently attached to the substrate or matrix (e.g., glass substrate). The term "covalent attachment to a solid support" should be interpreted accordingly to encompass this type of arrangement.

[0175] The pooled samples may be amplified on beads, each bead containing forward and reverse amplification primers. In one embodiment, a library of modified target nucleic acids is used to prepare clustered arrays of nucleic acid colonies by solid-phase amplification, more specifically solid-phase isothermal amplification, similar to those described in US Patent Application Publication No. 2005 / 0100900, US Patent No. 7,115,400, WO 00 / 18957 and WO 98 / 44151. The terms "cluster" and "colony" are used interchangeably herein to refer to distinct sites on a solid support that contain a plurality of identical immobilized nucleic acid strands and a plurality of identical immobilized complementary nucleic acid strands. The term "clustered array" refers to an array formed from such clusters or colonies. In this context, the term "array" should not be understood as requiring an ordered arrangement of clusters.

[0176] The terms "solid phase" or "surface" are used to mean either a planar array of beads attached to a flat surface, such as a glass, silica or plastic microscope slide, or similar flow cell device, or beads to which one or two primers are attached and the beads are amplified, or an array of beads on a surface after the beads have been amplified.

[0177] Clustered sequences may be prepared using a process of thermal cycling such as described in WO 98 / 44151, or a process in which the temperature is held constant and cycles of extension and denaturation are performed using changes in reagents. Such isothermal amplification methods are described in WO 02 / 46456 and U.S. Patent Application Publication No. 2008 / 0009420.

[0178] It will be understood that any of the amplification methods described herein or generally known in the art can be used with universal or target-specific primers to amplify the immobilized DNA fragments. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA), and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354. The above amplification methods may be used to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA, etc., may be used to amplify the immobilized DNA fragments. In some embodiments, primers specifically directed to the polynucleotide of interest are included in the amplification reaction.

[0179] Other methods suitable for amplifying polynucleotides may include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998)), and oligonucleotide ligation assay (OLA) techniques (see generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907; European Patent Nos. 0320308(B1); 0336731(B1); 0439182(B1); WO 90 / 01069; WO 89 / 12696; and WO 89 / 09835). It is understood that these amplification methods may be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method may include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction containing a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method may include a primer extension ligation reaction containing a primer specifically directed to the nucleic acid of interest. As non-limiting examples of primer extension and ligation primers that may be specifically designed to amplify the nucleic acid of interest, the amplification may include the primers used in the GoldenGate assay (Illumina, San Diego, Calif.), as exemplified by U.S. Pat. Nos. 7,582,420 and 7,611,869.

[0180] DNA nanoballs can also be used in combination with the methods, systems and compositions described herein.Methods for making and using DNA nanoblocks for genome sequencing can be found, for example, in U.S. Patents and Publications U.S. Patent No. 7,910,354, U.S. Patent Application Publication No. 2009 / 0264299, U.S. Patent Application Publication No. 2009 / 0011943, U.S. Patent Application Publication No. 2009 / 0005252, U.S. Patent Application Publication No. 2009 / 0155781, U.S. Patent Application Publication No. 2009 / 0118488, and can be found, for example, as described in Drmanac et al. (2010, Science 327(5961):78-81). Briefly, after generation of the modified target nucleic acid, the modified target nucleic acid is circularized and amplified by rolling circle amplification (Lizardi et al., 1998. Nat. Genet. 19:225-232; US Patent Application Publication No. 2007 / 0099208(A1)). The extended concatemeric structure of the amplicon promotes coiling and creates compact DNA nanoballs. The DNA nanoballs can be captured on a substrate and preferably form an ordered or patterned array such that the distance between each nanoball is maintained, thereby allowing sequencing of individual DNA nanoballs. In some embodiments, such as those used by Complete Genomics (Mountain View, Calif.), successive rounds of adaptor addition, amplification and digestion are performed prior to circularization to create a head-to-tail construct with several target nucleic acids separated by adaptor sequences.

[0181] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA), exemplified by, for example, Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification, exemplified, for example, by U.S. Pat. No. 6,214,587. Other non-PCR-based methods that can be used in the present disclosure include strand displacement amplification (SDA), as described, for example, in Walker et al., Molecular Methods for Virus Detection, Academic Press, 1995, U.S. Patent Nos. 5,455,166 and 5,130,238, and in Walker et al., Nucl. Acids Res. 20:1691-96 (1992), or hyperbranched strand displacement amplification, as described, for example, in Lage et al., Genome Res. 13:294-307 (2003). Isothermal amplification methods can be used, for example, with strand displacing Phi29 polymerase or Bst DNA polymerase large fragment, 5'->3' exo for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. The high processivity allows the polymerase to produce fragments of 10-20 kb in length. As mentioned above, polymerases with low processivity and polymerases with strand displacement activity, such as Klenow polymerase, may be used to generate smaller fragments under isothermal conditions. Further description of the amplification reaction, conditions and components are described in detail in the disclosure of U.S. Patent No. 7,670,810.

[0182] In some embodiments, isothermal amplification can be performed using kinetic exclusion amplification (KEA), also referred to as exclusion amplification (ExAmp). The nucleic acid library of the present disclosure can be made using a method that includes reacting amplification reagents to generate a plurality of amplification sites, each of which contains a substantially clonal population of amplicons from individual target nucleic acids seeded at the site. In some embodiments, the amplification reaction proceeds until a sufficient number of amplicons are generated to fill the capacity of each amplification site. In this way, filling a previously seeded site to capacity inhibits target nucleic acids from landing and amplifying at that site, thereby generating a clonal population of amplicons at that site. In some embodiments, apparent clonality can be achieved even if an amplification site is not filled to capacity before a second target nucleic acid arrives at the site. Under some conditions, amplification of a first target nucleic acid can proceed to a point where a sufficient number of copies are generated to effectively exceed or overwhelm the generation of copies from a second target nucleic acid transported to the site. For example, in an embodiment using a bridge amplification process on a circular feature less than 500 nm in diameter, it was determined that after 14 cycles of exponential amplification for a first target nucleic acid, contamination from a second target nucleic acid at the same site produces an insufficient number of contaminating amplicons to adversely affect sequence synthesis analysis on an Illumina sequencing platform.

[0183] In some embodiments, the amplification sites in the array can be, but are not necessarily, completely clonal. Rather, in some applications, each amplification site is populated primarily with amplicons from a first modified target nucleic acid, and can also have low levels of contaminating amplicons from a second modified target nucleic acid. An array can have one or more amplification sites with low levels of contaminating amplicons, as long as the level of contamination does not have an unacceptable effect on the subsequent use of the array. For example, when an array is used in a detection application, an acceptable level of contamination is one that does not affect the signal-to-noise ratio or resolution of the detection technique in an unacceptable manner. Thus, apparent clonality is generally related to the particular use or application of an array made by the methods described herein. Exemplary levels of contamination that may be acceptable in an individual amplification site for a particular application include, but are not limited to, up to 0.1%, 0.5%, 1%, 5%, 10%, or 25% contaminating amplicons. An array can include one or more amplification sites with these exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or even 100% of the amplification sites in an array may have some contaminating amplicons. It is understood that in an array or other collection of sites, at least 50%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the sites may be clonal or appear clonal.

[0184] In some embodiments, dynamic exclusion may occur when a process occurs at a rate fast enough to effectively exclude another event or process from occurring. Take as an example the creation of a nucleic acid array in which the sites of the array are randomly seeded with modified target nucleic acids from a solution, and copies of the modified target nucleic acids are generated in an amplification process to fill each of the seeded sites to capacity. According to the dynamic exclusion method of the present disclosure, the seeding and amplification processes can proceed simultaneously under conditions where the amplification rate exceeds the seeding rate. Thus, the relatively fast rate at which copies are generated at a site seeded by a first target nucleic acid effectively excludes a second nucleic acid from seeding that site for amplification. The binding equilibrium exclusion method can be carried out as described in detail in the disclosure of US Patent Application Publication No. 2013 / 0338042.

[0185] Dynamic exclusion can take advantage of the relatively fast rate of making subsequent copies of the modified target nucleic acid (or of the first copy of the modified target nucleic acid) versus the relatively slow rate of initiating amplification (e.g., the relatively slow rate of making the first copy of the modified target nucleic acid). In the example of the previous paragraph, dynamic exclusion occurs due to the relatively fast rate at which amplification occurs to fill the site with the seeded copies of the modified target nucleic acid versus the relatively slow rate of seeding of the modified target nucleic acid (e.g., the relatively slow diffusion or transport). In another exemplary embodiment, dynamic exclusion can occur due to the delay (e.g., delayed or slow activation) of the formation of the first copy of the modified target nucleic acid seeded at the site versus the relatively fast rate at which subsequent copies are made to fill the site. In this example, an individual site may be seeded with several different modified target nucleic acids (e.g., prior to amplification, there may be several modified target nucleic acids at each site). However, the first copy formation of any given modified target nucleic acid may be activated randomly, which results in a relatively slow average rate of first copy formation compared to the rate at which subsequent copies are generated. In this case, an individual site may be seeded with several different modified target nucleic acids, but dynamic exclusion allows amplification of only one of them. More specifically, when a first modified target nucleic acid is activated for amplification, the site is rapidly filled to capacity with copies of it, thereby preventing copies of a second modified target nucleic acid from being made at that site.

[0186] In one embodiment, the method simultaneously (i) transports modified target nucleic acid to the amplification site at an average transport rate and (ii) amplifies modified target nucleic acid at the amplification site at an average amplification rate, so that the average amplification rate exceeds the average transport rate (U.S. Pat. No. 9,169,513). Thus, in such an embodiment, binding equilibrium exclusion can be achieved by using a relatively slow transport rate. For example, a modified target nucleic acid with a sufficiently low concentration can be selected to achieve a desired average transport rate, since a low concentration results in a slow average transport rate. Alternatively or additionally, a high viscosity solution and / or the presence of a molecular crowding agent in the solution can be used to reduce the transport rate. Examples of useful molecular crowding agents include, but are not limited to, polyethylene glycol (PEG), ficoll, dextran, or polyvinyl alcohol. Exemplary molecular crowding agents and formulations are described in U.S. Pat. No. 7,399,590, which is incorporated herein by reference. Another factor that can be adjusted to achieve a desired transport rate is the average size of the target nucleic acid.

[0187] Amplification reagents may include additional components that facilitate amplicon formation, potentially increasing the rate of amplicon formation. One example is a recombinase. Recombinases can facilitate amplicon formation by allowing repeated infiltration / extension. More specifically, recombinases can facilitate the infiltration of modified target nucleic acids by polymerases using the modified target nucleic acids as templates for amplicon formation, and the extension of primers by the polymerase. This process can be repeated as a chain reaction in which the amplicons generated from each round of infiltration / extension serve as templates in subsequent rounds. This process can occur more quickly than standard PCR, since no denaturation cycles (e.g., by heating or chemical denaturation) are required. Thus, recombinase-facilitated amplification can be performed isothermally. In general, it is desirable to include ATP, or other nucleotides (or potentially non-hydrolyzable analogs thereof) in recombinase-facilitated amplification reagents to facilitate amplification. A mixture of recombinase and single stranded binding (SSB) protein is particularly useful, as SSB can further facilitate amplification. Representative formulations for recombinase-facilitated amplification include those sold as TwistAmp kits by TwistDx (Cambridge, UK). Useful components and reaction conditions for recombinase-facilitated amplification reagents are described in U.S. Patent Nos. 5,223,414 and 7,399,590.

[0188] Another example of a component that can be included in an amplification reagent to promote amplicon formation and possibly increase the rate of amplicon formation is a helicase. Helicase can promote amplicon formation by allowing a chain reaction of amplicon formation. This process can occur more quickly than standard PCR because no denaturation cycles (e.g., by heating or chemical denaturation) are required. Thus, helicase-promoted amplification can be carried out isothermally. A mixture of helicase and single-stranded binding (SSB) protein is particularly useful because SSB can further promote amplification. Representative formulations for helicase-promoted amplification include those commercially available as IsoAmp kits from Biohelix (Beverly, MA). Further examples of useful formulations that include helicase proteins are described in U.S. Patent Nos. 7,399,590 and 7,829,284.

[0189] Yet another example of a component that can be included in an amplification reagent to facilitate amplicon formation, and in some cases increase the rate of amplicon formation, is an origin binding protein.

[0190] Sequencing methods Following the binding of the modified target nucleic acid to the surface, the sequence of the immobilized and amplified modified target nucleic acid is determined. Sequencing can be performed using any suitable sequencing technology, and methods for determining the sequence of the immobilized and amplified modified target nucleic acid, including strand resynthesis, are known in the art and are described, for example, in Bignell et al. (U.S. Pat. No. 8,053,192), Gunderson et al. (WO 2016 / 130704), Shen et al. (U.S. Pat. No. 8,895,249), and Pipenburg et al. (U.S. Pat. No. 9,309,502).

[0191] The methods described herein can be used in conjunction with various nucleic acid sequencing techniques. Particularly applicable techniques are those in which the nucleic acids are attached to fixed positions in an array such that their relative positions do not change, and the array is repeatedly imaged. For example, embodiments in which images are obtained in different color channels that correspond to different labels used to distinguish one nucleotide base type from another are particularly applicable. In some embodiments, the process of determining the nucleotide sequence of the modified target nucleic acid can be an automated process. Preferred embodiments include sequencing-by-synthesis ("SBS") techniques.

