Methods and compositions for methylation-specific duplex sequencing

Abstract

The disclosure provides a powerful new approach to dual-strand high-throughput next-generation methylation-specific sequencing. The disclosed method provides an enzymatically deaminated strand concatenated to the reverse complement of the other strand, which is protected from conversion by using conversion-resistant dCTPs (e.g., 5- hydroxycytosine, 5-carboxycytosine or 5-propargylaminocytosine) in the strand linking step. This enables both strands to be sequenced together, with mutations detected with duplex sequencing accuracy from both strands, and methylation detected from the converted strand.

Description

114203-4000 (BI- 11308)METHODS AND COMPOSITIONS FOR METHYLATION-SPECIFIC DUPLEX SEQUENCINGCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 734,004, filed December 13, 2024, the entire content of which is incorporated herein by reference.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. CA221874 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] DNA molecules carry not only genetic information within their canonical nucleobases but also crucial epigenetic modifications. Together, these genetic and epigenetic alterations influence cellular behavior in a synergistic manner, and a comprehensive assessment of both is essential for effective disease detection and treatment. While duplex sequencing is considered the gold standard for highly accurate genetic sequencing, high numbers of reads are often required when both strands are sequenced separately. To overcome this issue, CODEC sequencing was developed which links both strands of each DNA duplex together and enables them to be sequenced together. In the realm of epigenetic analysis, methods such as Whole Genome Bisulfite Sequencing (WGBS) and Enzymatic Methyl Sequencing (EM- seq) are widely employed. Both methods convert non-methylated cytosine (C), which accounts for the majority of the total cytosine (> 95%), into uracil (U) and subsequently into thymine (T). This process results in 'three-letter' alignment, which can hinder alignment speed and efficiency. Recently, TET-assisted pyridine borane sequencing (TAPS) and direct methylation sequencing (DM-seq) have been introduced, offering improved mapping due to selectively converting 5-methylcytosine (5mC) instead of unmethylated C. However, a significant limitation of all these methods is their difficulty in distinguishing between OT mutations and C to T conversions caused by enzymatic or chemical deamination.14930-8958-8993.1114203-4000 (BI- 11308)

[0004] Recent advancements have aimed to develop technologies capable of simultaneous epigenetic and genetic sequencing. Third-generation sequencing technologies, such as Oxford Nanopore Technologies (ONT)™ and Pacific Biosciences™ (PACBIO™), enable direct readouts of both epigenetic and genetic sequences. However, their genetic sequencing accuracy is typically lower than that of short-read sequencing and falls far below the accuracy of duplex sequencing, with the exception of when consensus reads are utilized, which compromises throughput. Additionally, they often require high amounts of unamplified DNA, which constrains their applicability to samples with limited DNA, such as liquid biopsies.

[0005] For short-read sequencing, methods have been developed that make a copy of singlestranded DNA and use the original strand for methylation detection and the copied strand for mutation detection. Methods like Five-letter sequencing and Methyl-SNP-seq allow simultaneous sequencing of epigenetic and genetic data in a single workflow, but their detection of rare mutations is limited by single-strand accuracy (error rate ~10'4). MethylSaferSeqS, an amplicon-based approach, makes duplex copies to achieve duplex accuracy for genetic analysis while bisulfite converts the original duplexes. However, it results in two separate libraries, and is limited to a small number of targeted regions. For assessing regional aggregated methylation levels (RAMLs) alongside genome sequencing in a single experiment, RIMS-seq2 employs limited deamination to achieve 1% C to T transitions at 5mC sites. Although it minimally modifies standard library preparation, it does not provide single-base methylation resolution. Furthermore, RIMS-seq2 has significant limitations in detecting rare somatic OT mutations. At present, methods for simultaneous epigenetic and genetic sequencing are limited in breadth and accuracy.SUMMARY OF THE DISCLOSURE

[0006] An aspect of the disclosure is directed to a method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having: a first end, a second end and a central portion positioned between the first end and the second end,24930-8958-8993.1114203-4000 (BI- 11308) wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second singlestrand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5-34930-8958-8993.1114203-4000 (BI- 11308) propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

[0007] In some embodiments, the method further comprises:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0008] In some embodiments, the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double-stranded DNA molecule.

[0009] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas. In some embodiments, the DNA sample comprises cfDNA. In some embodiments, the DNA sample is from a diseased subject.

[0010] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.

[0011] Another aspect of the disclosure is directed to a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter comprising at least ten (10) regions (R01 -R10) in the following configuration:44930-8958-8993.1114203-4000 (BI- 11308)wherein ' — ' represents bonding, wherein R01, R02, and R03 comprise a first oligonucleotide, wherein R04 and R05 comprise a second oligonucleotide, wherein R06 and R07 comprise a third oligonucleotide, wherein R08, R09, and RIO comprise a fourth oligonucleotide, wherein R01 and R06 are annealed to one another, wherein R03 and R08 are annealed to one another, wherein R05 and RIO are annealed to one another, wherein R02 and R07 are not annealed to one another, and wherein R04 and R09 are not annealed to one another; wherein R02 comprises a single- stranded linker, afirst unique molecular identifier (UMI), and a first read primer site, and wherein R09 comprises a single-stranded linker, a second UMI, and a second read primer site, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the sequencing adapter to a dsDNA duplex as follows: ligating the 5' end of R01 to the 3' end of a first strand of the dsDNA duplex; ligating the 3' end of R05 to the 5' end of the first strand of the dsDNA duplex;54930-8958-8993.1114203-4000 (BI- 11308) ligating the 5' end of RIO to the 3' end of a second strand of the dsDNA duplex; and ligating the 3' end of R06 to the 5' end of the second strand of the dsDNA duplex; thereby forming a partially circularized DNA molecule comprising the target DNA molecule and the sequencing adapter;(c) synthesizing first and second single-strand DNA molecules by extending the free 3' end of R03 and 3' end of R08 on the sequencing adapter each using the opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5- propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

[0012] In some embodiments, the method further comprises:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double-stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0013] In some embodiments, the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the64930-8958-8993.1114203-4000 (BI- 11308) sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double-stranded DNA molecule.

[0014] In some embodiments:(1) R01 comprises a first adapter;(2) R03 comprises a first sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase;(3) R04 comprises a free 5' end comprising a first next-generation sequencing (NGS) adapter sequence;(4) R05 comprises a third adapter and a first sample index;(5) R06 comprises a second adapter and a second sample index;(6) R07 comprises a free 5' end comprising a second next-generation sequencing (NGS) adapter sequence;(7) R08 comprises a second sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; and / or(8) RIO comprises a fourth adapter, optionally wherein the first sequence and the second sequence further comprise the same or different primer binding sites, and optionally wherein the first primer site and the second primer site are oriented to initiate sequencing by addition in opposing directions.

[0015] In some embodiments, the ligating of step (b) comprises adding ligase.

[0016] In some embodiments, the synthesizing of step (c) comprises contacting the circular double-stranded DNA intermediate with a polymerase.

[0017] In some embodiments, the polymerase is a DNA-dependent DNA polymerase.

[0018] In some embodiments, the polymerase has a strand- displacement activity.

[0019] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

[0020] In some embodiments, the DNA sample comprises cfDNA.

[0021] In some embodiments, the DNA sample is from a diseased subject.74930-8958-8993.1114203-4000 (BI- 11308)

[0022] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.

[0023] An aspect of the disclosure is directed to a method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter comprises unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second single-strand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double-stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized doublestranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and standard dATP, dGTP, dCTP, and dTTP deoxynucleotides.

[0024] In some embodiments, the method further comprises:(d) deaminating methylated cytosines to uracil or dihydro-uracil;84930-8958-8993.1114203-4000 (BI- 11308)(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double-stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined.

[0025] In some embodiments, methylated cytosines are deaminated by a CpG specific deaminase selected from CseDaOl, MGYPDaO6, LbDaO2, CrDaOl, MGYPDa829, LbsDaOl, or combinations thereof.

[0026] In some embodiments, methylated cytosines are deaminated by a TET-assisted pyridine borane treatment.

[0027] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

[0028] In some embodiments, the DNA sample comprises cell-free DNA (cfDNA).

[0029] In some embodiments, the DNA sample is from a diseased subject.

[0030] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.

[0031] It should be appreciated that the foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various nonlimiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0033] FIGs. 1A-1B shows a Methyl-CODEC library preparation and analysis workflows. As seen in Fig. 1A, Methyl-CODEC captures both genetic and epigenetic information in a single workflow. Modified CODEC adapter quadruplex, which is resistant to conversion in94930-8958-8993.1114203-4000 (BI- 11308) its functional regions, is used for ligation. The strand-displacing extension is performed using Phi29 polymerase with conversion-resistant dCTP, instead of regular dCTP, followed by enzymatic conversion, producing two linearly linked products, each consisting of the converted sequence (Read 2, bottom dashed line) concatenated with the reverse complemented opposite strand with conversion protection (Read 1, line with grey background). UMIs are included in the adapters to allow for the two products to be reassociated as needed, although a single product is sufficient to achieve duplex accuracy. Fig. IB shows a Methyl-CODEC analysis workflow, i) Determine the strand information (protected vs. converted) for a given read, ii) For a given duplex read pair, the protected strand was aligned to standard 4-letter HG38 by BWA-mem. The local reference sequence was extracted from the alignment of the protected strand, iii) The converted strand was aligned to the extracted local reference sequence by BWA-SW using a custom setting that does not penalize C / T or A / G mismatches, iv) A, G, T, non-methylated C and 5mC were determined by the duplex informed deconvolution table from comparing the sequences of the two strands. Cases not covered by the deconvolution table are treated as errors and suppressed. Because of the use of complementary strands, methyl-CODEC is expected to have duplex accuracy for mutation detection.