[0192] SBS technology generally involves the enzymatic extension of nascent nucleic acid strand by repeated addition of nucleotides to template strand.In the conventional method of SBS, a single nucleotide monomer can be provided to target nucleic acid in the presence of polymerase in each delivery.However, in the method described herein, two or more kinds of nucleotide monomers can be provided to target nucleic acid in the presence of polymerase during delivery.

[0193] In one embodiment, the nucleotide monomer comprises a locked nucleic acid (LNA) or a bridged nucleic acid (BNA). The use of an LNA or BNA in the nucleotide monomer increases the hybridization strength between the nucleotide monomer and the sequencing primer sequence present on the immobilized modified target nucleic acid.

[0194] SBS can use nucleotide monomers with or without terminator moieties. Methods using nucleotide monomers without terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides, as described in more detail herein. In methods using nucleotide monomers without terminators, the number of nucleotides added in each cycle is largely variable and depends on the template sequence and the mode of nucleotide delivery. In SBS techniques using nucleotide monomers with terminator moieties, the terminator can be effectively irreversible under the sequencing conditions used, as in the case of conventional Sanger sequencing using dideoxynucleotides, or the terminator can be reversible, as in the case of the sequencing method developed by Solexa (now Illumina, Inc.).

[0195] SBS techniques can use nucleotide monomers with or without a label moiety. Thus, incorporation events can be detected based on the properties of the label, such as the fluorescence of the label, the properties of the nucleotide monomer, such as the molecular weight or charge, the by-products of nucleotide incorporation, such as the release of pyrophosphate, and the like. In embodiments in which two or more different nucleotides are present in the sequencing reagent, the different nucleotides may be distinguishable from each other, or the two or more different labels may be distinguishable under the detection technique used. For example, the different nucleotides present in the sequencing reagent can have different labels, which can be distinguished using appropriate optical systems, as exemplified by the sequencing method developed by Solexa (now Illumina).

[0196] A preferred embodiment includes the pyrosequencing technique, which detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375),363, U.S. Patent Nos. 6,210,891, 6,258,568, and 6,274,320. In pyrosequencing, the released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurase, and the level of ATP generated is detected via luciferase-generated photons. The nucleic acid to be sequenced can be bound to features in an array, and the array can be imaged to capture chemiluminescent signals generated by incorporation of nucleotides into the features of the array. Images can be obtained after treatment of the array with a particular nucleotide type (e.g., A, T, C, or G). Images obtained after addition of each nucleotide type differ with respect to which features in the array are detected. These differences in the images reflect the different sequence content of the features on the array. However, the relative position of each feature remains unchanged in the image. The images can be stored, processed, and analyzed using the methods described herein.For example, images obtained after treating the array with each different nucleotide type can be processed in the same manner as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.

[0197] In another exemplary type of SBS, cycle sequencing is achieved by stepwise addition of reversible terminator nucleotides containing cleavable or photobleachable dye labels, for example as described in WO 04 / 018497 and U.S. Pat. No. 7,057,026. This approach has been commercialized by Solexa (now Illumina) and is also described in WO 91 / 06678 and WO 07 / 123,744. The availability of fluorescently labeled terminators, both of whose ends can be reversed and whose fluorescent labels are cleaved, facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

[0198] In some reversible terminator-based sequencing embodiments, the label does not substantially inhibit extension under SBS reaction conditions. However, the detection label may be removable, for example, by cleavage or degradation. Images can be taken after incorporation of the label into the arrayed nucleic acid features. In certain embodiments, each cycle involves simultaneous delivery of four different nucleotide types to the array, each nucleotide type having a spectrally distinct label. Four images can then be obtained, each using a detection channel selective for one of the four different labels. Alternatively, different nucleotide types can be added sequentially, and images of the array can be obtained during each addition step. In such embodiments, each image shows nucleic acid features that incorporate a particular type of nucleotide. Different features are present or absent in different images because the sequence content of each feature is different. However, the relative positions of the features remain unchanged within the images. Images obtained from such reversible terminator-SBS methods can be stored, processed, and analyzed as described herein. Following the image taking step, the label can be removed, and the reversible terminator moiety can be removed for subsequent cycles of nucleotide addition and detection. Removal of the label after detection in a particular cycle and prior to the subsequent cycle has the advantage of reducing background signal and crosstalk between cycles. Examples of useful labeling and removal methods are described herein.

[0199] In certain embodiments, some or all of the nucleotide monomers may include a reversible terminator. In such embodiments, the reversible terminator / cleavable fluorophore may include a fluorophore attached to the ribose moiety via a 3' ester bond (Metzker, Genome Res. 15:1767-1776 (2005)). Other approaches have separated the terminator chemistry from the fluorescent label (Ruparel et al., Proc Natl Acad Sci USA 102:5932-7 (2005)). Ruparel et al. describe the development of a reversible terminator that uses a small amount of 3' allyl group to block extension, but can be easily unblocked by brief treatment with a palladium catalyst. The fluorophore was attached to the group via a photocleavable linker that can be easily cleaved by 30 seconds of exposure to long wavelength UV light. Thus, either disulfide reduction or photocleavage can be used as the cleavable linker. Another approach to reversible termination is the use of a natural terminus followed by placement of a bulky dye on the dNTP. The presence of a charged bulky dye on the dNTP can act as an effective terminator through steric and / or electrostatic hindrance. The presence of one incorporation event prevents further binding unless the dye is removed. Cleavage of the dye removes the fluorophore, effectively reversing the terminus. Examples of modified nucleotides are also described in U.S. Pat. Nos. 7,427,673 and 7,057,026.

[0200] Additional exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Application Publication Nos. 2007 / 0166705, 2006 / 0188901, 2006 / 0240439, 2006 / 0281109, 2012 / 0270305, and 2013 / 0260372, U.S. Patent No. 7,057,026, and WO 05 / 065814, U.S. Patent Application Publication No. 2005 / 0100900, and WO 06 / 064199 and WO 07 / 010,251.

[0201] Some embodiments may employ detection of four different nucleotides using fewer than four different labels. For example, SBS may be performed using the methods and systems described in incorporated document US Patent Publication No. 2013 / 0079232. As a first example, pairs of nucleotide types may be detected at the same wavelength but may be distinguished based on differences in intensity for one member of the pair, or based on a change to one member of the pair (e.g., via making a chemical modification, photochemical modification, or physical modification) that results in the appearance or disappearance of a distinct signal compared to the signal detected for the other member of the pair. As a second example, three of the four different nucleotide types may be detected under certain conditions, while the fourth nucleotide type may have no detectable label under those conditions or may be minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc.). Incorporation of the first three nucleotide types into a nucleic acid may be determined based on the presence of their corresponding signals, and incorporation of the fourth nucleotide type into a nucleic acid may be determined based on the absence or minimal detection of any signal. As a third example, one nucleotide type can include a label that is detected in two different channels, while the other nucleotide type is detected in no more than one of the channels. The three exemplary configurations above are not considered mutually exclusive and can be used in various combinations.An exemplary embodiment combining all three examples is a fluorescence-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g., dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a second nucleotide type that is detected in a second channel (e.g., dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and second channels (e.g., dTTP having at least one label that is detected in both channels when excited by the first and / or second excitation wavelengths), and a fourth nucleotide type that is not detected in any channel or that is minimally devoid of a label (e.g., unlabeled dGTP).

[0202] Furthermore, as described in US Patent Application Publication No. 2013 / 0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing methods, a first nucleotide type is labeled but the label is removed after the first image is generated, and a second nucleotide type is labeled only after the first image is generated. A third nucleotide type retains its label in both the first and second images, and a fourth nucleotide type remains unlabeled in both images.

[0203] Some embodiments may use sequencing by ligation techniques. Such techniques incorporate oligonucleotides using DNA ligases to identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that correlate with the identity of a particular nucleotide in the sequence to which the oligonucleotide hybridizes. As with other SBS methods, images can be obtained after treating an array of nucleic acid features with labeled sequencing reagents. Each image shows nucleic acid features that incorporate a particular type of label. Because the sequence content of each feature is different, different features are present or absent in different images, but the relative positions of the features remain unchanged within the images. Images obtained from ligation-based sequencing methods can be stored, processed, and analyzed as described herein. Exemplary SBS systems and methods that can be utilized with the methods and systems described herein are described in U.S. Patent Nos. 6,969,488, 6,172,218, and 6,306,597.

[0204] Some embodiments can use nanopore sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis." Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and JA Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope." Nat. Mater. 2:611-615 (2003)). In such embodiments, the modified target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. As the modified target nucleic acid passes through the nanopore, each base pair can be identified by measuring the variation in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni, GV & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am Chem. Soc. 130, 818-820 (2008). Data obtained from nanopore sequencing can be stored, processed, and analyzed as described herein.In particular, the data can be processed as an image according to the exemplary processing of optical and other images described herein.

[0205] Some embodiments can use methods involving real-time monitoring of DNA polymerase activity. Nucleotide incorporation can be detected via fluorescence resonance energy transfer (FRET) interactions between fluorophore-containing polymerases and γ-phosphate-labeled nucleotides, for example, as described in U.S. Pat. Nos. 7,329,492 and 7,211,414, or nucleotide incorporation can be detected using zero-mode waveguides, for example, as described in U.S. Pat. No. 7,315,019, and fluorescent nucleotide analogs and engineered polymerases, for example, as described in U.S. Pat. No. 7,405,281 and U.S. Patent Application Publication No. 2008 / 0108082. Illumination can be restricted to a zeptoliter-scale volume around the surface-tethered polymerase so that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, MJ et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science, 299, 682-686 (2003); Lundquist, PM et al. "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008)). Images obtained from such methods can be stored, processed, and analyzed as described herein.

[0206] Some SBS embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use commercially available electric detectors and related technology from Ion Torrent (Guilford, CT, a Life Technologies subsidiary), or the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082, 2009 / 0127589, 2010 / 0137143, and 2010 / 0282617. The methods described herein for amplifying target nucleic acids using kinetic exclusion can be easily adapted to substrates used to detect protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons used to detect protons.

[0207] The SBS method described above can be advantageously carried out in a multiplex format so that multiple different modified target nucleic acids are manipulated simultaneously. In certain embodiments, the different modified target nucleic acids can be processed in a common reaction vessel or on the surface of a particular substrate. This allows for convenient delivery of sequencing reagents, removal of unreacted reagents, and detection of incorporation events in a multiplexed manner. In embodiments using surface-bound target nucleic acids, the modified target nucleic acids can be in an array format. In an array format, the modified target nucleic acids can typically be bound to a surface in a spatially distinguishable manner. The modified target nucleic acids can be bound by direct covalent binding, binding to beads or other particles, or binding to a polymerase or other molecule bound to the surface. The array can include a single copy of the modified target nucleic acid at each site (also referred to as a feature), or multiple copies with the same sequence can be present at each site or feature. The multiple copies can be generated by amplification methods such as bridge amplification or emulsion PCR, which are described in more detail herein.

[0208] The methods described herein can be used to fabricate, for example, at least about 10 features / cm 2 , 100 features / cm 2, 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 , 5,000,000 features / cm 2 Arrays having features of any of a variety of densities, including 100 s, 100 s, 200 s, 300 s, 400 s, 500 s, 600 s, 700 s, 800 s, 900 s, 10 ...2000 s, 3000 s, 4000 s,

[0209] The advantage of the method described herein is that multiple cm 2 The object of the present invention is to provide rapid, efficient, and parallel detection of nucleic acids. Thus, the present disclosure provides an integrated system that can prepare and detect nucleic acids using techniques known in the art, such as those exemplified herein. Thus, the integrated system of the present disclosure can include fluidic components that can deliver amplification and / or sequencing reagents to one or more immobilized modified target nucleic acids, the system including components such as pumps, valves, reservoirs, fluid lines, etc. A flow cell can be configured and / or used in the integrated system for detecting target nucleic acids. Exemplary flow cells are described, for example, in U.S. Pat. Nos. 8,241,573 and 8,951,781. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used for amplification and detection methods. Taking the embodiment of nucleic acid sequencing as an example, one or more of the fluidic components of the integrated system can be used for delivery of sequencing reagents in the amplification methods described herein and in the sequencing methods as exemplified above. Alternatively, the integrated system can include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and determining the sequence of nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, Calif.) and the apparatus described in U.S. Pat. No. 8,951,781.

[0210] Although the embodiments presented herein are generally described using a sequencing platform (such as a sequencing by synthesis platform) as readout, those skilled in the art will recognize that nucleic acids modified by modified cytidine deaminases presented herein can also be detected using any other suitable readout methodology. For example, the location and identity of modified cytosines can be assessed using a microarray. Any of a variety of analyte arrays (also referred to as "microarrays") known in the art can be used in the methods or systems described herein. A typical array includes specimens, each of which has an individual probe or a population of probes. In the latter case, the population of probes in each specimen is typically homogenous, with a single type of probe. For example, in the case of nucleic acid sequences, each specimen can have multiple nucleic acid molecules, each with a common sequence. However, in some embodiments, the population in each specimen of the array can be heterogeneous. Similarly, protein sequences can have specimens with a single protein or a population of proteins, typically, but not necessarily, with the same amino acid sequence. The probes can be attached to the surface of the array, for example, by covalently binding the probes to the surface or through non-covalent interactions between the probes and the surface. In some embodiments, the probes, such as nucleic acid molecules, can be attached to the surface through a gel layer, see, for example, U.S. Patent Application Serial No. 13 / 784,368 and U.S. Patent Application Publication No. 2011 / 0059865 A1.