[0034] FIGs. 2A-2B show a concordance of 5-methylcytosine readout between Methyl- CODEC and EM-seq or WGBS. Fig. 2A shows a genome-wide methylation level at 3 different sequence contexts (CpG (right column), CHG (left column) and CHH (middle column)). H is an IUPAC code which stands for A, C or T. Fig. 2B shows scatter plots comparing methylation level per CpG island (n=25,368) between Methyl-CODEC and EMseq, and between Methyl-CODEC and WGBS. The middle lines were the best linear fits of the data. Pearson correlations and adjusted R-squares were shown on the top left comer.

[0035] FIGs. 3A-3B show Methyl-CODEC achieves duplex sequencing level accuracy (> Q60). Fig. 3A shows a scatter plot comparing Residual SNV frequencies of Methyl-CODEC and standard CODEC in NA12878 (raw deduplicated depth at 25x, duplex depth at 5x), both using a commercial end-repair / dA-tailing protocol. Methyl-CODEC data is split into C&T as the reference and G&A as the reference, while CODEC data represents the average across both strands. Error bars indicate 95% binomial confidence intervals by Wilson method. Mutations that produce methylated cytosines (5mC) and unmethylated cytosines (umC) are labeled next to the corresponding dots. Fig. 3B shows a hypothetical double strand DNA 104930-8958-8993.1114203-4000 (BI- 11308) example demonstrating how strand information in Methyl-CODEC can be used to achieve duplex accuracy for OI7G>A context and explains how T>umC can result from sequencing errors, “rc” indicates reverse complement. If sequenced deep enough to get both products 1 and 2, duplex coverage will be obtained at all positions.

[0036] FIGs. 4A-4B show an expanded mutation calling of methyl-CODEC in NA12878. Fig. 4A shows a bar plot showing, from left to right, the total number of rare duplex SNVs, the number of rare duplex SNVs mutating to C, and the number of rare duplex SNVs creating novel CpG sites, excluding OT and T>umC which are not at duplex accuracy. Fig. 4B shows plots showing the count of rare duplex SNVs mutating to C across 48 trinucleotide contexts, separated by 5mC and umC as the alternative bases. Reference tri-nucleotide contexts when a mutation creating new CpG are indicated on the X-axis. T>umC was dropped due to lack of duplex accuracy.

[0037] FIGs. 5A-5C show a Methyl-CODEC detailed adapter and product structure. Fig. 5A shows the adapter quadruplex, which is resistant to conversion in its functional regions (indicated by black bullets in the adapter structure), is used for ligation, resulting in a loopshaped ligated DNA. The strand- displacing extension is performed using Phi29 polymerase with conversion-resistant dCTP, instead of regular dCTP, producing a linear ligated DNA. Each strand consists of the original sequence (solid line) concatenated with the extended protected sequence (dashed line). The former retains the methylation pattern of the input DNA, while the latter remains intact during enzymatic conversion. Following enzymatic conversion, each strand allows for both duplex-accuracy genetic variant calling and methylation analysis. Fig. 5B shows TET2 and BGT enzymes convert methyl-dCTP and hydroxy-methyl-dCTP into carboxy-dCTP and glucosyl- hydroxy-methyl-dCTP, which are resistant to conversion due to structural modifications that prevent recognition by the APOBEC enzyme. In addition to methyl-dCTP, hydroxy-methyl-dCTP, and carboxy-dCTP, propargylamino-dCTP, a synthetic variant not naturally occurring in the body, was also tested. Methyl- CODEC results indicate that propargylamino-dCTP is also a conversionresistant dCTP variant. Fig. 5C shows Duplex deconvolution table: the bases A, G, T, unmethylated C and methylated C are determined by comparing the complementary bases from the two strands, according to whether the read pair corresponds to product 1 vs product 2. Cases not addressed by the table are considered as errors and hence suppressed.114930-8958-8993.1114203-4000 (BI- 11308)

[0038] FIGs. 6A-6E show additional advantages of preserving original DNA for methylation sequencing. Fig. 6A shows comparing alignments of NA12878 Methyl-CODEC correct products (duplicates excluded) using Methyl- CODEC and Bismark, inferred by distances of the two alignments less than 500 bp; bismark_not_align: reads were aligned by Methyl- CODEC but not by Bismark; discordant: reads aligned to different regions in the genome by Methyl-CODEC and Bismark (at least 500 bp apart from each other); fail mapq bismark: reads below mapping quality cutoff for Bismark alignment but satisfying mapping quality cutoff for Methyl-CODEC alignment; fail mapq mscodec: reads below mapping quality cutoff for Methyl-CODEC alignment but satisfying mapping quality cutoff for Bismark alignment; fail mapq both: reads below mapping quality cutoff for both Methyl-CODEC and Bismark alignments. Mapping quality cutoff was determined by the highest mapping quality of the respective aligner (Bowtie2 for Bismark and BWA for Methyl-CODEC) and was required to be a multiple of 10 (Bismark: 40, Methyl-CODEC: 60).

[0039] FIG. 6B shows bar plot of numbers of total CpGs in HG38, CpGs overlapping any HCT116 germline mutations (including Indels and SNVs) and CpGs overlapping HCT116 germline C>T and G>A mutations.

[0040] FIGs. 6C-6E shows comparison of methylation calling results from HCT116 Methyl- CODEC vs simulated EM-seq WGMS. Paired-end EM-seq WGMS was simulated by in- silico methyl-converting the protected strands of the Methyl-CODEC samples and then aligned by Bismark (default and local modes) Fig. 6C shows average sample level CpG methylation for unmethylated control, methylated control and 1 to 19 ratio mixing of the two. Fig. 6D shows average CpG methylation per sample when excluding CpGs overlapping germline C>T and G>A mutations for the unmethylated control, methylated control and 1 to 19 ratio mixing of the two. Fig. 6E shows average CpG methylation at CpGs overlapping germline C>T and G>A mutations for the unmethylated control, methylated control and 1 to 19 ratio mixing of the two.

[0041] FIG. 7 shows a scatter plot comparing residual SNV frequency of standard CODEC and 4 different versions of Methyl-CODEC (using 4 different conversion-resistant dCTP variants) in NA12878, all using a commercial end-repair protocol. Different shadings are used to indicate C&T and G&A as the reference bases, respectively. Mutations that produce unmethylated cytosines were indicated as well. Whereas mutations that produce methylated124930-8958-8993.1114203-4000 (BI- 11308) cytosines (5mC) are distinguished by slightly different shadings and are labeled next to the corresponding dots.

[0042] FIGs. 8A-8M. Fig. 8A is a schematic showing a variant Concatenated Duplex Sequencing (CODEC). Fig. 8B is a schematic showing the integration of CODEC with an Illumina workflow. Fig. 8C shows the long duplex with mismatch bubbles variant. Fig. 8D shows the modular duplex with mismatch bubbles variant. Fig. 8E shows the half adapter complex variant. Fig. 8F shows the UMI variant. Fig. 8G shows the variant with regions 2 and 3 as partial read primer binding sites. Fig. 8H shows the variant with regions 2 and 3 as complete read primer binding sites. Fig. 81 is a schematic showing formation of a variant with region 1 as indices. Fig. 8J is a schematic showing the mechanism by which CODEC adapter complex creates the concatenated structure. Fig. 8K is a schematic showing CODEC. Fig. 8L is a schematic showing that CODEC structure ignores single insert byproducts which impact NGS quality. Fig. 8M shows the mechanism of single-insert byproduct formation during bridge amplification, leading to mixed clusters.DETAILED DESCRIPTION

[0043] DNA mutations and aberrations in methylation patterns often contribute to disease development in a synergistic manner. While duplex sequencing is recognized as the most accurate method for detecting DNA mutations, it typically lacks the capability to simultaneously assess methylation and comes at high cost. The present disclosure provides a novel methylation-specific DNA sequencing method referred to herein as “Methyl Concatenating Original Duplex for Error Correction” or “Methyl-CODEC” that improves upon duplex sequencing, as well as compositions for conducting said novel sequencing method (e.g., a multi-oligonucleotide adapter for library production, adapter constructs, and sequencing libraries), methods for making the adapters, methods for library construction, and duplex sequencing methods that improve the accuracy of duplex sequencing and at a lower cost. In various aspects, library preparation using Methyl-CODEC adapters results in each DNA molecule becoming self-sufficient for forming a duplex consensus, facilitating the identification of methylated cytosines, as well as true mutations and avoiding false mutations.