[0211] Exemplary arrays include, but are not limited to, BeadChip arrays available from Illumina, Inc. (San Diego, Calif.) or others, such as those in which probes are attached to beads present on a surface (e.g., beads in wells on a surface), as described in U.S. Pat. Nos. 6,266,459, 6,355,431; 6,770,441; 6,859,570; or 7,622,294, or WO 00 / 63437. Further examples of commercially available microarrays that can be used include, for example, Affymetrix® GeneChip® microarrays or other microarrays synthesized according to a technology sometimes referred to as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis) technology. Spotted microarrays can also be used in the methods or systems according to some embodiments of the present disclosure. An exemplary spotted microarray is the CodeLink™ Array available from Amersham Biosciences. Another useful microarray is one manufactured using inkjet printing techniques, such as SurePrint™ Technology available from Agilent Technologies.

[0212] In certain embodiments, the modified cytidine deaminase provided herein can be used to provide a sample of DNA suspected of containing single-stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5CaC), or a combination thereof; contacting the DNA with the modified cytidine deaminase under conditions suitable for conversion of 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than conversion of cytosine (C) to uracil (U) by deamination to obtain converted single-stranded DNA, wherein 5-methylcytosine (5mC) can be converted to thymidine (T) by deamination as described herein, such as by conversion of 5mC, 5hmC, 5fC, and / or 5CaC to T.

[0213] In certain embodiments, the modified cytidine deaminase of the present disclosure can be used to detect 5-hydroxymethylcytosine (5hmC) as described herein, for example, by providing a sample of DNA suspected of containing single stranded DNA having at least one 5-hydroxymethylcytosine (5hmC), and contacting the DNA with the modified cytidine deaminase under conditions suitable for conversion of unmodified cytosine to uracil and conversion of 5mC to thymidine and no detectable conversion of 5hmC to 5hmU.

[0214] The converted single stranded DNA can then be treated as necessary to facilitate hybridization to the microarray. For example, the converted DNA can be amplified. Any one of a number of amplification methods known in the art can be performed. For example, whole genome amplification or amplification using universal primers that hybridize to common regions in the converted DNA, such as adapter sequences, can be used. Additionally or alternatively, the converted DNA can be fragmented. Fragmentation can be performed before or after amplification, or in the absence of amplification. Any one of a number of fragmentation methods known in the art can be performed. As an example, fragmentation can be performed using an enzymatic process, such as a restriction endonuclease or other enzyme that can cleave the converted DNA. As another example, fragmentation can be performed using mechanical means, such as shearing, using an ultrasonicator, such as one supplied by Covaris. The fragmented converted DNA can then be precipitated and / or resuspended in a buffer suitable for hybridization to the microarray. After hybridization, the methylation status of a region of interest, such as a specific CpG locus or loci, can be examined at a specific location on the microarray. Methods for preparing converted DNA for microarray analysis are known in the art. One example of such a method is described in the methylation protocol guide for the Infinium HD assay from Illumina (San Diego, CA). Although such protocol guides may describe the use of microarrays designed for the investigation of bisulfite converted DNA, it will be understood that the array features, specifically the probe sequences, can be specifically designed for DNA that is not bisulfite converted. As an example, commercially available microarrays such as the Infinium Methylation EPIC BeadChip (Illumina) are specifically designed to hybridize with reduced-complexity DNA fragments in which most, if not all, cytosines are converted to thymidines, as found in bisulfite converted DNA.Thus, for example, the same CpG sites can be examined in non-bisulfite converted DNA by using a microarray containing probes designed to hybridize to the same regions of natural non-bisulfite converted DNA. Such microarrays may be easily obtained by those skilled in the art. In one embodiment, a custom array can be designed using an array manifest such as the Infinium Methylation EPIC BeadChip by using a "forward sequence" to identify probe sequences that contain native DNA sequences covering similar or identical sequence regions of the allele-specific probe sequences designed to hybridize to DNA sequences in which most or all cytosines have been converted to thymidines. Using such an array designed to hybridize to natural (non-bisulfite converted) DNA sequences, methylated CpG sites in sample DNA could be identified by following the methodology and analysis methods described in Illumina's Methylation Protocol Guide for the Infinium HD Assay (San Diego, CA).

[0215] composition The present disclosure also provides compositions comprising modified cytidine deaminase as described herein. The compositions can include one or more additional other components in addition to modified cytidine deaminase. For example, the other components can include a single-stranded DNA or RNA substrate that contains or is suspected to contain at least one modified cytosine, such as 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine (5fC), 5-carboxycytosine (5CaC), or a combination thereof. In another example, the single-stranded DNA or RNA substrate can be one that contains one or more known modified cytosines, such as a single-stranded DNA or RNA substrate that can be used as a control to measure conversion efficiency. In another example, the other components can include a buffer having a pH as described herein. In another example, the other components can include a buffer as described herein, such as a citrate buffer, a sodium acetate buffer, or a Bis-Tris buffer. In another example, the other components can include a reducing agent, including, but not limited to, DTT and / or TCEP, and Zn.

[0216] The composition can also include a polynucleotide encoding a modified cytidine deaminase as described herein. The polynucleotide can be present in a vector, such as a plasmid or a viral vector. The vector containing the polynucleotide can be present in a host cell, such as E. coli.

[0217] kit The present disclosure also provides a kit for determining the methylation status of DNA or RNA. The kit includes at least one modified cytidine deaminase as described herein and one or more other components in a suitable packaging material in an amount sufficient for at least one reaction. Examples of other components include a positive control polynucleotide, such as a single-stranded DNA containing one or more known modified cytosines for use in measuring conversion efficiency, or a negative control polynucleotide, such as a single-stranded DNA containing unmodified cytosines. Another component can be a glucosyltransferase, such as T4-β glucosyltransferase. Optionally, other reagents, such as buffers and solutions required for using the modified cytidine deaminase and nucleotide solution, are also included. Typically, instructions for use of the packaged components are also included.

[0218] As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is preferably constructed by known methods to provide a sterile, contaminant-free environment. The packaging material has a label indicating that the components can be used to determine the methylation state of DNA or RNA. Additionally, the packaging material includes instructions indicating how to use the materials in the kit to perform a reaction with modified cytidine deaminase. As used herein, the term "package" refers to a solid matrix or material, such as glass, plastic, paper, foil, etc., capable of holding a polypeptide within certain limits. "Instructions for use" typically include specific language describing at least one assay method parameter, such as reagent concentrations, or the relative amounts of reagents and sample to mix, the duration of the reagent / sample mixture, temperature, buffer conditions, etc.

[0219] The present invention is defined in the claims. However, the following provides a non-exhaustive list of non-limiting exemplary aspects. Any one or more of the features of these aspects can be combined with any one or more features of other examples, embodiments, or aspects described herein.

[0220] Exemplary Aspects Embodiment 1 is an engineered cytidine deaminase that includes amino acid substitution mutations of the cytidine deaminase at positions functionally equivalent to (Tyr / Phe)130 and Tyr132 in the wild-type APOBEC3A protein.

[0221] Aspect 2 is a modified cytidine deaminase comprising an amino acid substitution mutation of cytidine deaminase at a position functionally equivalent to (Tyr / Phe)130 of a wild-type APOBEC3A protein, wherein the substitution mutation is (Tyr / Phe)130Trp.

[0222] Aspect 3 is the modified cytidine deaminase according to aspect 1 or 2, wherein (Tyr / Phe)130 is Tyr130 and the wild-type APOBEC3A protein is SEQ ID NO:3.

[0223] Aspect 4 is a modified cytidine deaminase according to any one of Aspects 1 to 3, wherein the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to Ala, Val, or Trp.

[0224] Aspect 5 is a modified cytidine deaminase according to any one of Aspects 1 to 4, wherein the substitution mutation at a position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln, or Lys.

[0225] Aspect 6 is a modified cytidine deaminase according to any one of Aspects 1 to 5, wherein the modified cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is faster than the conversion of cytosine (C) to uracil (U) by deamination.

[0226] Aspect 7 is a modified cytidine deaminase according to any one of aspects 1 to 6, wherein the rate is at least 100-fold greater.

[0227] Aspect 8 is a modified cytidine deaminase according to any one of Aspects 1 to 7, wherein the modified cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is faster than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

[0228] Aspect 9 is a modified cytidine deaminase according to any one of aspects 1 to 8, which has no detectable conversion of 5hmC to 5hmU by deamination.

[0229] Aspect 10 is the modified cytidine deaminase of any one of Aspects 1 to 9, wherein the modified cytidine deaminase is a member of the AID subfamily, the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3B subfamily, the APOBEC3C subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, the APOBEC3G subfamily, the APOBEC3H subfamily, or the APOBEC4 subfamily.

[0230] Embodiment 11 is a method for determining whether the modified cytidine deaminase has a ZDD motif H-[P / A / V]-EX [23-28] -PCX [2-4] -C (SEQ ID NO: 12).

[0231] Embodiment 12 is a method for determining whether the modified cytidine deaminase is a member of the APOBEC3A subfamily and has the ZDD motif HXEX 24 SW(S / T)PCX [2-4] CX6FX8LX5R(L / I)YX [8-11] LX2LX

[10] M (SEQ ID NO: 13), and the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is the Tyr (Y) amino acid of the ZDD motif.

[0232] Embodiment 13 is a method for treating a cytidine deaminase comprising the steps of:

[0233] [Table 6]

[0234] 13. The modified cytidine deaminase according to any one of aspects 1 to 12, comprising:

[0235] Aspect 14 is the modified cytidine deaminase according to any one of Aspects 1 to 13, wherein the modified cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 59.

[0236] Aspect 15 is a modified cytidine deaminase according to any one of Aspects 1 to 14, wherein the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine, or threonine.

[0237] A sixteenth aspect is a polynucleotide encoding the modified cytidine deaminase according to any one of the first to fifteenth aspects.

[0238] Aspect 17 is a composition comprising the modified cytidine deaminase according to any one of aspects 1 to 16 and a buffer solution.

[0239] Example 18 is the composition of any one of Examples 1 to 17, wherein the composition can further comprise at least one of: (i) a sample comprising DNA including at least one modified cytosine, the modified cytosine being 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), or a combination thereof; or (ii) a buffer having a pH of less than 7; or (iii) a combination thereof.

[0240] Example 19 is the composition of any one of Examples 1 to 18, wherein the DNA comprises single-stranded DNA.

[0241] Example 20 is a composition according to any one of Examples 1 to 19, wherein the modified cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr132 in a wild-type APOBEC3A protein.

[0242] Example 21 is the composition according to any one of Examples 1 to 20, wherein the sample comprises genomic DNA or cell-free DNA.

[0243] Example 22 is the composition according to any one of Examples 1 to 21, wherein the genomic DNA is from a single cell or is a mixture from multiple cells.

[0244] Example 23 is a composition according to any one of Examples 1 to 22, wherein the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine, or threonine, and wherein the modified cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a faster rate than it converts cytosine (C) to uracil (U) by deamination.

[0245] Example 24 is the composition of any one of Examples 1-23, wherein the rate is at least 100 times greater.

[0246] Example 25 is a composition according to any one of Examples 1 to 24, wherein the substitution mutation at a position functionally equivalent to (Tyr / Phe)130 comprises a mutation to Trp, and at least one 5-hydroxymethylcytosine (5hmC) is present in the DNA, and the modified cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is greater than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

[0247] Example 26 is a composition according to any one of Examples 1 to 25, wherein there is no detectable conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

[0248] Aspect 27 includes providing a sample of DNA suspected of containing single-stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5CaC), or a combination thereof; and (i) converting 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is faster than the conversion of cytosine (C) to uracil (U) by deamination, thereby obtaining converted single-stranded DNA. or (ii) contacting the single-stranded DNA with a modified cytidine deaminase under conditions suitable for deamination conversion of C to U and deamination conversion of 5mC to T at a rate greater than deamination conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU), where the modified cytidine deaminase is a modified cytidine deaminase according to any one of aspects 1 to 26, and processing the converted single-stranded DNA to produce a sequencing library.

[0249] Example 28 is the method according to any one of the preceding examples, wherein the method further comprises, prior to providing, denaturing double-stranded DNA present in the sample to provide single-stranded DNA.

[0250]

[0033] Aspect 29 includes providing a sample of DNA suspected of containing double-stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5caC), or combinations thereof, treating the double-stranded DNA to produce a sequencing library; denaturing the sequencing library to produce single-stranded DNA; (i) converting 5-methylcytosine (5mC) to cytosine (C) by deamination at a rate that is faster than the conversion of cytosine (C) to uracil (U) by deamination. (ii) conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination at a rate greater than conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination to obtain converted single-stranded DNA, wherein the modified cytidine deaminase is the modified cytidine deaminase of embodiment 1 or 2, and converting the converted single-stranded DNA into a converted double-stranded DNA sequencing library.

[0251] Example 30 is the method according to any one of Examples 1 to 29, wherein the processing comprises fragmentation or tagmentation of the double-stranded DNA, and addition of universal sequences to the double-stranded DNA fragments.

[0252] Example 31 is the method according to any one of examples 1 to 30, wherein the universal sequence is part of an adaptor added to the double stranded DNA fragment. Example 32 is the method according to any one of examples 1 to 31, wherein converting comprises amplifying the converted single stranded DNA into converted double stranded DNA.

[0253] Example 33 is the method according to any one of Examples 1 to 32, wherein the sample is a biological sample.

[0254] Example 34 is the method according to any one of Examples 1 to 33, wherein the biological sample comprises cell-free DNA.

[0255] Example 35 is the method of any one of Examples 1 to 34, wherein the biological sample comprises a fluid selected from blood or serum.

[0256] Example 36 is the method of any one of Examples 1 to 35, wherein the sample comprises a single cell or an isolated nucleus.

[0257] Example 37 is the method of any one of Examples 1 to 36, wherein the biological sample comprises a tissue.

[0258] Example 38 is the method of any one of examples 1 to 37, wherein the tissue comprises tumor tissue. Example 39 is the method of any one of examples 1 to 38, further comprising providing a surface comprising a plurality of amplification sites, the amplification sites comprising at least two populations of bound single-stranded capture oligonucleotides having free 3' ends, and contacting the surface comprising the amplification sites with a sequencing library under conditions suitable to produce a plurality of amplification sites, each comprising a clonal population of amplicons derived from an individual member of the sequencing library.

[0259] Example 40 is the method of any one of Examples 1 to 39, further comprising providing a surface comprising a plurality of amplification sites, the amplification sites comprising at least two populations of bound single-stranded capture oligonucleotides having free 3' ends, and contacting the surface comprising amplification sites with the converted double-stranded DNA sequencing library under conditions suitable to produce a plurality of amplification sites, each comprising a clonal population of amplicons derived from an individual member of the converted double-stranded DNA sequencing library.

[0260] Example 41 is a method for detecting the location of a modified cytosine in a target nucleic acid, the method comprising: (a) contacting a target nucleic acid suspected of containing at least one modified cytosine with a modified cytidine deaminase described in any one of Examples 1 to 40 to produce a converted nucleic acid containing at least one converted cytosine; and (b) detecting the at least one converted cytosine in the converted nucleic acid of (a).

[0261] Example 42 is the method of any one of Examples 1 to 41, wherein the modified cytidine deaminase has cytosine-deficient deaminase activity, and detecting comprises identifying thymidine nucleotides in the converted nucleic acid to determine the position of 5mC nucleotides in the target nucleic acid.

[0262] Example 43 is a method according to any one of Examples 1 to 42, wherein the modified cytidine deaminase has 5hmC deficient deaminase activity and detecting comprises identifying cytosine nucleotides in the converted nucleic acid to determine the position of the 5hmC nucleotide in the target nucleic acid.

[0263] Example 44 is the method of any one of Examples 1 to 43, wherein detecting comprises sequencing the converted nucleic acid or hybridizing a nucleic acid probe to the converted nucleic acid.

[0264] Example 45 is the method of any of the preceding examples, wherein the detecting comprises sequencing the converted nucleic acid, and the method further comprises (c) comparing the sequence of the converted nucleic acid to an untreated reference sequence to determine which cytosines in the target nucleic acid are modified.

[0265] Example 46 is the method according to any one of Examples 1 to 45, wherein a predetermined sequence of the untreated reference sequence and a predetermined sequence of the converted nucleic acid are compared.

[0266] Example 47 is the method according to any one of Examples 1 to 46, wherein the predetermined sequence comprises a CpG island or a promoter.

[0267] Example 48 is the method of any one of examples 1 to 47, wherein the detecting comprises hybridizing the converted nucleic acid to a nucleic acid probe, the nucleic acid probe being present on an analyte array, and the method further comprises sequencing the hybridized converted nucleic acid.

[0268] Example 49 is the method of any one of examples 1 to 48, wherein the detecting comprises hybridizing the converted nucleic acid to a nucleic acid probe, and the method further comprises amplifying the converted nucleic acid, wherein the nucleic acid probe comprises two primers for amplification of a predetermined sequence, and wherein the primers anneal with higher affinity to regions of the converted nucleic acid that contain at least one converted cytosine than to regions of the converted nucleic acid where at least one cytosine is not a converted cytosine, and the presence of an amplification product is indicative of a modified cytosine in the target nucleic acid.

[0269] Example 50 is the method of any one of examples 1 to 49, wherein the detecting comprises hybridizing the converted nucleic acid to a nucleic acid probe, and the method further comprises cleaving a single-stranded DNA (ssDNA) reporter substrate by a CRISPR-based system, the ssDNA reporter substrate comprising a fluorophore and a quencher, and the presence of fluorescence indicates a modified cytosine in the target nucleic acid.

[0270] Example 51 is the method of any one of the preceding examples, wherein the CRISPR-based system comprises a guide RNA sequence that anneals to a predetermined sequence of a nucleic acid that includes at least one converted cytosine and anneals with a lower affinity to the predetermined sequence of a nucleic acid if the at least one cytosine is unconverted.

[0271] Example 52 is a method according to any one of Examples 1 to 51, wherein the CRISPR-based system comprises CRISPR-Cas12.

[0272] Example 53 is the method of any one of examples 1 to 52, wherein detecting comprises hybridizing the converted nucleic acid to a nucleic acid probe, wherein the converted nucleic acid is present in fixed cells, and wherein the nucleic acid probe comprises a fluorescently labeled probe, and wherein the nucleic acid probe anneals with higher affinity to a predetermined sequence of the converted nucleic acid containing at least one converted cytosine than to a region of the converted nucleic acid where at least one cytosine is not a converted cytosine, and wherein the presence of cell-associated fluorescence is indicative of a modified cytosine in the target nucleic acid.

[0273] Example 54 is the method according to any one of Examples 1 to 53, wherein the untreated reference sequence is a predetermined sequence.

[0274] Example 55 is the method according to any one of Examples 1 to 54, wherein at least one modified cytosine is 5mC or 5hmC.

[0275] Example 56 is the method of any one of Examples 1 to 55, further comprising providing a target nucleic acid, wherein providing comprises preparing single-stranded (ss) DNA from a sample comprising the target nucleic acid.

[0276] Example 57 is the method according to any one of Examples 1 to 56, wherein the target nucleic acid is genomic DNA or cell-free DNA.

[0277] Example 58 is the method according to any one of Examples 1 to 57, wherein the genomic DNA is from a single cell or is a mixture from multiple cells.

[0278] Example 59 is the method of any one of Examples 1 to 58, wherein the sequencing comprises processing the converted nucleic acid to generate a sequencing library.

[0279] Example 60 is the method of any one of examples 1 to 59, further comprising providing a surface comprising a plurality of amplification sites, the amplification sites comprising at least two populations of bound single-stranded capture oligonucleotides having free 3' ends, and contacting the surface comprising the amplification sites with a sequencing library under conditions suitable to produce a plurality of amplification sites, each comprising a clonal population of amplicons derived from an individual member of the sequencing library.

[0280] Example 61 is a method according to any one of examples 1 to 60, wherein the target nucleic acid is obtained from the subject, and the detecting comprises obtaining a pattern of cytosine modifications in the converted nucleic acid, and the method further comprises comparing the pattern of cytosine modifications in the converted nucleic acid with a pattern of cytosine modifications in a reference nucleic acid.

[0281] Example 62 is the method according to any one of Examples 1 to 61, wherein the subject has or is at risk of having a disease or condition and the reference nucleic acid is from a normal subject.

[0282] Example 63 is a method according to any one of the preceding examples, wherein the pattern of cytosine modifications is linked in cis to a coding region that correlates with the disease or condition.

[0283] Example 64 is the method of any one of the preceding examples, wherein the pattern of cytosine modifications is linked in cis to the coding region, and wherein the coding region in the reference nucleic acid is transcriptionally active or transcriptionally inactive, and wherein comparing further comprises determining whether the pattern of cytosine modifications in the converted nucleic acid indicates that the coding region is transcriptionally active or transcriptionally inactive in the subject.

[0284] Example 65 is a method according to any one of examples 1 to 64, wherein transcription of the coding region correlates with a disease or condition.

[0285] Example 66 is the method of any one of the preceding examples, wherein the subject has a disease or condition and is undergoing treatment for the disease or condition, and the method further comprises determining whether the treatment correlates with a change in the pattern of cytosine modification in the subject.

[0286] Example 67 is a method according to any one of the preceding examples, wherein the subject previously had the disease or condition, and the method further comprises comparing the pattern of cytosine modification in the subject with the pattern of cytosine modification in the subject when the subject had the disease or condition. EXAMPLES

[0287] The present disclosure is illustrated by the following examples, it being understood that the specific examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the present disclosure described herein.

[0288] Example 1 Experimental assay for cytidine deaminase activity Deamination by cytidine deaminase was monitored using a gel-based assay with the restriction enzyme SwaI (Figure 4).

[0289] Deamination of C to U or 5mC to T results in a mismatched DNA substrate that can be cleaved by SwaI. These products are visualized as two distinct species on denaturing polyacrylamide gels.

[0290] Incubation of APOBEC3A wild type or its mutants with 5' fluorescein amidite (FAM) labeled oligonucleotide substrates containing C or 5mC results in deamination and no deamination depending on the enzyme substrate preference. Subsequent introduction of a complementary oligonucleotide may result in perfect match or mismatch base pairing. If a mismatch is present, SwaI cleaves the double-stranded oligonucleotide. The original or cleaved 5'-FAM labeled oligonucleotides can be visualized by the extent of migration using 15% urea-PAGE with a FAM filter. Although FAM is used here, essentially any label can be used, and either the 5' or 3' end can be labeled.

[0291] This assay was adapted and modified from Schutsky et al., Nucleic Acid Research, 45, 7655-7665, 2017. doi:10.1093 / nar / gkx345. Modifications to Schutsky et al. included the following: Instead of performing DNA precipitation and redissolving the DNA substrate in a SwaI-compatible buffer, 1 μL of modified cytidine deaminase APOBEC3A(Y130A) deamination reaction mixture was aliquoted into 9 μL SwaI-compatible buffer for restriction enzyme digestion for gel assay. Appropriate controls were performed to determine that SwaI restriction enzyme digestion efficiency was not compromised by the APOBEC reaction buffer. Instead of introducing a 1.5-fold excess of complementary strand prior to overnight SwaI restriction enzyme digestion, a 3-fold excess of complementary strand was introduced. Instead of running a preheated 20% acrylamide / Tris-borate-EDTA (TBE) / urea gel as reported by Schutsky et al., we performed gel runs at room temperature using a 15% acrylamide / Tris-borate-EDTA (TBE) / urea gel and observed good separation between cleaved (deaminated) and uncleaved (unreacted) oligo substrates.

[0292] The SwaI assay was first validated using FAM-labeled DNA oligonucleotides containing C, 5mC, U, or T residues (Figure 5). Oligonucleotides were purchased from Integrated DNA Technologies (IDT) and visualized by 15% urea-PAGE using FAM filters. Synthesized oligonucleotide oLB1609 contains substrate C and oLB1610 contains its corresponding deaminated product U. oLB1611 contains substrate 5mC and oLB1612 contains its corresponding deaminated product T. oLB1679 is a complementary oligonucleotide to oLB1609, oLB1610, oLB1611, and oLB1612 (Figure 5A). As shown in Figure 5B, annealing of oLB1609 / oLB1679 and oLB1611 / oLB1679 resulted in perfect base pairing of the oligonucleotides. Addition of SwaI did not result in cleavage of this substrate. However, single base mismatch substrates formed by annealing oLB1610 / oLB1679 (U / G mismatch) or oLB1612 / oLB1679 (T / G mismatch) yielded cleavage products upon addition of SwaI. Because specific cleavage products were observed in the presence of U or T, but not C or 5mC, the SwaI assay can serve as a readout for C to U and 5mC to T deamination by APOBEC3A.

[0293] As a further control, the C- and 5mC-containing DNA oligonucleotides oLB1609 (C oligo) and oLB1612 (5mC oligo) were incubated with APOBEC3A enzyme purchased from New England Biolabs (NEBNext® Enzymatic Methyl-seq Kit (catalog no. E7120)), which was reported to efficiently deaminate both C and 5mC (Figure 6). The deamination reaction mixtures (5 μL, 2 μL, and 1 μL) were then added directly to SwaI assay buffer containing SwaI, resulting in a total volume of 10 μL. 1 μL of the deamination reaction was sufficient to observe a cut band, allowing quantification of the extent of deamination. Treatment with NEB APOBEC3A and subsequent digestion with SwaI resulted in the formation of cleavage products with similar mobility as both the U- and T-containing substrates in Figure 5A. These data further support the idea that SwaI digestion can be used to monitor deaminase activity of APOBEC3A and other cytidine deaminases.

[0294] Example 2 Purification of APOBEC3A(Y130X) mutant protein We systematically evaluated the impact of all possible amino acid substitutions at position 130 of APOBEC3A on the deaminase activity of this enzyme. To this end, 19 different His-tagged APOBEC3A constructs were cloned, each encoding a different amino acid at position 130 relative to the wild-type tyrosine. The corresponding proteins were expressed in BL21(DE3) cells, purified using Ni-NTA agarose beads, and desalted / concentrated using spin columns into storage buffer (50 mM Tris pH 7.5, 200 mM NaCl, 5% (v / v) glycerol, 0.01% (v / v) Tween-20, 0.5 mM DTT). This resulted in APOBEC3A(Y130X) mutant protein preparations with a purity of 80–85% as judged by SDS-PAGE analysis (Figure 7A).