[0044] Methyl-CODEC enables simultaneous methylation sequencing and duplex sequencing of single DNA duplexes using single read pairs, based on CODEC. CODEC links both strands of each DNA duplex to enable 1000-fold more accurate sequencing using single read pairs.134930-8958-8993.1114203-4000 (BI- 11308)Methyl-CODEC, on the other hand, provides an enzymatically deaminated strand concatenated to the reverse complement of the other strand, which was protected from conversion by using conversion-resistant dCTPs in the strand linking step. This enables both strands to be sequenced together, with mutations detected with duplex sequencing accuracy from both strands, and methylation detected from the converted strand. Methyl-CODEC showed high concordance in genome-wide methylation compared to Enzymatic Methyl-Seq (EM-seq) and whole genome bisulfite sequencing (WGBS). However, unlike EM-seq and WGBS, it uniquely preserves the original DNA sequence which improves genetic sequencing accuracy, enables better NGS alignment, and distinguishes C>T mutations from unmethylated Cs. From single read pairs, Methyl-CODEC could identify rare mutations, interestingly those that produced 5-methylcytosine (5mC), and those somatic 5mC mutations were substantially enriched in CpG contexts. In all, Methyl-CODEC offers a streamlined approach for highly accurate and efficient epigenetic and genetic sequencing of single DNA duplexes.

[0045] All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.Definitions

[0046] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present disclosure unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.144930-8958-8993.1114203-4000 (BI- 11308)

[0047] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0048] The headings provided herein are not limitations of the various aspects or embodiments of the invention. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3D ED., John Wiley and Sons, New York (2006), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference. The meaning and scope of the terms are clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this disclosure, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0050] The terms “approximately” or “about,” as may be used interchangeably herein, and as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of ( / .< ., percentage greater than or percentage less than) the stated reference value unless otherwise stated or otherwise evident from the context (for example, when such number would exceed 100% of a possible value).

[0051] The terms “percent identity,” “sequence identity,” “% identity,” “% sequence identity,” and % identical,” as they may be interchangeably used herein, refer to a quantitative measurement of the similarity between two sequences (e.g., nucleic acid or amino acid). The percent identity of genomic DNA sequence, intron and exon sequence, and 154930-8958-8993.1114203-4000 (BI- 11308) amino acid sequence between humans and other species varies by species type, with chimpanzee having the highest percent identity with humans of all species in each category.

[0052] Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0053] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073164930-8958-8993.1114203-4000 (BI- 11308)(1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0054] When a percent identity is stated, or a range thereof (e.g., at least, more than, etc.), unless otherwise specified, the endpoints shall be inclusive and the range (e.g., at least 70% identity) shall include all ranges within the cited range (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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 95.5%, at least 96%, at least 96.5%, at least 97%, at least97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity) and all increments thereof (e.g., tenths of a percent (e.g., 0.1%), hundredths of a percent (e.g., 0.01%), etc.).

[0055] The term “substantially,” as may be used herein, when used to describe the degree or abundance of an activity, generally refers to the value of the activity as being an amount which is achievable without undue effort. As can be appreciated, this amount may vary depending on the activity being performed, with simpler activities requiring a higher threshold and more complex activities requiring a lower threshold. For example, without limitation, when referring to substantially eliminating or removing reagents, dNTPs, or enzymes from a mixture, a substantial amount, may refer to 50% or more removal. In some embodiments, substantial refers to at least 50% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.95%, 99.99%, or more) and to all values of the variable that are within the experimental error (e.g., within the 95% confidence interval for the mean) or within + / - 10% of the indicated value, whichever is greater. In some embodiments, substantially refers to at least 75% of the target being removed. In some embodiments, substantially refers to at least 80% of the target being removed. In some embodiments, substantially refers to at least 85% of the target being removed. In some embodiments, substantially refers to at least 90% of the target being174930-8958-8993.1114203-4000 (BI- 11308) removed. In some embodiments, substantially refers to at least 95% of the target being removed.

[0056] The terms “wild type” and “native,” as may be used interchangeably herein, are terms of art understood by skilled artisans and mean the typical form of an item, organism, strain, gene, or characteristic as it occurs in nature as distinguished from engineered, mutant, or variant forms.

[0057] The term “dA-tailing,” as may be used herein, refer to the status, or to a characteristic, of a nucleic acid (e.g., DNA, RNA) as having a “tail” comprising a non-templated adenosine (A) (e.g., adenosine monophosphates). By “tail” it is meant that the adenosines (e.g., AAAAA) at the 3' end of the nucleic acid (e.g., DNA, RNA), comprises an overhang beyond the 5' terminal nucleotide of the complementary strand. The term e.g., dA-tail) may be used as a verb (e.g., dA-tailing) to describe the process by which the adenosine is added to the 3' end of a nucleic acid. In some embodiments, dA-tailing is performed using Taq polymerase. In some embodiments, dA-tailing is performed using KI enow Fragment lacking 3' to 5' exonuclease activity.

[0058] The term “downstream,” as may be used herein, refers to the location of a nucleotide in relation to a landmark in a given sequence of multiple nucleotides (e.g., a nucleic acid), such that downstream shall mean “more 3'” (in the case of a nucleic acid) than the landmark. For example, a nucleotide is downstream from a landmark if it is closer to the 3' end (and thus further from the 5' end) of the nucleic acid than the landmark. Conversely, the term “upstream,” as may be used herein, refers to the location of a nucleotide in relation to a landmark of a given sequence of multiple nucleotides (e.g., a nucleic acid), such that upstream shall mean “more 5'” (in the case of a nucleic acid) than the landmark. For example, a nucleotide is upstream from a landmark if it is closer to the 5' end (and thus further from the 3' end) of the nucleic acid than the landmark.

[0059] The term “overhang,” as may be used herein, is a term of art known to the skilled artisan to refer to a portion of a double-stranded nucleic acid which extends (e.g., protrudes) beyond the end (e.g., terminal nucleotide) of the opposing strand (e.g., complementary strand). For example, without limitation, a 5' overhang will refer to the portion of a strand of a nucleic acid which extends beyond the 3' end (3' terminal nucleotide) of the opposing strand (e.g., complementary strand) with which it forms a double-stranded nucleic acid duplex. As an additional example, without limitation, a 3' overhang will refer to the portion 184930-8958-8993.1114203-4000 (BI- 11308) of a strand of a nucleic acid which extends beyond the 5' end (5' terminal nucleotide) of the opposing strand (e.g., complementary strand) with which it forms a double-stranded nucleic acid duplex. As will be appreciated by the skilled artisan, a double-stranded duplex, may comprise both a 5' and 3' overhang, a single 5' overhang, two 5' overhangs, a single 3' overhang, two 3' overhangs, an overhang (e.g., 5' or 3') and a blunt end, or two blunt ends. As used herein, the term “blunt end,” refers the quality of double-stranded duplex, wherein the two strands forming the duplex terminate at the same pair of nucleotides and thus has no overhang at that end of the duplex (e.g., the end is blunt).

[0060] The term “exonuclease,” as may be used herein, refers to the term of art generally known to the skilled artisan to refer to an enzyme that has at least the activity of cleaving nucleotides from the end of a nucleic acid (e.g., polynucleotide, oligonucleotide). In some embodiments, an exonuclease will cleave the nucleotides one at a time. An exonuclease may cleave nucleotides in either direction (e.g., from either the 5' or 3' end) of a nucleic acid. When describing such activity, often the notation is shown to be 5' to 3' exonuclease activity, when referring to an exonuclease that cleaves nucleotides starting from the 5' end of a nucleic acid (e.g., the 5' nucleotide that is distal to the 3' end) or 3' to 5' exonuclease activity, when referring to an exonuclease that cleaves nucleotides starting from the 3' end of a nucleic acid (e.g., the 3' nucleotide that is distal to the 5' end). In some embodiments, an exonuclease has 5' to 3' exonuclease activity. In some embodiments, the exonuclease can be Exo VII.

[0061] The terms “complementary” and “complementarity,” as may be used interchangeably herein, refer a property of a nucleotide (e.g., A, C, G, T, U) in a nucleic acid (e.g., RNA, DNA) in a strand (e.g., oligonucleotide) to pair with another particular nucleotide in a nucleic acid strand of the opposite orientation (e.g., strands running parallel, but in the reverse direction (z.e., 5'-3 ' aligns with 3 '-5', and 3'-5' with 5'-3 ')) (z.e., Watson-Crick base-pairing rules). With respect to deoxyribonucleic acids (DNA), the base pairings that are complementary are adenine (A) and thymine (T) (e.g., A with T, T with A) and guanine (G) and cytosine (C) (e.g., G with C, C with G) and with respect to ribonucleic acid (RNA), the base pairings that are complementary are A and uracil (U) (e.g., A with U, U with A) and G and C (e.g., G with C, C with G). This occurs because of the ability of each base pair to form an equivalent number of hydrogen bonds with its complementary base (e.g., A-T / U, T / U-A, C-G, G-C), for example the bond between guanine and cytosine shares three hydrogen bonds compared to the A-T / U bond which always shares two hydrogen bonds.194930-8958-8993.1114203-4000 (BI- 11308)

[0062] When every base in at least one strand of a pair of nucleic acids is found opposite its complementary base pair, such strand is considered fully complementary to its sequence in the other strand. When one or more bases of such a strand is found in a position where it is opposite any other base excepting its complementary base pair, that base is considered “mismatched” and the strand is considered partially complementary. Accordingly, strands can be varying degrees of partially complementary, until no bases align, at which point they are non- complementary. Other non-standard nucleotides (e.g., 5 -methylcytosine, 5- hydroxymethylcytosine) are known in the art and their properties and complementarity will be readily apparent to the skilled artisan.

[0063] The term “adapter ligation,” as may be used herein, refers to the term as known to the skilled artisan to generally refer to the process of attaching (e.g., ligating) known sequences of nucleotides e.g., nucleic acids, oligonucleotides, e.g., adapters) to one or more ends of one or more nucleic acids (e.g., DNA fragments, complementary strands of DNA). Often adapters contain specific sequences that are complementary to the nucleic acid fragments they are intended to attach to, for example, without limitation in the event nucleic acids are dA-tailed, an adapter may have a “T” overhang, wherein the “T” refers to a nucleotide comprising a thymine nucleobase. The T overhang is complementary to the dA-tail, thus facilitating ligation.

[0064] The term “polymerase,” as may be used herein, is a term of art known to the skilled artisan to refer generally to an enzyme which aids in, or synthesizes nucleic acids (e.g., DNA polymerase, RNA polymerase) and polymers. There are known a multitude of polymerases, for example, without limitation and which are all contemplated herein, DNA polymerase I (Pol gamma, Pol theta, Pol nu), DNA polymerase II (Pol alpha, Pol delta, Pol epsilon, Pol zeta), DNA polymerase III holoenzyme, DNA polymerase IV (DinB) (SOS repair polymerase, Pol beta, Pol lambda, Pol mu), DNA polymerase V (SOS polymerase, Pol eta, Pol iota, Pol kappa), Reverse transcriptase, and RNA polymerase (RNA Pol I, RNA Pol II, RNA Pol III, T7 RNA Pol, RNA replicase, Primase). Additionally, as is further contemplated, are polymerases from bacterium (e.g., Thermus aquaticus). For example, Taq from Thermus aquaticus is a common DNA polymerase used in polymerase chain reactions (PCR). In some embodiments, a polymerase is a Taq polymerase. In some embodiments, a polymerase lacks 3' to 5' exonuclease activity. In some embodiments, a polymerase is a KI enow fragment. In some embodiments, a polymerase is a KI enow fragment lacking 3' to 5' 204930-8958-8993.1114203-4000 (BI- 11308) exonuclease activity. In some embodiments, a polymerase is a human variant of any of the polymerases described herein.

[0065] The term “unique molecular identifier (UMI),” refers to a short oligonucleotide molecular barcode that provides error correction and increased accuracy during sequencing.

[0066] The terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides,” as may be used interchangeably herein, refer to a string of at least two, nucleobase-sugar-phosphate combinations (e.g., nucleotides) and includes, among others, single stranded and double stranded DNA, DNA that is a mixture of single stranded and double stranded regions, single stranded and double stranded RNA, and RNA that is mixture of single stranded and double stranded regions, hybrid molecules comprising DNA and RNA that may be single stranded or, more typically, double stranded or a mixture of single stranded and double stranded regions. In addition, the terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides,” (e.g., nucleic acid, et al.) as used herein can refer to triple stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions can be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple helical region often referred to as an oligonucleotide.

[0067] The terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides,” (e.g., nucleic acid, et al.) also encompass such chemically, enzymatically, or metabolically modified forms of nucleic acids, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells. For instance, the terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides,” (e.g., nucleic acid, et al.) as used herein can include DNA or RNA as described herein that contain one or more modified bases. The nucleic acids may also include natural nucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2 aminoadenosine, 2 thiothymidine, inosine, pyrrolo pyrimidine, 3 methyl adenosine, 5 methylcytidine, C5 bromouridine, C5 fluorouridine, C5 iodouridine, C5 propynyl uridine, C5 propynyl cytidine, C5 methylcytidine, 7 deazaadenosine, 7 deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, 0(6) methylguanine, 4 acetylcytidine, 5 (carboxyhydroxymethyl)uridine, dihydrouridine, methylpseudouridine, 1 214930-8958-8993.1114203-4000 (BI- 11308) methyl adenosine, 1 methyl guanosine, N6 methyl adenosine, and 2 thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2' fluororibose, ribose, 2' deoxyribose, 2' O methylcytidine, arabinose, and hexose), or modified phosphate groups (e.g., phosphorothioates and 5' N phosphoramidite linkages). Thus, DNA or RNA including unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acids as the term is used herein. The terms “nucleic acid,” “nucleotide sequence,” “polynucleotide,” “oligonucleotide,” and “polymer of nucleotides,” (e.g., nucleic acid, et al.) also includes peptide nucleic acids (PNAs), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids. Natural nucleic acids have a phosphate backbone. Artificial nucleic acids can contain other types of backbones, but contain the same bases. Thus, DNA or RNA with backbones modified for stability or for other reasons are nucleic acids as that term is intended herein.

[0068] As used herein, 5 -hydroxy cytosine refers to an oxidized form of cytosine, having the following chemical formula:

[0069] As used herein, 5-carboxy cytosine refers to a cytosine analog having the following chemical formula:

[0070] As used herein, 5-propargylaminocytosine refers to a cytosine analog having the following chemical formula:224930-8958-8993.1114203-4000 (BI- 11308)

[0071] The term “nucleobase,” as used herein, refers to a nitrogenous base, which is a nitrogen-containing biological compound that forms a component of a nucleoside, which is itself a component of a nucleotide. The nucleobases (also referred to herein as simply a base), are one of the basic building blocks of nucleic acids (e.g., DNA, RNA) as they possess the ability to form base pairs and to stack one upon another forming the long-chain helical structures. There are five canonical nucleobases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), with A, C, G, and T being found in DNA and A, C, G, and U being found in RNA.

[0072] The term “nucleoside,” as may be used herein, refers to glycosylamines (e.g., N- glycosides) that are generally known to be nucleotides without a phosphate group. A nucleoside consists of a nucleobase (e.g., a nitrogenous base) and a five-carbon sugar (e.g., pentose). The five-carbon sugar can be either ribose or deoxyribose. Nucleosides are the biochemical precursors of nucleotides, which are the constituent components of RNA and DNA. Examples of nucleosides include cytidine (C), uridine (U), adenosine (A), guanosine (G), thymidine (T), and inosine (I), but includes variants (e.g., modified or synthetic nucleosides, nucleosides containing modified or synthetic nucleobases).

[0073] The term “nucleotide,” as may be used herein is a term of art known to the skilled artisan to generally refer to those compositions comprising a nucleobase, sugar, and phosphate (e.g., a nucleoside and a phosphate) (which compositions (e.g., nucleotides) are separated into purines and pyrimidines). Nucleotides are components of nucleic acids that can be copied using a polymerase. Nucleosides, cytidine (C), uridine (U), adenosine (A), guanosine (G), thymidine (T), and inosine (I), along with a phosphate group, represent the canonical nucleotides, and may be referred to in DNA form (e.g., with a deoxyribose) as dATP, dGTP, dCTP, and dTTP when referring to individual nucleotides used in a synthesis reaction (e.g., nucleotide with 3 phosphate groups (e.g., “tri-phosphate”)). Two of the phosphate groups may be hydrolyzed to yield a monophosphate nucleotide for use in the234930-8958-8993.1114203-4000 (BI- 11308) polymerization of a nucleic acid. Generally, dATP, dGTP, dCTP, and dTTP may be referred to as dNTPs, wherein “N” represents the ambiguity as to the nature of the nucleoside. Thus, a mixture of dNTPs may include a concentration of all or some of each. Nucleotides contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been damaged (e.g., bases that have oxidized, methylated, acylated, deadenylated, etc.). The term is well-known in the art and will be readily appreciated by the skilled artisan.

[0074] As used herein, a “mammal,” refers to any animal constituting the class Mammalia (e.g., a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Marmoset, Macaque)). In some embodiments, a mammal is a human.

[0075] The term “subject,” as used herein, refers to any organism in need of treatment or diagnosis using the subject matter herein. For example, without limitation, subjects may include mammals and non-mammals.