[0295] Example 3 DNA deaminase activity of APOBEC3A(Y130X) mutant protein The deaminase activity of all purified APOBEC3A (Y130X) proteins was then analyzed using a SwaI assay with a 37°C / 2h reaction time and NEB APOBEC3A as a positive control (Figure 8). Y130X recombinant enzymes at final concentrations of 10-20 μM were incubated with oLB1609 (C oligo, top panel) and oLB1612 (5mC oligo, bottom panel) for 2h at 37°C. NEBAPOBEC3A enzymes were purchased from the NEBNext® Enzymatic Methyl-seq Kit (catalog no. E7120). Wild-type APOBEC3A completely deaminated 5mC and C substrates, in agreement with previous literature. Different mutants showed a wide range of reactivity towards 5mC and C substrates, with some showing preference for either substrate. Notably, APOBEC3A(Y130A) (box 1) almost completely deaminated the 5mC substrate (94.2%) but only slightly (29.4%) the corresponding C substrate. Other mutants such as APOBEC3A(Y130P) and APOBEC3A(Y130T) also showed more complete deamination of 5mC than C substrate, although to a lesser extent than APOBEC3A(Y130A). In contrast, APOBEC3A(Y130L) (box 2) deaminated about half (56%) of the C substrate but almost no 5mC substrate (6.8%). The deaminase activities of all APOBEC3A(Y130X) mutants were quantified and are summarized in Figures 8, 9, and 10.

[0296] Because these SwaI assays were performed as single end-point measurements (2 h), it is possible that the respective deamination reactions may have already saturated. Therefore, a time course analysis of APOBEC3A(Y130A) deaminase activity was performed. By incubating approximately 10-20 μM APOBEC(Y130A) with 500 nM C and 5mC oligonucleotide substrates, the extent of C and 5mC deamination was monitored at 0, 5, 10, 30, 60, and 120 min (Figure 11). At t ≤ 30 min, a larger difference in the extent of 5mC vs. C deamination was observed.

[0297] The kinetics of deamination by wild-type APOBEC3A and mutant APOBEC3A(Y130A) were quantitatively compared. Initial deamination reaction rates were measured over a range of DNA substrate concentrations and used to construct Michaelis-Menten curves for 5mC and C substrates, respectively. The resulting Km and Kcat values ​​were then derived from these data. The catalytic efficiency of APOBEC3A(Y130A) was approximately 100-fold higher on 5mC than on C substrates (Figure 12), confirming the endpoint SwaI assays shown in Figures 8, 9, and 10.

[0298] Example 4 Purification of APOBEC3A(Y130A-Y132H) double mutant protein APOBEC3A(Y130A-Y132H) protein was expressed in BL21(DE3) cells, purified using Ni-NTA agarose beads, and desalted / concentrated using spin columns into storage buffer (50 mM Tris pH 7.5, 200 mM NaCl, 5% (v / v) glycerol, 0.01% (v / v) Tween-20, 0.5 mM DTT). This resulted in an APOBEC3A(Y130A-Y132H) mutant protein preparation with a purity of 90-95% as judged by SDS-PAGE analysis (Figure 7B).

[0299] Example 5 DNA deaminase activity of APOBEC3A(Y130A-Y132H) double mutant protein The deaminase activity of the purified APOBEC3A(Y130A-Y132H) double mutant protein was then analyzed using a SwaI assay with a 37°C / 2 hour reaction time and NEB APOBEC3A as a positive control. The conditions used were the same as those described in Example 3, except that the SwaI assay used 40 mM sodium acetate pH 5.2, 37°C, 1 hour to 16 hours reaction conditions. The DNA substrates are shown in Figure 12. After the deaminase reaction, the deaminated oligo substrates were PCR amplified and sequenced, and the number of C and 5mC deamination events per read was counted. The DNA oligonucleotide substrates used for the experiments with APOBEC3A(Y130A-Y132H) are shown in Figure 13. APOBEC3A(Y130A-Y132H) showed higher levels of deamination at all methylated sites compared to unmethylated sites. This was consistent across both CpG and non-CpG contexts and robust to variations in reaction time (Figures 14, 15). The difference in deamination levels between methylated and unmethylated sites was significantly higher for APOBEC3A(Y130A-Y132H) than for APOBEC3A(Y130A), indicating that APOBEC3A(Y130A-Y132H) achieved better discrimination of methylated sites than APOBEC3A(Y130A). Furthermore, APOBEC3A(Y130A-Y132H) deaminates methylated sites more efficiently than unmethylated sites across all xCpGx motifs (Figure 16).

[0300] Example 6 Purification of APOBEC3A(Y130W) mutant protein Recombinant human His-tagged APOBEC3A(Y130W) protein was expressed in E. coli BL21(DE3) cells, purified using Ni-NTA affinity chromatography, and desalted / concentrated using spin columns into storage buffer (50 mM Tris pH 7.5, 200 mM NaCl, 5% (v / v) glycerol, 0.01% (v / v) Tween-20, 0.5 mM DTT).

[0301] Example 7 DNA deaminase activity of APOBEC3A(Y130W) mutant protein Using the SwaI assay, we measured the activity of APOBEC3A(Y130W) on C, 5mC, and 5hmC substrates over a 90 min time interval. Most of C and 5mC were deaminated, but no detectable activity was observed for 5hmC (Figure 17).

[0302] To better understand the substrate preferences of APOBEC3A(Y130W), the deaminase activity against C, 5mC, and all oxidized derivatives of 5mC was assayed (Figure 18). A SwaI restriction enzyme assay was performed with a reaction time of 90 min to measure the deaminase activity of APOBEC3A wild-type and Y130W mutant enzymes. A control without protein was included to account for potential degradation of the oligonucleotide substrate and to account for non-specific activity of SwaI during the course of the assay. APOBECY130A(Y130W) showed no detectable deamination of 5hmC, 5fC, and 5caC, whereas wild-type APOBEC retained significant deaminase activity against 5hmC and showed residual activity against 5fC and 5caC. Both enzymes efficiently deaminate C and 5mC.

[0303] Example 8 Detection of 5mC The following examples are generally known in the art, as exemplified by the method disclosed in Schutsky et al., Nature biotechnology, 10.1038 / nbt.42048 Oct. 2018, doi:10.1038 / nbt.4204. Provide genomic DNA (gDNA) from an organism suspected of having 5hmC. Then, process 20ng of the gDNA mixture as follows:

[0304] To provide single-stranded DNA and facilitate deamination activity, 1 µL of DMSO is added, samples are denatured at 95 °C for 5 min, and quenched by transferring to a PCR tube rack pre-incubated at -80 °C. Prior to thawing, reaction buffer is overlaid to a final concentration of 20 mM MES pH 6.0 + 0.1% Tween, and a modified cytidine deaminase as described herein is added to the samples to a final concentration of 5 µM in a total volume of 10 µL. The deamination reaction is incubated under linear ramp temperature conditions from 4 to 50 °C over a 2 h period.

[0305] After deamination, samples are prepared for Illumina sequencing using the Accel Methyl-NGS kit (Swift Biosciences). Specifically, the reaction is purified using the Zymo Oligo Clean and Concentrator Kit and eluted in 15 μL of elution buffer (10 mM Tris pH 8.0). The Accel-NGS Methyl-Seq kit (Swift Biosciences) is then used for library preparation of single-stranded DNA according to the manufacturer's instructions. After library purification, 1–5 ng of amplified DNA is run on an Agilent Bioanalyzer High Sensitivity DNA Chip to confirm appropriate library fragment sizes. The resulting ACE-Seq library is sequenced in single-end mode on a NextSeq 500 sequencer (Illumina) at 1.9 pM using the NextSeq 500 / 500 High Output kit v2 (150 cycles).

[0306] Sequencing data from the samples is analyzed to detect 5mC at 500 CpG alleles suspected of 5mC modification. These CpG sites that sequence as thymidines are characterized as likely to contain 5mC modifications in the original sample.

[0307] Example 9 Detection of 5hmC. The following examples are generally known in the art, as exemplified by the method disclosed in Schutsky et al., Nature biotechnology, 10.1038 / nbt.42048 Oct. 2018, doi:10.1038 / nbt.4204. Provide genomic DNA (gDNA) from an organism suspected of harboring 5hmC. Provide two aliquots of 20 ng each. Glucosylate one of the aliquots of the 20 ng gDNA mixture using UDP-glucose and T4 β-glucosyltransferase (βGT, NEB). Specifically, mix 0.5 μL of 10× Cutsmart Buffer (NEB), 0.1 μL of 50× UDP-Glucose, 0.5 μL of T4 βGT (NEB) with 20 ng gDNA, and add water to bring the total volume to 5 μL. Assemble a control reaction using another 20 ng aliquot of gDNA and mix the reaction components as above but add 0.5 µL of water instead of T4βGT. Incubate the reaction at 37 °C for 1 h.

[0308] To provide single-stranded DNA and facilitate deamination activity, 1 µL of DMSO is added, samples are denatured at 95 °C for 5 min, and quenched by transferring to a PCR tube rack pre-incubated at -80 °C. Prior to thawing, reaction buffer is overlaid to a final concentration of 20 mM MES pH 6.0 + 0.1% Tween, and modified cytidine deaminase is added to each sample to a final concentration of 5 µM in a total volume of 10 µL. Deamination reactions are incubated under linear temperature ramp conditions from 4 to 50 °C over a 2 h period.

[0309] After deamination, samples are prepared for Illumina sequencing using the Accel Methyl-NGS kit (Swift Biosciences). Specifically, reactions are purified using the Zymo Oligo Clean and Concentrator Kit and eluted in 15 μL of elution buffer (10 mM Tris pH 8.0). The Accel-NGS Methyl-Seq kit (Swift Biosciences) is used for library preparation according to the manufacturer's instructions. After library purification, 1–5 ng of amplified DNA is run on an Agilent Bioanalyzer High Sensitivity DNA Chip to confirm appropriate library fragment sizes. The resulting ACE-Seq library is sequenced in single-end mode on a NextSeq 500 sequencer (Illumina) at 1.9 pM using the NextSeq 500 / 500 High Output kit v2 (150 cycles).

[0310] The sequencing data from the sample and the control are analyzed to detect 5hmC and 5mC in 500 CpG alleles suspected of 5mC modification. Specifically, any CpG site that sequences as cytosine in the control sample is compared with the same CpG site in the sample treated with βGT. Those sites that sequence as thymidine are characterized as likely to contain 5mC modification in the original sample.

[0311] Example 10 Detection of 5mC and 5hmC The following examples are generally known in the art, as exemplified by the method disclosed in Schutsky et al., Nature biotechnology, 10.1038 / nbt.42048 Oct. 2018, doi:10.1038 / nbt.4204. Provide genomic DNA (gDNA) from an organism suspected of having 5mC and 5hmC. Provide two aliquots of 20ng each. Then, process the gDNA mixture of each aliquot of 20ng as follows:

[0312] To provide single-stranded DNA and promote deamination activity, 1 μL of DMSO is added, samples are denatured at 95 °C for 5 min, and quenched by transferring to a PCR tube rack pre-incubated at -80 °C. Prior to thawing, reaction buffer is overlaid to a final concentration of 20 mM MES pH 6.0 + 0.1% Tween. To one of the aliquots and the modified cytidine deaminase described herein (Y130W) is added to the sample to a final concentration of 5 μM in a total volume of 10 μL. To the other aliquot, wild-type cytidine deaminase is added to the sample to a final concentration of 5 μM in a total volume of 10 μL. The deamination reaction is incubated under linear temperature ramp conditions from 4 to 50 °C for 2 h.

[0313] After deamination, samples are prepared for Illumina sequencing using the Accel Methyl-NGS kit (Swift Biosciences). Specifically, the reaction is purified using the Zymo Oligo Clean and Concentrator Kit and eluted in 15 μL of elution buffer (10 mM Tris pH 8.0). The Accel-NGS Methyl-Seq kit (Swift Biosciences) is then used for library preparation of single-stranded DNA according to the manufacturer's instructions. After library purification, 1–5 ng of amplified DNA is run on an Agilent Bioanalyzer High Sensitivity DNA Chip to confirm appropriate library fragment size. The resulting APOBEC-associated epigenetic sequencing (ACE-seq) library is sequenced at 1.9 pM in single-end mode on a NextSeq 500 sequencer (Illumina) using the NextSeq 500 / 500 High Output kit v2 (150 cycles).

[0314] Sequencing data from the samples is analyzed to detect 5mC at 500 CpG alleles suspected of 5mC and / or 5hmC modifications. CpG sites that sequence predominantly as thymidine in the wild-type sample but predominantly as cytosine in the Y130W sample are characterized as likely to contain a 5hmC modification in the original sample.

[0315] Example 11 Detection of 5mC and 5hmC Human genomic DNA is combined with fully unmethylated lambda control DNA (New England Biolabs) and enzymatically CpG-methylated pUC19 control DNA and mechanically sheared to obtain fragments of approximately 300 bp. This sheared DNA (10-100 ng) is then subjected to end repair, A-tailing and adaptor ligation according to standard Illumina library preparation procedures. The sample is then split into two aliquots to allow for differential processing.