[0076] The term “mutation,” as may be used herein, refers to a change, alteration, or modification to a nucleotide in a nucleic acid as compared to its wild-type sequence. For example, without limitation, mutations may include substitutions, insertions, deletions, or any combination of the same. Mutations, which as described hereinabove, are regions (e.g., sections, portions, nucleobases, nucleosides, nucleotides) of a given nucleic acid (e.g., DNA, RNA) which differ as compared to their wild-type nucleic acid, will most often be reflected in each strand of a nucleic acid. That is to say that, when a mutation is present in a sample it and its complement will be observed in each strand of the nucleic acid when sequenced. This presents a problem however, when considering that a sample may contain single-stranded portions (e.g., gaps, overhangs), or areas which may instigate strand resynthesis (e.g., nicks). This problem presents because if a damaged base is present in such single-stranded region, or other region which is resynthesized, a damaged base may instruct the synthesis of its complementary strand to include a base which was not originally present in the nucleic acid from which the sample was generated (because damaged bases can affect non-canonical base pairings). The same could happen if one strand contains mismatched bases. In such instances, the mismatch will show a paired match in the re-synthesized complement instead of its native mismatched base. When this happens, a sequencing of both strands will read a mutation in each of the strands, thus showing a mutation, however, this mutation may not be a true reflection of the original nucleic acid. Such mutations are termed “false mutations,”244930-8958-8993.1114203-4000 (BI- 11308) herein. False mutations are mutations which result from the resynthesis of complementary strands of nucleic acid, which do not represent the original (e.g., native, wild-type) complementary strand of nucleic acid from which the sample was obtained.

[0077] The term “contacted,” as may be used herein, is used to describe the exposure of one substance (e.g., enzyme, reagent, dNTP) to another substance (e.g., sample, mixture), in an amount and with the intention that the two substances interact in a way to effectuate activity of one of the substances on, or to interact with, the other (e.g., an enzyme acting upon a sample). The term is not to be construed to require physical contact between the two substances, but further does not prohibit physical contact either. For example, proximity may be sufficient to affect the interaction and / or activity of the substances with one another. In some embodiments, contact is accomplished by introducing the substances into the same container (e.g., reaction vessel). In some embodiments, contact is accomplished by introducing the substances into the same reaction vessel. In some embodiments, contact is accomplished by introducing substance A (e.g., reagent, dNTP, enzyme, etc.) into a reaction vessel, which either contains substance B (e.g., sample), to which substance B is simultaneously introduced, or to which substance B is later introduced. In some embodiments, contact is accomplished when substances physically touch one another (e.g., interact physically). In some embodiments, contact is accomplished when substances chemically interact with one another. In some embodiments, contact is accomplished when substances enzymatically interact with one another. In some embodiments, contact is accomplished when substances are proximal to one another.

[0078] As used herein, “TET-assisted pyridine borane sequencing” or “TAPS” refers to a bi sulfite-free, base-resolution DNA methylation sequencing method that directly converts methylated cytosines into thymine-equivalent signals for sequencing. Specifically, TAPS employs Ten-Eleven Translocation (TET) family dioxygenases (e.g. TET1 / TET2 / TET3 enzymes) to oxidize 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA to 5-carboxylcytosine (5caC). The oxidized DNA is then treated with pyridine borane, a mild borane reducing agent, which selectively reduces 5caC (and any 5-formylcytosine, 5fC) to dihydrouracil (DHU). During subsequent PCR amplification and sequencing, DHU is read as a thymine (T), whereas unmodified cytosine (which is not altered by this process) remains read as cytosine (C). Accordingly, originally methylated cytosines (5mC or 5hmC) are detected as C^T transitions in the sequencing data, while unmethylated cytosines are 254930-8958-8993.1114203-4000 (BI- 11308) retained as C, enabling direct, quantitative mapping of DNA methylation at single-base resolution.

[0079] As used herein, “TET-assisted pyridine borane treatment” refers to using Ten-Eleven Translocation (TET) family dioxygenases (e.g. TET1 / TET2 / TET3 enzymes) to oxidize 5- methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in DNA to 5-carboxylcytosine (5caC) and treating the oxidized DNA with pyridine borane, which selectively reduces 5caC (and any 5-formylcytosine, 5fC) to dihydrouracil (DHU).MODES FOR CARRYING OUT THE DISCLOSUREMethylation-Specific CODEC Sequencing

[0080] Disclosed herein is “Methylation-specific CODEC Sequence” (aka., “Methyl- CODEC” or “Methylation-specific CDS”), a method that can be used for performing improved mutation and methylation sequencing of DNA samples.

[0081] The methods disclosed herein enable extraction of information about DNA methylation, as well as mutation, from the interrogated DNA sample. There has been increasing interest in extracting DNA methylation information from clinical samples in several fields, including cancer. For example, extracting cancer-specific fingerprints of methylated DNA from liquid biopsies have recently led to approaches for early detection of multiple cancers.

[0082] To enable extraction of methylation information from a DNA sample and to perform methylation-sensitive sequencing, in most cases a chemical or enzymatic de-amination step is applied to the sample prior to performing sample amplification. This step enables selective conversion of un-methylated cytosines to uracils, while methylated cytosines remain unchanged. Following this step, amplification of the sample with standard deoxynucleotides (dNTPs) results in conversion of unmethylated cytosines to thymidines, while methylated cytosines become cytosines. Subsequent sequencing enables one to infer which cytosines in the original sample were methylated or un-methylated.

[0083] To enable CODEC to retain and report DNA methylation information, the following protocol has been developed.

[0084] The protocol involves the following steps:264930-8958-8993.1114203-4000 (BI- 11308)(a) Synthesize the CODEC adaptor-complex to contain 5 -hydroxy cytosine (aka., “5- hydroxy-dCTP”), 5-carboxycytosine (“5-carboxy-dCTP”) or 5- propargylaminocytosine (“5-propargylamino-dCTP”) instead of regular, unmethylated cytosine. In this way, protected cytosines are refractory to subsequent de-amination and can be amplified with the described primers. Applicant surprisingly found that 5- hydroxycytosine, 5-carboxycytosine and / or 5 -propargylaminocytosine are superior over other deamination-resistant cytosine analogues and provides almost perfect protection from deamination in the following steps.(b) Following ligation of the modified CODEC adaptors (“methyl-CODEC adaptors”), a copy of the opposite DNA strand is generated using 5 -hydroxy cytosine, 5-carboxycytosine or 5-propargylaminocytosine (or a combination / mix of 5- hydroxycytosine, 5-carboxycytosine and 5-propargylaminocytosine) nucleotides along with standard dATP, dGTP and dTTP nucleotides. In this way, the copy of the original strand is always protected at cytosine positions and becomes refractory to subsequent de-amination. In some embodiments, TET2 and BGT enzymes are used to convert a methyl-dCTP or a hydroxy-methyl-dCTP nucleotide into a carboxy-dCTP or a glucosyl-hydroxy-methyl-dCTP nucleotide which are resistant to conversion due to structural modifications that prevent recognition by the APOB EC enzyme.(c) Conduct a deamination step to convert un-methylated cytosines to uracils in the linearized double-stranded DNA molecule. The deamination of cytosines can be performed with one of several approaches, such as the standard bisulfite-de- amination; or enzymatic deamination using enzymatic methyl-seq (EM-seq) technique, which uses enzymatic steps by TET2 and AP0BEC2 enzymes to differentiate between methylated and un-methylated cytosine.(d) Following the deamination step, amplification using the CODEC adaptor primers is applied.(e) Conduct duplex sequencing. See, FIGS. 1A-1B.

[0085] By generating a copy of the original DNA strand, which is insensitive to deamination, while retaining the methylation / unmethylation information in the original strand, it is now possible to infer methylation sequencing information as well as mutation information by comparison of the sequencing results obtained from the two strands. For example, if a C is present in the copied strand and a C is also present in the original strand, 274930-8958-8993.1114203-4000 (BI- 11308) one can infer that this sequence position was methylated in the original sample. While if there is a T in the original strand then this sequence position was probably un-methylated in the original sample (FIG. IB). (To exclude the possibility that the T appears because of a sequencing error, additional analysis may need to be done. For example, one can observe the nucleotide context in which this T appears on the original strand. If additional Ts also appear nearby, then the T likely represents an unmethylated C; if it is an isolated T then there is a good probability the T is a result of sequencing error).

[0086] Creating a methylation-insensitive second DNA strand copy in the CODEC protocol along with the original methylation-sensitive DNA strand has several advantages and possible practical applications.

[0087] For example, since the copied DNA strand by preserving the cytosines at all positions is not ‘cytosine poor’ it can be used for unambiguous alignment during sequencing, thus enabling enhanced mapping of sequence reads. Also, the methylation insensitive strand can be used for improved hybrid capture since DNA strands with multiple un-methylated sites are often problematic for hybrid capture. Also, it can be used to improve proof-reading of sequence calls and for general duplex sequencing correction on other bases. Finally, it can be used to create libraries that preserve both mutation and methylation information for subsequent combined ‘methyl - mutation’ sequencing using a single DNA sample (instead of using two separate samples, one for mutation and another for methylation analysis).

[0088] Synthesizing the opposite strand using 5 -hydroxy cytosine, 5-carboxycytosine or 5- propargylaminocytosine and followed by de-amination of un-methylated cytosines has some advantages such as: 1) unambiguous alignment, since all 4 bases are present, this preserves sequence diversity and enhances the ability to align sequences, 2) improved hybrid capture, even for un-methylated sites which are often a problem, 3) improved proof-reading of sequence calls on the methylation-sensitive portion and for general duplex sequencing correction on other bases, and 4) creating a library for subsequent combined methyl — mutation sequencing using a single DNA sample (instead of two separate samples).

[0089] An aspect of the disclosure is directed to a method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having:284930-8958-8993.1114203-4000 (BI- 11308) a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second singlestrand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and294930-8958-8993.1114203-4000 (BI- 11308) wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5 -carboxy -dCTP or 5-propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

[0090] In some embodiments, the reverse complement of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the originalDNA duplex contain 5-hydroxy cytosine, 5-carboxy cytosine or 5- propargylaminocytosine rather than unmethylated cytosine.

[0091] In some embodiments, there is a linker sequence between the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex.

[0092] In some embodiments, there is a linker sequence between the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex.

[0093] In some embodiments, the method further comprises:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0094] In some embodiments, TET2 and / or BGT enzymes are used to convert a methyl- dCTP or a hydroxy-methyl-dCTP nucleotide into a carboxy-dCTP or a glucosyl-hydroxy- methyl-dCTP nucleotide which are resistant to conversion due to structural modifications that prevent recognition by the APOBEC enzyme.

[0095] Another aspect of the disclosure provides the ability to detect a mutation (e.g., a C to T, a T to C, an A to G or a G to A) in addition to the DNA methylation status. Among the mutations, C to T mutations are particularly difficult to detect and verify using prior art methods because, in the prior art methods, when a T is detected in place of a C, one304930-8958-8993.1114203-4000 (BI- 11308) cannot know whether it is a converted unmethylated C or a sequence read error. Methyl- CODEC solves this problem because there is redundancy in the sequencing, wherever there is a C on one strand, there is a G in the corresponding strand and vice versa, which can be used to correctly call C to T mutations confidently. In the case of a C to T mutation the corresponding strand will have a G to A mutation, which will confirm that it is a bona fide mutation, not a sequencing error. The present method allows T to C, an A to G or a G to A mutations in the same manner.

[0096] In some embodiments, the method further comprises detecting a C to T, a T to C, an A to G or a G to A mutation by comparing the sequence of the top strand to the sequence of the first linearized double-stranded DNA molecule, the bottom strand of the first linearized double-stranded DNA molecule, the sequence of the top strand to the sequence of the second linearized double-stranded DNA molecule, and the bottom strand of the second linearized double-stranded DNA molecule..

[0097] In some embodiments, the first duplex is 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 28 bp, 29 bp, 30 bp, 31 bp, 32 bp, 33 bp, 34 bp, 35 bp, 36 bp, 37 bp, 38 bp, 39 bp, or 40 bp in length. In some embodiments, the first duplex has hybridization free energy of about -10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, or about -35 kcal / mol. In some embodiments, the second duplex is 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, or 25 bp in length. In some embodiments, the second duplex has hybridization free energy of about -10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, or about -35 kcal / mol. In some embodiments, the third duplex is 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, or 25 bp in length. In some embodiments, the third duplex has hybridization free energy of about -10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, or about -35 kcal / mol.

[0098] In some embodiments, the single stranded regions are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.314930-8958-8993.1114203-4000 (BI- 11308)

[0099] In some embodiments, the first oligonucleotide comprises a free 5’ end comprising a first next-generation sequencing (NGS) flow cell binding region. In some embodiments, the third oligonucleotide comprises a free 5’ end comprising a second next-generation sequencing (NGS) flow cell binding region. In some embodiments, the first duplex has a first free 5’ end and the second duplex has a second free 5’ end. In some embodiments, the third duplex comprises a free 5’ end on each strand of the duplex. In some embodiments, the first and second 3’ ends can prime DNA synthesis by a DNA-dependent DNA polymerase.

[0100] In some embodiments, the linearized double-stranded DNA molecule configured for next generation sequencing and having first and second ends comprises the following structure: first end - [a first next generation flow cell adapter] - [a first duplex region comprising the original top strand paired with a copy of the original bottom strand] - [a second duplex region comprising the central portion of the next-generation sequencing adapter] - [a third duplex region comprising a copy of the original top strand paired with the original bottom strand] - [a second next generation flow cell adapter] - second end.

[0101] In some embodiments, the first next generation flow cell adapter is an Illumina P5 or P7 adapter sequence. In some embodiments, the second next generation flow cell adapter is an Illumina P5 or P7 adapter sequence. In some embodiments, the second duplex region comprises first and second read primer binding sites, wherein each first and second read primer site is further associated with a unique molecule identifier (UMI) and a sample index sequence.

[0102] In some embodiments, the first and second read primer binding sites are orientated outwardly towards the ends of the linearized double-stranded DNA molecule. In some embodiments, a first read primer can be used to obtain a sequence read comprising a UMI, sample index, and the original top strand, or portion thereof, of the sample DNA duplex to be sequenced. In some embodiments, a second read primer can be used to obtain a sequence read comprising a UMI, sample index, and the original bottom strand, or portion thereof, of the sample DNA duplex to be sequenced. In some embodiments, the method is used in place of a commercial next-generation library construction kit. In some embodiments, the ligating of the first step described above comprises adding ligase. In some embodiments, the synthesizing of the second step described above comprises adding a DNA polymerase. In some embodiments, the polymerase has a strand-displacement activity. In some324930-8958-8993.1114203-4000 (BI- 11308) embodiments, the methods further comprise the step of obtaining the sequence of the original top and original bottom strands by conducting next generation sequencing with the first and second read primers.

[0103] Another aspect of the present disclosure relates to a linearized double-stranded DNA molecule configured for next generation sequencing obtained by the method described herein, wherein the linearized double-stranded DNA molecule comprises first and second ends and has the following structure: first end - [a first next generation flow cell adapter] - [a first duplex region comprising the original top strand paired with a copy of the original bottom strand] - [a second duplex region comprising the central portion of the next-generation sequencing adapter] - [a third duplex region comprising a copy of the original top strand paired with the original bottom strand] - [a second next generation flow cell adapter] - second end.

[0104] In some embodiments, the first next generation flow cell adapter is an Illumina P5 or P7 adapter sequence. In some embodiments, the second next generation flow cell adapter is an Illumina P5 or P7 adapter sequence. In some embodiments, the second duplex region comprises first and second read primer binding sites, wherein each first and second read primer site is further associated with a unique molecule identifier (UMI) and a sample index sequence. In some embodiments, the first and second read primer binding sites are orientated outwardly towards the ends of the linearized doublestranded DNA molecule. In some embodiments, a first read primer can be used to obtain a sequence read comprising a UMI, sample index, and the original top strand, or portion thereof, of the sample DNA duplex to be sequenced. In some embodiments, a second read primer can be used to obtain a sequence read comprising a UMI, sample index, and the original bottom strand, or portion thereof, of the sample DNA duplex to be sequenced.

[0105] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas. In some embodiments, the DNA sample comprises cfDNA. In some embodiments, the DNA sample is from a diseased subject.

[0106] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.334930-8958-8993.1114203-4000 (BI- 11308)

[0107] In some embodiments, the biological sample is blood. In some embodiments, the biological sample is a sample of tissue from liver, kidney, brain, heart, skin, lung, colon, or pancreas. In some embodiments, the biological sample is a sample of a diseased tissue from liver, kidney, brain, heart, skin, lung, colon, or pancreas. In some embodiments, the diseased tissue is a proliferative disease. In some embodiments, the diseased tissue is a tumor. In some embodiments, the sequencing error rate is similar to a control based on Duplex Sequencing, but wherein the number of reads required is decreased by at least 100-fold.

[0108] Another aspect of the disclosure is directed to methods that convert methylated C to T. Such methods include TET-assisted pyridine borane treatment (from TET-assisted pyridine borane sequencing “TAPS” as defined in US12410467B2, which is incorporated herein in its entirety) as well as enzymatic approaches that achieve methylated C to T conversion in a single step (CpG-specific deaminases as described in WO2023097226 A2, and Vaisvila, Romualdas, et al. (BioRxiv (2023): 2023-06) which are incorporated herein in their entireties). TET-assisted pyridine borane treatment and CpG-specific deaminases convert methylated C to U and to T when it is amplified, the key differences are that (1) one would use a standard (not methylated) NGS adapter and (2) one would not use conversion resistant dCTPs during the strand displacing extension. Otherwise, the result would be the same where one could determine methylation and mutations from both strands that are linked and sequenced together.

[0109] Such methods that convert methylated C to T comprise preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter comprises unmethylated cytosine;344930-8958-8993.1114203-4000 (BI- 11308)(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second single-strand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double-stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized doublestranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and standard dATP, dGTP, dCTP, and dTTP deoxynucleotides.

[0110] In some embodiments, the method further comprises:(d) deaminating methylated cytosines to uracil or dihydro-uracil;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double-stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in the sequencing step.[OHl] In some embodiments, methylated cytosines are deaminated by a CpG specific deaminase selected from CseDaOl, MGYPDaO6, LbDaO2, CrDaOl, MGYPDa829, LbsDaOl (as described in WO2023097226A2 and Vaisvila, Romualdas, et al. (BioRxiv (2023): 2023- 06) which are incorporated herein in their entireties), or combinations thereof.

[0112] In some embodiments, methylated cytosines are deaminated by a TET-assisted pyridine borane treatment (as defined in US12410467B2, which is incorporated herein in its entirety).