[0316] One aliquot of the adaptor-ligated DNA is glucosylated using UDP-glucose and T4β-glucosyltransferase (βGT, NEB). Specifically, the sample is treated with 0.5U / uL of T4βGT (NEB) in 1× CutSmart buffer (NEB) containing 40 μM UDP-glucose for 3 hours at 37° C. A control reaction is set up using another aliquot of the DNA sample, omitting the T4βGT enzyme. The sample is then optionally SPRI purified and then denatured by incubation in 0.02N sodium hydroxide at 50° C. for 10 minutes. The ssDNA sample is then enzymatically deaminated in 50 mM Bis-Tris (pH 6.5), 10 μg / mL RNAse A containing cytidine deaminase (200 nM) for 25 minutes at 37° C. The library is then PCR amplified using 9 cycles of PCR using unique double-index primers and Q5U (New England Biolabs). Samples are sequenced on a NovaSeq6000 and analysis is performed using the DRAGEN Methylation Pipeline. To determine which sites are hydroxymethylated, methylation calls between the two treatment conditions (+ / -T4βGT) are compared. Bases detected as methylated in both sample treatments are assigned to mC, whereas bases detected as methylated in the -βGT condition and unmethylated in the +βGT condition are assigned to hmC (Figure 19).

[0317] Example 12 A generalized method for generating methylation sequencing libraries Sheared DNA or cfDNA (10-100 ng) was first subjected to end repair, A-tailing and adapter ligation according to standard library preparation procedures. Adapters suitable for this method can have unmodified C or pyrrolo-C modifications to avoid deamination of the adapter sequences. The adapter-ligated DNA was then denatured to ssDNA. This ssDNA sample was subsequently enzymatically deaminated in a buffer solution containing an engineered deaminase (APOBEC3A-Y130A-Y132H, 50 nM-1000 nM) at incubation temperatures ranging from 20 °C to 55 °C for 5 min to 3 h. The deaminated libraries were optionally SPRI-purified using either a uracil-resistant polymerase (e.g., Q5U®, KapaU™) or a uracil-non-resistant polymerase (e.g., Q5 HiFi®, KAPA HiFi™) using 9-12 cycles of PCR prior to PCR amplification with unique double-index primers. Libraries were then sequenced on a NextSeq550 or NovaSeq 6000 and analysis was performed using the DRAGEN Methylation Pipeline.

[0318] Denaturation Method Various methods for denaturation are known to those skilled in the art, including, but not limited to, (i) heating in the presence of NaOH or high pH buffer at moderate temperatures (e.g., 0.02N sodium hydroxide at 50°C for 10 minutes), (ii) heating at high temperatures (e.g., 95°C for 10 minutes), (iii) heating in the presence of DMF (e.g., 50% DMF at 95°C for 10 minutes), (iv) heating in the presence of formamide (e.g., 50% formamide at 95°C for 10 minutes), and (v) heating in the presence of DMSO (e.g., 50% DMSO at 95°C for 10 minutes).

[0319] Suitable buffer for deamination Typically, the deamination buffer is added to the denatured DNA sample, therefore any additives that promote denaturation are present at a lower (diluted) concentration in the deamination reaction.

[0320] Several buffer systems have been identified in which deamination can occur. A buffer 50 mM Bis Tris, pH 6.5 may be used, and other pH levels from 5 to 7.5 may be feasible. Additionally, other buffer strengths (concentrations) may also be feasible. Those skilled in the art will recognize that if NaOH is used for denaturation, appropriate buffer strength / pH can be used to ensure that the final pH is within the ideal range for the deaminase. Alternative buffer systems including MES (e.g., tcichemicals.com / US / en / c / 10367) may also provide good results.

[0321] In some embodiments, other buffer systems have been shown to have poorer performance when used for deamination of genomic DNA (gDNA) samples. Examples include citrate buffer and sodium acetate buffer (Figure 20). These buffer systems may have poorer performance due to sodium content that is believed to be detrimental to the activity of the modified deaminase. APOBEC3A is a zinc-dependent enzyme (Marx et al., Scientific Reports 5, no. December (2015): 1-9. https: / / doi.org / 10.1038 / srep18191), and therefore the presence of metal cations (e.g., sodium, magnesium) may generally be detrimental. Another explanation is that metal ions can affect the formation of secondary structures in nucleic acids (Einert et al., Biophysical Journal 100, no. 11 (2011): 2745-53, doi.org / 10.1016 / j.bpj.2011.04.038), which may also affect the activity of deaminases on substrates, as APOBEC3A is unable to deaminate Cs in double-stranded DNA such as hairpins. It is therefore anticipated that any buffer system that maintains the pH in the desired range can be used, and in some embodiments, buffers with minimal sodium can be used.

[0322] Selectivity of engineered cytidine deaminases for genomic DNA The modified cytidine deaminase maintains some activity towards unmethylated cytidine deamination, and therefore it can be useful to adjust the activity to maintain the desired selectivity towards methylated cytosine. For example, high concentrations of enzyme, long incubation times, or buffers that promote high activity of the enzyme may result in undesirable levels of unmethylated cytidine deamination. Typically, the enzyme concentration may be 50 nM to 1000 nM, and the reaction may occur for 5 minutes to 3 hours at incubation temperatures ranging from 20°C to 55°C. In particular, it may be useful to adjust the enzyme concentration based on the purification method of the deaminase and how much activity a given enzyme preparation contains.

[0323] As an example, libraries were prepared, deaminated, and sequenced according to the general method described above. Varying the concentration of APOBEC-Y130A-Y132H resulted in observable differences in activity levels and off-target cytidine deamination levels (Figure 20A). Furthermore, while one might expect that commercially available buffers for APOBEC activity would work well for this application, testing of the APOBEC buffer included in the EM-seq™ kit (New England Biolabs) resulted in undesirably high levels of activity against cytidine substrates and elevated conversion of unmethylated C nucleobases in lambda DNA (Figure 20B).

[0324] Methods for determining suitable conditions for deamination To determine whether conditions were suitable for selective deamination on genomic DNA, DNA mixtures containing NA12878 (human) DNA, fully CpG methylated pUC19, and fully unmethylated lambda were prepared. After adapter ligation, samples were subjected to various deamination conditions and prepared as sequencing libraries using the general method described above. Methylation levels of pUC19 and lambda were used to select conditions with the desired 5mC activity and selectivity.

[0325] Example 13 Illustrative examples of conditions used for library preparation and deamination with engineered cytidine deaminases.

[0326] In all methods described in Examples 13-20 below, the modified cytidine deaminase specifically refers to APOBEC3A-Y130A-Y132H. Various human genomic DNA samples were tested for different application evaluations.

[0327] Method A: Human genomic DNA was combined with fully unmethylated lambda control DNA and enzymatically CpG methylated pUC19 control DNA and mechanically sheared to obtain fragments of approximately 300 bp. This sheared DNA (10-100 ng) was then subjected to end repair, A-tailing and adapter ligation according to standard Illumina library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02 N sodium hydroxide at 50 °C for 10 min. The ssDNA samples were then enzymatically deaminated in 50 mM Bis-Tris (pH 6.5), 10 μg / mL RNAse A containing cytidine deaminase (200 nM) for 25 min at 37 °C. The library was then PCR amplified using 9 cycles of PCR using unique dual index primers and Q5U (New England Biolabs). Samples were sequenced on a NovaSeq6000 and analysis was performed using the DRAGEN Methylation Pipeline.

[0328] Method B: Human genomic DNA was combined with fully unmethylated lambda control DNA and enzymatically CpG methylated pUC19 control DNA and mechanically sheared to obtain fragments of approximately 300 bp. This sheared DNA (10-100 ng) was then subjected to end repair, A-tailing and adapter ligation according to standard Illumina library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02 N sodium hydroxide at 50 °C for 10 min. The ssDNA samples were then enzymatically deaminated in 50 mM Bis-Tris (pH 6.5), 10 μg / mL RNAse A containing modified cytidine deaminase (200 nM) for 15 min at 37 °C. The library was then PCR amplified using 9 cycles of PCR using unique dual index primers and Q5U (New England Biolabs). Samples were sequenced on a NovaSeq6000 and analysis was performed using the DRAGEN Methylation Pipeline.

[0329] Method C: Human genomic DNA was combined with fully unmethylated lambda control DNA and enzymatically CpG methylated pUC19 control DNA and mechanically sheared to obtain fragments of approximately 300 bp. This sheared DNA (10-100 ng) was then subjected to end repair, A-tailing and adapter ligation according to standard Illumina library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02 N sodium hydroxide at 50°C for 10 min. The ssDNA samples were then enzymatically deaminated in 50 mM Bis-Tris (pH 6.5), 10 μg / mL RNAse A containing modified cytidine deaminase (200 nM) for 15 min at 37°C. The library was then PCR amplified using 9 cycles of PCR using unique dual index primers and a Q5 HiFi (New England Biolabs). Samples were sequenced on a NovaSeq6000 and analysis was performed using the DRAGEN Methylation Pipeline.

[0330] Method D: Human genomic DNA was combined with fully unmethylated lambda control DNA and enzymatically CpG methylated pUC19 control DNA and mechanically sheared to obtain fragments of approximately 300 bp. This sheared DNA (10-100 ng) was then subjected to end repair, A-tailing and adapter ligation according to standard Illumina library preparation procedures. The adapter-ligated DNA was denatured by incubation in 0.02 N sodium hydroxide at 50°C for 10 min. The ssDNA samples were then enzymatically deaminated in 50 mM Bis-Tris (pH 6.5) containing modified cytidine deaminase (200 nM) for 25 min at 37°C. The library was then PCR amplified using 9 cycles of PCR using unique dual index primers and Q5U (New England Biolabs). Samples were sequenced on a NovaSeq6000 and analysis was performed using the DRAGEN Methylation Pipeline.

[0331] Use of RNAse A The literature suggests that RNAse may increase the activity of cytidine deaminases by removing contaminating RNA (Bransteitter et al., Proceedings of the National Academy of Sciences of the United States of America 100, no. 7 (2003): 4102-7. https: / / doi.org / 10.1073 / pnas.0730835100). However, testing of RNAse A showed that contrary to this hypothesis, RNAse A reduced the activity of modified cytidine deaminases and had a more pronounced effect on reducing off-target cytosine deamination, thus resulting in more 5mC selectivity (Figure 21).

[0332] Example 14 Deamination of 5hmC To assess 5hmC deamination, oligos were designed with C, 5mC, or 5hmC modifications in defined contexts. These oligos were assembled together using ligation, and the oligos were constructed such that the resulting ligated fragments contained the necessary handles for subsequent amplification and sequencing (Figure 22A). The assembled control oligo was spiked into an adaptor-ligated DNA library and processed according to method A (Example 12). Analysis of the methylation reported from this control oligo indicated that 5mC was the most preferred substrate for APOBEC Y130A-Y132H, although there was still significant activity towards 5hmC (Figure 22B).

[0333] Example 15 Determination of methylation on CpG islands Libraries were prepared using NA12878 gDNA and deaminated according to method A (Example 13). A comparative data set was also generated using EM-Seq™ conversion (New England Biolabs). To evaluate the methylation performance on the human genome, regional methylation values ​​for CpG islands across the human genome were calculated using methylpy. The per-region methylation values ​​were plotted against the per-region methylation values ​​derived from the EM-seq™ data set (Figure 23). The regional analysis demonstrates a high correlation between the use of Y130A-Y132H deaminase and EM-Seq™, a commercially available methylation detection method. This indicates that the 5mC selective deamination assay described herein can effectively detect and report methylation in CpG islands.

[0334] Example 16 Mutant calling against methylation libraries Libraries from human genome samples NA12878, NA24385, and NA24631 were prepared and deaminated according to method A (Example 13). A comparative data set was also generated without conversion by proceeding directly to PCR after adapter ligation. After variant calling analysis, comparison with the truth set for each genome using hap.py showed that libraries subjected to methylation conversion (modified cytidine deaminase) provided good SNV / indel calling performance approaching that of the no-conversion control (Figure 24). Although SNV / indel calling is discussed here, other types of variant calling are feasible, including copy number variation (CNV) and short tandem repeats (STR), as well as structural variants (SV).

[0335] Example 17 Variable methylation region (DMR) calling Variable methylation region analysis is commonly used for comparison of methylomes across different diseases, tissues, and cell types (Chen et al., Briefings in Functional Genomics 15, no. 6 (2016): 485-90. https: / / doi.org / 10.1093 / bfgp / elw018). Libraries were prepared and deaminated according to Method A (Example 13) using genomic DNA isolated from HCC2218 tumor and normal cell types (CRL-2363D, CRL-2343D, ATCC) and HCC1187 tumor and normal cell types (CRL-2323D, CRL-2322D, ATCC). Comparative data sets were also generated using EM-Seq™ conversion (New England Biolabs) and bisulfite conversion (EZ DNA Methylation-Gold Kit-Zymo Research). For variably methylated region analysis, we used HOME, a program for identifying DMRs (Srivastava et al., BMC Bioinformatics 20, no. 1 (2019): 1-15, doi.org / 10.1186 / s12859-019-2845-y). For each methylation assay, a comparison of methylation between tumor and normal samples was performed. As shown in Figure 25, the assay was able to detect the relevant variably methylated regions of the ZNF-154 gene (Almeida et al., BMC Cancer 19, no. 1 (2019): 1-12. https: / / doi.org / 10.1186 / s12885-019-5403-0). Furthermore, quantitative evaluation of DMR calling performance using methods A, B, and C (Example 13) showed that the modified cytidine deaminase-based method detected the predicted DMRs with high precision and recall (Figure 26).