[0113] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.354930-8958-8993.1114203-4000 (BI- 11308)

[0114] In some embodiments, the DNA sample comprises cell-free DNA (cfDNA).

[0115] In some embodiments, the DNA sample is from a diseased subject.

[0116] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.

[0117] An aspect of the disclosure is directed to a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter comprising at least ten (10) regions (R01 -R10) in the following configuration:wherein ' — ' represents bonding, wherein R01 , R02, and R03 comprise a first oligonucleotide, wherein R04 and R05 comprise a second oligonucleotide, wherein R06 and R07 comprise a third oligonucleotide, wherein R08, R09, and R10 comprise a fourth oligonucleotide, wherein R01 and R06 are annealed to one another, wherein R03 and R08 are annealed to one another, wherein R05 and R10 are annealed to one another, wherein R02 and R07 are not annealed to one another, and wherein R04 and R09 are not annealed to one another; wherein R02 comprises a single- stranded linker, afirst unique molecular identifier (UMI), and a first read primer site, and364930-8958-8993.1114203-4000 (BI- 11308) wherein R09 comprises a single-stranded linker, a second UMI, and a second read primer site, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the sequencing adapter to a dsDNA duplex as follows: ligating the 5' end of R01 to the 3' end of a first strand of the dsDNA duplex; ligating the 3' end of R05 to the 5' end of the first strand of the dsDNA duplex; ligating the 5' end of RIO to the 3' end of a second strand of the dsDNA duplex; and ligating the 3' end of R06 to the 5' end of the second strand of the dsDNA duplex; thereby forming a partially circularized DNA molecule comprising the target DNA molecule and the sequencing adapter;(c) synthesizing first and second single-strand DNA molecules by extending the free 3' end of R03 and 3' end of R08 on the sequencing adapter each using the opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with aDNA polymerase and 5-hydroxy-dCTP, 5 -carboxy -dCTP or 5-propargylamino- dCTP along with standard dATP, dGTP and dTTP deoxynucleotide.

[0118] In some embodiments, the method further comprises:(d) deaminating unmethylated cytosines to uracils;374930-8958-8993.1114203-4000 (BI- 11308)(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e)

[0119] In some embodiments, the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double- stranded DNA molecule.

[0120] In some embodiments:(1) R01 comprises a first adapter;(2) R03 comprises a first sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase;(3) R04 comprises a free 5' end comprising a first next-generation sequencing (NGS) adapter sequence;(4) R05 comprises a third adapter and a first sample index;(5) R06 comprises a second adapter and a second sample index;(6) R07 comprises a free 5' end comprising a second next-generation sequencing (NGS) adapter sequence;(7) R08 comprises a second sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; and / or(8) RIO comprises a fourth adapter, optionally wherein the first sequence and the second sequence, further comprise the same or different primer binding sites, and optionally wherein the first primer site and the second primer site are oriented to initiate sequencing by addition in opposing directions.384930-8958-8993.1114203-4000 (BI- 11308)

[0121] In some embodiments, the ligating of step (b) comprises adding ligase.

[0122] In some embodiments, the synthesizing of step (c) comprises contacting the circular double-stranded DNA intermediate with a polymerase.

[0123] In some embodiments, the polymerase is a DNA-dependent DNA polymerase.

[0124] In some embodiments, the polymerase has a strand- displacement activity.

[0125] In some embodiments, the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

[0126] In some embodiments, the DNA sample comprises cfDNA.

[0127] In some embodiments, the DNA sample is from a diseased subject.

[0128] In some embodiments, the diseased subject suffers from a proliferative disease or a tumor.Methyl-CODEC Adapters, Library Preparation, and Sequencing

[0129] In various aspects, the disclosure provides compositions required for and / or produced by Methyl-CODEC sequencing, including adapters (referred to herein in various embodiments as “Methyl-CODEC adapters”), circularized intermediates each comprising a Methyl-CODEC adapter ligated to both ends of a DNA fragment to be sequenced (referred to herein in various embodiments as “Methyl-CODEC circularized intermediates”), and linearized double-stranded products comprising concatenated top and bottom strands of the single DNA fragments to be sequenced (referred to herein in various embodiments as “the Methyl-CODEC library” or individually as “Methyl-CODEC library members”). In some embodiments, the Methyl-CODEC adapter is modified to contain a deamination-resistant cytosine in place of unmethylated cytosine. In some embodiments, the Methyl-CODEC adapter is modified to contain a 5-hydroxy cytosine, 5 -carboxy cytosine or 5- propargylaminocytosine in place of unmethylated cytosine. In various embodiments, the Methyl-CODEC adapter includes NGS adapters for NGS workflow (e.g., cluster amplification on NGS flow cell), sequencing read primer sites for reading both strands of a DNA fragment, and optionally one or more sample indices and one or more unique molecular identifiers (UMIs).394930-8958-8993.1114203-4000 (BI- 11308)

[0130] According to the methods disclosed herein, two products are produced during the preparation of the Methyl-CODEC library from an original DNA duplex. In some instances, one strand of the linearized double-stranded DNA molecule comprises the top strand of the original DNA duplex concatenated to the reverse complement sequence of the bottom strand of the original DNA duplex while the other stand of the linearized double-stranded DNA molecule will have the reverse complement sequence of the top strand of the original DNA duplex concatenated to the bottom strand of the original DNA duplex.

[0131] In some embodiments, a Methyl-CODEC adapter complex consists of four hybridized oligonucleotides, which include every element required for both concatenation and adapter attachment. In some embodiments, the Methyl-CODEC adapter complex comprises at least ten regions (RO 1 -RIO) in the following configuration:

[0132] In some embodiments, ‘ - ’ represents bonding. In some embodiments, R01, R02, and R03 comprise the first oligonucleotide, R04 and R05 comprise the second oligonucleotide, R06 and R07 comprise the third oligonucleotide, R08, R09, R10 comprise the fourth oligonucleotide. In some embodiments, R01 and R06 are annealed to one another, R03 and R08 are annealed to one another, R05 and R10 are annealed to one another, R02 and R07 are not annealed to one another, and R04 and R09 are not annealed to one another.

[0133] In some embodiments, a CODEC adapter complex is ligated (adapter ligation) with one end of a target duplex (target DNA molecule), followed by ligation between the other ends to produce circularized product.

[0134] In some embodiments, R01 comprises a first concatenated duplex sequencing (CODEC) adapter; R02 comprises a single-stranded linker, first unique molecular identifier (UMI), and a first read primer site; R03 comprises a first sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; R04 comprises a404930-8958-8993.1114203-4000 (BI- 11308) free 5' end comprising a first next-generation sequencing (NGS) adapter sequence; R05 comprises a third CODEC adapter and a first sample index; R06 comprises a second CODEC adapter and a second sample index; R07 comprises a free 5' end comprising a second nextgeneration sequencing (NGS) adapter sequence; R08 comprises a second sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; R09 comprises a single-stranded linker, a second UMI, and a second read primer site; and / or RIO comprises a fourth CODEC adapter.

[0135] In some embodiments, a polymerase is a Taq polymerase. In some embodiments, a polymerase lacks 3' to 5' exonuclease activity. In some embodiments, a polymerase is a KI enow fragment. In some embodiments, a polymerase is a KI enow fragment lacking 3' to 5' exonuclease activity. In some embodiments, a polymerase is a human variant of any of the polymerases described herein.

[0136] In various embodiments, the four Methyl-CODEC adapter oligonucleotides may be annealed before (i.e., pre-annealed) ligation with DNA fragments to be sequenced. In various other embodiments, the four Methyl-CODEC adapter oligonucleotides may be annealed during or contemporaneous to the ligation step.

[0137] The advantage of pre-annealing four oligonucleotides before ligation is that both ends always get different adapters, whereas ligation without hybridization results in 50% of the target ligating to the same adapter on both sides, which cannot be circularized. In some embodiments, a single A / T overhang is added at ligation sites to improve the yield. In some embodiments, DNA blunt ends or DNA sticky ends are added. In some embodiments, singlestranded DNA regions are incorporated into the Methyl-CODEC complex to add flexibility for circularization.

[0138] In some embodiments, the first sequence and second sequence, further comprise the same or different primer binding sites. In some embodiments, the first and second primer sites are oriented to initiate sequencing by addition in opposing directions. In some embodiments, the first and second UMI are distinct.

[0139] In some embodiments, R01 comprises between 12 and 30 nucleotides, R02 comprises between 14 and 75 nucleotides, R03 comprises between 12 and 99 nucleotides, R04 comprises between 20 and 49 nucleotides, R05 comprises between 12 and 30 nucleotides, R06 comprises between 12 and 30 nucleotides, R07 comprises between 20 and 49414930-8958-8993.1114203-4000 (BI- 11308) nucleotides, R08 comprises between 12 and 99 nucleotides, R09 comprises between 14 and 75 nucleotides, and / or RIO comprises between 12 and 30 nucleotides.

[0140] In some embodiments, R01 and R06 comprise a hybridization free energy of about - 10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, or about -35 kcal / mol; R03 and R08 comprise a hybridization free energy of about -10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, about -35 kcal / mol, about -40 kcal / mol, about -45 kcal / mol, about -50 kcal / mol, about -55 kcal / mol, about -60; and / or R05 and R10 comprise a hybridization free energy of about -10 kcal / mol, about -15 kcal / mol, about -20 kcal / mol, about -25 kcal / mol, about -30 kcal / mol, or about -35 kcal / mol.