[0336] Example 18 Promoter methylation The methylation status of promoter regions can be correlated with both histone status as well as gene expression activity. To evaluate whether the modified cytidine deaminase assay can be used to examine promoter methylation status, a library from human genome sample NA12878 was prepared and deaminated according to method A (Example 13). A comparative library was also generated using EM-seq™ conversion. Methylation levels at known histone marker sites, including H3K36me3 and H3K27ac, were quantified from the resulting data. H3K36me3 sites are predicted to be inactive promoters with high histone and DNA methylation, while H3K27ac sites are predicted to be active promoters with high histone acetylation and low DNA methylation. The modified cytidine deaminase assay was able to report these methylation trends as expected (Figure 27).

[0337] Example 19 Detection of tumor samples in a background of normal samples Libraries from human genomic samples with either HCC2218 normal DNA or 10% spike-in of HCC2218 tumor DNA into a background of HCC2218 normal DNA were generated using appropriate adaptors. Evaluation of methylation levels for the 10% spike-in samples was performed in comparison to established methylation methods for CpGs within regions also targeted by the PanSeer cancer panel (Chen, et al. Nature Communications 11, no. 1 (2020): 1-10, https: / / doi.org / 10.1038 / s41467-020-17316-z). Libraries subjected to modified cytidine deaminase reflected methylation changes between HCC2218 normal DNA and 10% spike-in of HCC2218 tumor DNA into HCC2218 normal DNA. Methods A, B, and C (Example 13) produced higher tumor signals in 10% tumor spike-in samples compared to samples without tumor DNA spike-in (Figure 28). This reflects the ability of modified cytidine deaminase to detect low levels of tumor DNA, which can be applied to cell-free DNA (cfDNA) samples for early cancer detection (Jamshidi et al., Cancer Cell 40, no. 12 (2022): 1537-1549. e12. doi.org / 10.1016 / j.ccell.2022.10.022.) or minimal residual disease (MRD) testing (see Jin et al., Proceedings of the National Academy of Sciences of the United States of America 118, no. 5 (2021): 1-8, doi.org / 10.1073 / pnas. 2017421118.).

[0338] Example 20 concentrated Most commercially available methylation assays convert C>T, resulting in extensive C>T conversion and low complexity sequences. This makes enrichment very difficult as probes need to be designed for all possible methylation states / strands (www.twistbioscience.com / sites / default / files / resources / 2020-02 / AppNote_Methylation-singles.pdf). This makes hybrid enrichment sequencing of methyl-converted libraries expensive and more difficult to optimize. An alternative is amplicon-based targeted sequencing, but advantages of hybridization-based targeted sequencing include the ability to easily sequence both unenriched (whole genome sequencing (WGS)) and enriched samples, as well as targeted sequencing over multiple contiguous regions of interest (Singh et al., Diagnostics (Basel, Switzerland) 12, no. 7 (June 24, 2022): 1539. doi.org / 10.3390 / diagnostics12071539.). Because of the 5mC>T conversion enabled by the modified cytidine deaminase, enrichment of methyl-converted libraries can be achieved using less complex panels while still maintaining high quality methylation information (Figure 29). Since only a small percentage of cytosines are expected to be methylated and converted, standard probe designs prepared for non-converted libraries may be used. Furthermore, hybridization probes tolerate mismatches (Paskey et al., BMC Genomics, 20(1). doi.org / 10.1186 / S12864-019-5543-2), and therefore some methylation conversion can be tolerated by standard panels. In the case of high CpG density methylated regions of the genome, it may be beneficial to design extra probes to account for mismatches to optimize performance.

[0339] To evaluate enrichment performance, we utilized the Illumina Methyl Capture EPIC panel, a probe panel typically used prior to bisulfite-based conversion. This panel is designed to target unconverted DNA due to challenges with enrichment of converted DNA, as discussed above. However, the Illumina Methyl Capture EPIC kit requires high input (500 ng) for enrichment upstream of the conversion step. In contrast, in the following examples, samples were converted, amplified, and then concentrated to allow for lower input (Figure 30A-C).

[0340] A library containing NA12878 genomic DNA was prepared according to method A (Example 13). As a control, a library that was not deaminated was also prepared (no conversion control). After conversion and amplification, the library was sequenced to generate whole genome methylation control data. Samples were also enriched using the Illumina Methyl Capture EPIC panel according to the protocol described in the RNA Prep with Enrichment Reference Guide (support.illumina.com / content / dam / illumina-support / documents / documentation / chemistry_documentation / illumina_prep / RNA / illumina-rna-prep-reference-guide-1000000124435-03.pdf). Read enrichment was assessed using Picard CollectHsMetrics, which showed that the deaminated library achieved similar read enrichment compared to the non-converted library (Figure 31A). CpG island methylation was assessed using the method described in Example 15. Comparison of sequencing data from the unenriched library with the enriched library showed good agreement between methylation levels reported with and without enrichment, suggesting that enrichment can be used to target and report methylation data (Figure 31B).

[0341] Example 21 Modified cytidine deaminase-mediated detection of the 5mC locus by qPCR The conversion of 5mC to T can be read by PCR using mismatch-sensitive primers. Upon conversion of 5mC to T, these primers can anneal without mismatches to the substrate DNA. Unmethylated DNA substrates are not converted by modified cytidine deaminase, and therefore these substrates show mismatches within the primer annealing sequence, resulting in poor PCR amplification. Using qPCR to measure amplification relative to a standard curve provides a quantitative readout of the methylation rate at the target locus.

[0342] A 78 nt ssDNA oligo containing 5mC and C was incubated with APOBEC3A(Y130A / Y132H) for 15 min at 37° C. to allow 5mC deamination to occur (FIG. 32). The deamination reaction was then heat inactivated at 95° C. for 5 min and a small aliquot was added directly to a qPCR master mix containing PCR buffer, dNTPs, primers, and Q5 Hot Start DNA Polymerase. The resulting mixture was cycled in a standard qPCR instrument. (Note: Q5 hot-start DNA polymerase was used in the qPCR assay as it is selective for uracil-containing templates, thus providing an additional layer of discrimination against spurious C to U conversion by APOBEC3A(Y130A / Y132H).) Cq values ​​obtained with APOBEC3A(Y130A / Y132H) and the slightly less selective mutant (Y130A) were significantly lower when compared to wild-type APOBEC3A [which is non-selective for 5mC]. Importantly, when a catalytically inactive mutant, APOBEC3A(Y130A / Y132H_E72A), was used for deamination, Cq values ​​were consistent with the no-enzyme control (BSA), confirming that the observed qPCR detection of 5mC is dependent on the evolved APOBEC mutant.

[0343] Because the qPCR primers are selective for 5mC-converted DNA, significant qPCR amplification can be detected even if only a small portion of the substrate is deaminated. A 15-minute reaction time was sufficient to observe a significant decrease in the Cq value of APOBEC3A(Y130A / Y132H) (Figure 33). Based on this initial success, we anticipate that alternative iterations of this detection methodology will be compatible with any number of DNA amplification-based diagnostic approaches, including (1) LAMP, (2) recombinase-polymerase amplification, and (3) other isothermal amplification methodologies.

[0344] Example 22 Detection of the 5mC locus using engineered cytidine deaminase and CRISPR-Cas12 CRISPR-Cas systems have emerged as promising tools for rapid and specific quantification of nucleic acid sequences. Technologies such as DETECTR (Chen et al., Science. 2018; 360: 436-439) and CDetection (Teng et al., Genome Biol. 2019; 20: 132) use Cas12 family enzymes to sensitively detect single nucleotide polymorphisms in analyte DNA. Cas12 family enzymes bind and cleave DNA substrates determined by their complementarity to the guide RNA that forms a complex with Cas12. Mismatches between the guide RNA and the target DNA inhibit this process. Importantly, Cas12 orthologs exhibit "collateral cleavage" activity: target DNA binding leads to activation of a highly processive nonspecific nuclease activity, resulting in cleavage of ssDNA in trans. This collateral cleavage activity can be visualized using a separate ssDNA reporter substrate that contains a fluorophore and a quencher. RNA-guided engagement of target DNA by Cas12 results in trans-cleavage of the ssDNA reporter. The subsequent release of the fluorophore from the quencher can be visualized with a fluorometer. Because the trans-cleavage activity of Cas12 is highly catalytic, subattomole concentrations of target DNA can be detected (Teng et al., Genome Biol. 2019;20:132).

[0345] The concomitant cleavage activity of Cas12 can be exploited for sensitive detection of 5mC (Figure 34). The workflow involves 1) analyte DNA denaturation, 2) conversion of 5mC to T using a modified cytidine deaminase, and 3) application of the Cas12-guide RNA complex to the sample. The guide RNA is perfectly complementary to the DNA converted from 5mC to T, thus allowing Cas12 to bind to the analyte DNA only if it contains 5mC. Subsequent activation of Cas12-mediated trans-cleavage cleaves the reporter ssDNA and increases fluorescence. This effect is measured using a standard plate reader instrument.

[0346] Cas12 family enzymes exhibit different requirements for target DNA binding, being specific for either dsDNA or ssDNA. The use of ssDNA-specific Cas12b or Cas12f is advantageous for the above workflow, since it avoids the need for target DNA rehybridization before applying Cas12-gRNA. Cas12b / f-gRNA and APOBEC can then be combined in a one-pot reaction, as Cas12b / f engages with target ssDNA after APOBEC-mediated 5mC to T conversion has occurred. This accelerates the development of this workflow as a point-of-care (POC) diagnostic for DNA methylation.

[0347] Example 23 Spatial detection of 5mC using fluorescence in situ hybridization (FISH) FISH is routinely used to detect the abundance and spatial localization of DNA sequences of interest in fixed cells and tissue sections using fluorescently labeled probes. It is increasingly used in IVD applications to diagnose cellular dysplasias such as chromosomal translocations, deletions and copy number alterations. For example, deletions of chromosome 5q are associated with poor prognosis in acute myeloid leukemia and therefore have been assayed as biomarkers using FISH probes (molecular.abbott / us / en / products / oncology / vysis-egr1-fish-probe-kit).

[0348] Although detection of DNA sequences by FISH is well established, extending this technique to detect DNA modifications such as 5mC remains challenging due to the technical difficulties of in situ bisulfite treatment of cells or tissue sections. Modified cytidine deaminase-mediated 5mC to T conversion enables such a workflow due to the mild conditions required for APOBEC enzyme activity, e.g., 37°C, pH 5.2-6.5. Furthermore, we discovered that the 5mC-selective APOBEC3A (Y130A / Y132H) enzyme is active in citrate buffer pH 6.0 (Figure 35), a buffer commonly used in FISH protocols to increase total probe signal intensity (Yu et al., Exp Ther Med. 2021;22:1480). A contemplated APOBEC-FISH protocol involves 1) permeabilization and denaturation of fixed cells or tissue sections, 2) 5mC to T conversion using APOBEC, and 3) hybridization of a probe specific for the deaminated DNA sequence (Figure 36).

[0349] Example 24 Array-based detection of 5mC ILMN methylation array products typically involve selective hybridization of bisulfite-converted DNA to bead-based probes. The use of engineered cytidine deaminases for direct 5mC to T conversion maintains the overall four-base genomic complexity, thus lowering DNA input requirements and simplifying probe design.

[0350] In the case of a 4-base genome, the array probes are more specific, and therefore quantification of methylated samples is expected to be more accurate than that of unmethylated samples. Hybridization efficiency for the 4-base genome is better than that for the degenerate 3-base genome due to the conservation of complexity between DNA and the probe, and can result in better resolution.

[0351] The complete disclosures of all patents, patent applications, and publications, and electronically available materials cited herein (e.g., nucleotide sequence submissions in GenBank and RefSeq, amino acid sequence submissions in SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, figures, materials and methods, and / or experimental data) are likewise incorporated by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure of a document incorporated by reference herein, the disclosure of this application shall prevail. The foregoing detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be understood therefrom. The disclosure is not limited to the exact details shown and described, since variations obvious to one skilled in the art are included in the disclosure defined by the claims.

[0352] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood in all instances to be modified by the term "about". Thus, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the scope of the claims to the doctrine of equivalents, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0353] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain ranges necessarily resulting from the standard deviation found in their respective testing measurements.

[0354] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless specifically stated.

Claims

1. A modified cytidine deaminase comprising amino acid substitution mutations of the cytidine deaminase at positions functionally equivalent to (Tyr / Phe)130 and Tyr132 in the wild-type APOBEC3A protein.

2. 1. A modified cytidine deaminase comprising an amino acid substitution mutation of said cytidine deaminase at a position functionally equivalent to (Tyr / Phe)130 of a wild-type APOBEC3A protein, said substitution mutation being (Tyr / Phe)130Trp.

3. The modified cytidine deaminase of claim 1 or 2, wherein the (Tyr / Phe)130 is Tyr130 and the wild-type APOBEC3A protein is SEQ ID NO:

3.

4. 3. The modified cytidine deaminase of claim 1 or 2, wherein the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to Ala, Val, or Trp.

5. 5. The modified cytidine deaminase of claim 2 or 4, wherein the substitution mutation at a position functionally equivalent to Tyr132 comprises a mutation to His, Arg, Gln, or Lys.

6. 2. The modified cytidine deaminase of claim 1, wherein the modified cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate that is faster than the conversion of cytosine (C) to uracil (U) by deamination.

7. The modified cytidine deaminase of claim 6, wherein the rate is at least 100-fold greater.

8. 3. The modified cytidine deaminase of claim 2, wherein the modified cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

9. The modified cytidine deaminase of claim 8, wherein conversion of 5hmC to 5hmU by deamination is undetectable.

10. 3. The modified cytidine deaminase of claim 1 or 2, wherein the modified cytidine deaminase is a member of the AID subfamily, the APOBEC1 subfamily, the APOBEC2 subfamily, the APOBEC3A subfamily, the APOBEC3B subfamily, the APOBEC3C subfamily, the APOBEC3D subfamily, the APOBEC3F subfamily, the APOBEC3G subfamily, the APOBEC3H subfamily, or the APOBEC4 subfamily.

11. The modified cytidine deaminase has the ZDD motif H-[P / A / V]-E-X ［２３－２８］ -P-C-X ［２－４］ The modified cytidine deaminase of claim 1 or 2, comprising -C (SEQ ID NO: 12).

12. The modified cytidine deaminase is a member of the APOBEC3A subfamily and has the ZDD motif HXEX ２４ SW(S / T)PCX ［２－４］ C.X. ６ Forex ８ LX ５ R(L / I)YX ［８－１１］ LX ２ LX ［１０］ 3. The modified cytidine deaminase of claim 1 or 2, comprising: M (SEQ ID NO: 13), and wherein the amino acid substitution mutation at a position functionally equivalent to (Tyr / Phe)130 of the wild-type APOBEC3A protein is the Tyr(Y) amino acid of the ZDD motif.

13. the modified cytidine deaminase is a member of the APOBEC3A subfamily; 【Table 1】 The modified cytidine deaminase of claim 1 or 2, comprising:

14. 3. The modified cytidine deaminase of claim 1 or 2, wherein the modified cytidine deaminase is a member of the APOBEC3A family and comprises SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:

59.

15. The modified cytidine deaminase of claim 14, wherein the substitution mutation at a position functionally equivalent to Tyr130 includes a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine, or threonine.

16. A polynucleotide encoding the modified cytidine deaminase of any one of claims 1 to 15.

17. A composition comprising the modified cytidine deaminase of any one of claims 1 to 15 and a buffer solution.

18. The composition, At least one (i) a sample comprising DNA containing at least one modified cytosine, the modified cytosine being 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC), or a combination thereof; or (ii) a buffer having a pH of less than 7, or (iii) The composition of claim 17 further comprising a combination thereof.

19. The composition of claim 18 , wherein the DNA comprises single-stranded DNA.

20. 19. The composition of claim 18, wherein the modified cytidine deaminase comprises an amino acid substitution mutation at a position functionally equivalent to Tyr132 in a wild-type APOBEC3A protein.

21. The composition of claim 18 , wherein the sample comprises genomic DNA or cell-free DNA.

22. 22. The composition of claim 21, wherein the genomic DNA is from a single cell or is a mixture from multiple cells.

23. 18. The composition of claim 17, wherein the substitution mutation at a position functionally equivalent to Tyr130 comprises a mutation to alanine, glycine, phenylalanine, histidine, glutamine, methionine, asparagine, lysine, valine, aspartic acid, glutamic acid, serine, cysteine, proline, arginine, or threonine, and wherein the modified cytidine deaminase converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a faster rate than it converts cytosine (C) to uracil (U) by deamination.

24. 24. The composition of claim 23, wherein the rate is at least 100 times greater.

25. 19. The composition of claim 18, wherein the substitution mutation at a position functionally equivalent to (Tyr / Phe)130 comprises a mutation to Trp, and wherein at least one 5-hydroxymethylcytosine (5hmC) is present in the DNA, and wherein the modified cytidine deaminase converts cytosine (C) to uracil (U) by deamination and converts 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination.

26. 26. The composition of claim 25, wherein conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination is undetectable.

27. 1. A method comprising: providing a sample of DNA suspected of containing single stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5CaC), or combinations thereof; (i) converting 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the conversion of cytosine (C) to uracil (U) by deamination to obtain a converted single-stranded DNA, or (ii) contacting said single-stranded DNA with a modified cytidine deaminase under conditions suitable for converting C to U and 5mC to T by deamination at a rate greater than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination, contacting the single-stranded DNA with a modified cytidine deaminase, wherein the modified cytidine deaminase is the modified cytidine deaminase of claim 1 or 2; and treating the converted single-stranded DNA to generate a sequencing library.

28. 28. The method of claim 27, wherein the method further comprises denaturing double-stranded DNA present in the sample to provide single-stranded DNA prior to said providing.

29. 1. A method comprising: providing a sample of DNA suspected of containing double stranded DNA containing at least one 5-methylcytosine (5mC), at least one 5-hydroxymethylcytosine (5hmC), at least one 5-formylcytosine (5fC), at least one 5-carboxycytosine (5caC), or combinations thereof; treating said double stranded DNA to generate a sequencing library; denaturing said sequencing library to produce single stranded DNA; (i) converting 5-methylcytosine (5mC) to thymidine (T) by deamination at a rate greater than the conversion of cytosine (C) to uracil (U) by deamination, or (ii) converting 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination at a rate greater than the conversion of 5-hydroxymethylcytosine (5hmC) to 5-hydroxymethyluracil (5hmU) by deamination, to obtain a converted single-stranded DNA; Obtaining a converted single-stranded DNA, wherein the modified cytidine deaminase is the modified cytidine deaminase according to claim 1 or 2; and converting said converted single-stranded DNA into a converted double-stranded DNA sequencing library.

30. 30. The method of claim 29, wherein said processing comprises fragmenting or tagmenting the double-stranded DNA and adding universal sequences to the double-stranded DNA fragments.

31. 31. The method of claim 30, wherein the universal sequence is part of an adaptor that is added to the double-stranded DNA fragment.

32. 30. The method of claim 29, wherein said converting comprises amplifying said converted single stranded DNA into said converted double stranded DNA.

33. 28. The method of claim 27, wherein the sample is a biological sample.

34. 34. The method of claim 33, wherein the biological sample comprises cell-free DNA.

35. 34. The method of claim 33, wherein the biological sample comprises a fluid selected from blood or serum.

36. 34. The method of claim 33, wherein the sample comprises a single cell or an isolated nucleus.

37. 34. The method of claim 33, wherein the biological sample comprises a tissue.

38. 38. The method of claim 37, wherein the tissue comprises tumor tissue.

39. providing a surface comprising a plurality of amplification sites, providing, the amplification site comprising at least two populations of linked single-stranded capture oligonucleotides having free 3′ ends; 28. The method of claim 27, further comprising contacting the surface comprising amplification sites with the sequencing library under conditions suitable to generate a plurality of amplification sites each comprising a clonal population of amplicons from an individual member of the sequencing library.

40. providing a surface comprising a plurality of amplification sites, providing, the amplification site comprising at least two populations of linked single-stranded capture oligonucleotides having free 3′ ends; 30. The method of claim 29, further comprising contacting the surface containing amplification sites with the converted double-stranded DNA sequencing library under conditions suitable to generate a plurality of amplification sites each containing a clonal population of amplicons from an individual member of the converted double-stranded DNA sequencing library.

41. 1. A method for detecting the location of a modified cytosine in a target nucleic acid, the method comprising: (a) contacting a target nucleic acid suspected of containing at least one modified cytosine with a modified cytidine deaminase according to any one of claims 1 to 15 to produce a converted nucleic acid containing at least one converted cytosine; (b) detecting said at least one converted cytosine in said converted nucleic acid of (a).

42. 42. The method of claim 41, wherein the modified cytidine deaminase has cytosine-deficient deaminase activity and the detecting comprises identifying thymidine nucleotides in the converted nucleic acid to determine the position of a 5mC nucleotide in the target nucleic acid.

43. 42. The method of claim 41, wherein the modified cytidine deaminase has 5hmC deficient deaminase activity and the detecting comprises identifying cytosine nucleotides in the converted nucleic acid to determine the position of a 5hmC nucleotide in the target nucleic acid.

44. 42. The method of claim 41 , wherein said detecting comprises sequencing the converted nucleic acid or hybridizing a nucleic acid probe to the converted nucleic acid.

45. said detecting comprising sequencing said converted nucleic acid, said method comprising: (c) comparing the sequence of the converted nucleic acid to an untreated reference sequence to determine which cytosines in the target nucleic acid have been modified.

45. The method of claim 44.

46. 46. ​​The method of claim 45, wherein a predetermined sequence of the untreated reference sequence and a predetermined sequence of the converted nucleic acid are compared.

47. 47. The method of claim 46, wherein the predetermined sequence comprises a CpG island or a promoter.

48. 45. The method of claim 44, wherein the detecting comprises hybridizing the converted nucleic acid to the nucleic acid probe, optionally wherein the nucleic acid probe is present on an analyte array, and the method further comprises sequencing the hybridized converted nucleic acid.

49. 45. The method of claim 44, wherein said detecting comprises hybridizing said converted nucleic acid to said nucleic acid probe, said method further comprising amplifying said converted nucleic acid, said nucleic acid probe comprising two primers for amplification of a predetermined sequence, said primers annealing with higher affinity to regions of the converted nucleic acid containing at least one converted cytosine than to regions of the converted nucleic acid in which at least one cytosine is not a converted cytosine, and the presence of an amplification product indicates a modified cytosine in the target nucleic acid.

50. 45. The method of claim 44, wherein the detecting comprises hybridizing the converted nucleic acid to the nucleic acid probe, the method further comprising cleaving a single-stranded DNA (ssDNA) reporter substrate with a CRISPR-based system, the ssDNA reporter substrate comprising a fluorophore and a quencher, the presence of fluorescence indicating a modified cytosine in the target nucleic acid.

51. 51. The method of Claim 50, wherein the CRISPR-based system comprises a guide RNA sequence that anneals to a predetermined sequence of a nucleic acid that contains at least one converted cytosine and anneals with a lower affinity to the predetermined sequence of the nucleic acid if the at least one cytosine is unconverted.

52. The method of claim 50, wherein the CRISPR-based system comprises CRISPR-Cas12.

53. 45. The method of claim 44, wherein said detecting comprises hybridizing said converted nucleic acid to said nucleic acid probe, said converted nucleic acid being present in a fixed cell, said nucleic acid probe comprising a fluorescently labeled probe, said nucleic acid probe anneals with higher affinity to a predetermined sequence of the converted nucleic acid containing at least one converted cytosine than to a region of the converted nucleic acid in which at least one cytosine is not a converted cytosine, and the presence of cell-associated fluorescence indicates a modified cytosine in said target nucleic acid.

54. 46. ​​The method of claim 45, wherein the unprocessed reference sequence is a predetermined sequence.

55. 42. The method of claim 41, wherein the at least one modified cytosine is 5mC or 5hmC.

56. 42. The method of claim 41, further comprising providing the target nucleic acid, said providing comprising preparing single-stranded (ss) DNA from a sample containing the target nucleic acid.

57. 42. The method of claim 41 , wherein the target nucleic acid is genomic DNA or cell-free DNA.

58. 58. The method of claim 57, wherein the genomic DNA is from a single cell or is a mixture from multiple cells.

59. 45. The method of claim 44, wherein said sequencing comprises processing the converted nucleic acid to generate a sequencing library.

60. providing a surface comprising a plurality of amplification sites, providing, the amplification site comprising at least two populations of linked single-stranded capture oligonucleotides having free 3′ ends; 60. The method of claim 59, further comprising contacting the surface comprising amplification sites with the sequencing library under conditions suitable to generate a plurality of amplification sites each comprising a clonal population of amplicons from an individual member of the sequencing library.

61. 42. The method of claim 41 , wherein the target nucleic acid is obtained from a subject, and the detecting comprises obtaining a pattern of cytosine modifications in the converted nucleic acid, the method further comprising comparing the pattern of cytosine modifications in the converted nucleic acid with a pattern of cytosine modifications in a reference nucleic acid.

62. 62. The method of claim 61, wherein the subject has or is at risk of having a disease or condition and the reference nucleic acid is from a normal subject.

63. 63. The method of claim 62, wherein the pattern of cytosine modifications is linked in cis to a coding region that correlates with a disease or condition.

64. 62. The method of claim 61 , wherein the pattern of cytosine modifications is linked in cis to a coding region, and the coding region in the reference nucleic acid is transcriptionally active or transcriptionally inactive, and said comparing further comprises determining whether the pattern of cytosine modifications in the converted nucleic acid indicates that the coding region is transcriptionally active or transcriptionally inactive in the subject.

65. 65. The method of claim 64, wherein transcription of the coding region correlates with a disease or condition.

66. 63. The method of claim 62, wherein the subject has the disease or condition and is undergoing treatment for the disease or condition, the method further comprising determining whether the treatment correlates with a change in the pattern of cytosine modification in the subject.

67. 67. The method of claim 66, wherein the subject previously had the disease or condition, the method further comprising comparing the pattern of cytosine modifications in the subject with the pattern of cytosine modifications in the subject when the subject had the disease or condition.

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