[0141] In some embodiments, R01 and R06 each comprise the same number of nucleotides, optionally wherein R06 has a one nucleotide overhang to facilitate ligation; R03 and R08 each comprise the same number of nucleotides; and / or R05 and R10 each comprise the same number of nucleotides, optionally wherein R05 has a one nucleotide overhang to facilitate ligation.

[0142] In some embodiments, R01 and R06 comprise sequences with at least 90% complementarity; R03 and R08 comprise sequences with at least 90% complementarity; and / or R05 and R10 comprise sequences with at least 90% complementarity.

[0143] In some embodiments, each R01, R06, R05, and R10 comprise the same number of nucleotides, optionally wherein R06 and R05 each have a one nucleotide overhang to facilitate ligation.

[0144] In some embodiments, R01 comprises a first concatenated duplex sequencing (CODEC) adapter; R02 comprises a single-stranded linker; R03 comprises a 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; R04 comprises a first UMI; R05 comprises a third CODEC adapter; R06 comprises a second CODEC adapter; R07 comprises a second UMI; R08 comprises a 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; R09 comprises a single-stranded linker; and R10 comprises a fourth CODEC adapter.

[0145] In some embodiments, the 5' end of R01 is ligated to the 3' end of a first strand of a target DNA duplex; the 3' end of R05 is ligated to the 5' end of the first strand of the target DNA duplex; the 5' end of R10 is ligated to the 3' end of a second strand of the target DNA duplex; the 3' end of R06 is ligated to the 5' end of the second strand of the target DNA 424930-8958-8993.1114203-4000 (BI- 11308) duplex; forming a circularized DNA duplex or optionally a partially double-stranded circular DNA.

[0146] In some embodiments, the Methyl-CODEC adapter complex may be prepared for NGS and used for a research or clinical purpose (e.g., identification of a mutation in a subject, diagnosis of a disease). In some embodiments, a subject is mammalian. In some embodiments, a subject is non-mammalian. In some embodiments, a mammal is a human.

[0147] In some embodiments, there at least one mutation. In some embodiments, there are more than one mutation. In some embodiments, where there is more than one mutation, the mutations are distinct (e.g., not of the same type (e.g., substitutions, insertions, deletions)). In some embodiments, where there is more than one mutation, the mutations are the same (e.g., not of the same type (e.g., substitutions, insertions, deletions)). Additionally, in some embodiments, the mutations result in a frameshift.

[0148] In some embodiments, the method or preparation of the Methyl-CODEC adapter complex may be a method of preparing a double-stranded DNA molecule (dsDNA duplex) for use in next-generation sequencing (NGS) of a target DNA molecule, comprising ligating the complex of any one of claims 1-21 to the dsDNA duplex as follows: ligating the 5' end of R01 to the 3' end of a first strand of the dsDNA duplex; ligating the 3' end of R05 to the 5' end of the first strand of the dsDNA duplex; ligating the 5' end of RIO to the 3' end of a second strand of the dsDNA duplex; and ligating the 3' end of R06 to the 5' end of the second strand of the dsDNA duplex; thereby forming a circular double-stranded DNA intermediate comprising the target DNA molecule and the complex; extending a first DNA strand from the 3' end of R03; extending a second DNA strand from the 3' end of R08; and optionally annealing the first and second DNA strands to form a double-stranded DNA molecule for use in next-generation sequencing (NGS) of the target DNA molecule. In some embodiments, the double-stranded DNA molecule comprises two copies of the target DNA molecule. In some embodiments, the ligating step comprises adding ligase. In some embodiments, the synthesizing steps comprise contacting the circular double-stranded DNA intermediate with a polymerase.

[0149] In some embodiments, the polymerase is a DNA-dependent DNA polymerase. In some embodiments, wherein the polymerase has a strand-displacement activity. In some embodiments, the next-generation sequencing (NGS) is a short-read strategy. In some434930-8958-8993.1114203-4000 (BI- 11308) embodiments, the method comprises sequencing double-stranded DNA molecule by nextgeneration sequencing.

[0150] In some embodiments, the Methyl-CODEC adapter sequence can be integrated to Illumina NGS library construction workflow by making R05 and R06 Illumina adapters. Indices are attached to demultiplex samples that have been pooled for NGS.

[0151] In other embodiments, the Methyl-CODEC adapters described herein may include one or more modifications. Without limitation, the following represent modifications that may be used in connection with CODEC sequencing methods described herein:1. Long duplex with mismatch bubbles

[0152] This variant, shown in FIG. 8A, works the same as the basic version except it needs to be cleaved to separate Regions 4, 5, and 6 after ligation. With only two oligos initially, it would be easier to hold all the components together.2. Modular duplex with mismatch bubbles

[0153] This variant, shown in FIG. 8B, works the same as Variant 4 except it needs to be ligated first to assemble the intact adapters.3. Half adapter complexes

[0154] Pre-annealing all four oligos is not necessary for CODEC. Annealing them into two half adapter complexes followed by ligation will theoretically result in 50% with both Region 4 and 4’. Once such structure is formed, Region 4 and 4’ will eventually hybridize with each other at some point during ligation or strand displacing extension (FIG. 8C).4. UMI

[0155] Unique molecular identifiers (UMI) can be introduced at ligation sites as a part of Region 1 (FIG. 8D).444930-8958-8993.1114203-4000 (BI- 11308)5 A. Regions 2 and 3 as partial read primer binding sites

[0156] Although the main purpose of Regions 2 and 3 is adding flexibility for circularization, they can be repurposed to have other functions as well. FIG. 8E shows using them as partial read primer binding sites to read only correct products with Regions 2, 3, and 4.

[0157] This is because some byproducts have only a single insert just like conventional NGS samples, and utilizing Regions 2 and 3 prevents them from hybridizing with read primers. (FIG. 8E, “Single insert (byproduct)”).

[0158] However, both regular CODEC adapter and this variant 5A may suffer from having two sites in a strand where the 3 ’-end of a read primer can hybridize (FIG. 8E, “Dual Fluorescence”). This can cause two different primers to generate dual fluorescence, which complicates data analysis. The variant shown in FIG. 8F solves this issue.5B. Regions 2 and 3 as complete read primer binding sites

[0159] This variant addresses the dual fluorescence issue by moving read primer binding sites completely into Regions 2 and 3 (FIG. 8F). The read primers now do not hybridize with Region 1, so their 3 ’-end sequences are unique.

[0160] Another advantage of this version is the low cost of introducing UMI. Both regular NGS adapters and CODEC adapters variant 1 need it at the end of double-stranded adapter regions before ligation with a target fragment. If UMI is 3 bp long, 43 = 64 pairs of adapter oligos have to be synthesized and annealed separately to avoid any UMI mismatch, which is expensive in terms of money and time. This variant can place UMI in single-stranded Regions 2 and 3 to avoid this requirement. With mixed bases at UMI positions, any length of UMI can be synthesized in a single batch.

[0161] Because the new read primer binding regions do not overlap with Region 1, base diversity at each sequencing cycle will be low if every read enters Region 1 at the same time. This can be solved by mixing four oligos with different lengths of UMI or using the next variant.6. Region 1 as indices454930-8958-8993.1114203-4000 (BI- 11308)

[0162] An adapter complex does not necessarily have the same Region 1 on both sides; there can be independent Region la and lb (FIG. 8G). Combined with the variant 5B, this variant can use Region la and lb as sample indices, eliminating needs for indexed primers. This example directly attaches an index next to a target sequence to reduce cross-talk between samples known as index hopping.

[0163] Using Region 1 as indices can also address the base diversity issue mentioned earlier. When multiple indices collectively have all four bases at every position, a pooled NGS library will get perfect base diversity throughout Region 1.(1) Overcoming “mixed clusters” to achieve highly-accurate, direct-repeat sequencing

[0164] Although successful concatenation of two strands may look sufficient for highly accurate and affordable NGS, byproducts comprising one strand (herein, referred as singleinsert, SI) with the same adapter sequences on either end could form (FIG. 8J). The danger of this is two-fold: (1) if sequencing read primers are directed against the end adapter regions, forward and reverse reads from SI vs. CODEC molecules would be difficult to discern, and(2) considering the high error rate from SI library molecules (0.1-1%), misclassification of even just a small fraction of SI reads as CODEC reads could be detrimental.

[0165] It has been found here that SI byproducts can form by three major mechanisms: (A) Phi29 extension if adapter ligation is incomplete, i.e., if not all four phosphodiester bonds form (e.g., FIG. 8H), (B) PCR jumping in library amplification, considering the homology between the direct repeat sequences in the CODEC product, and (C) PCR jumping in bridge amplification on the flow cell (FIG. 8K). (A) and (B) can be mitigated in part by size selection prior to sequencing and requiring ‘evidence’ of the linker sequence, e.g., using long enough reads to detect it after the insert. However, neither are sufficient to address (C). Indeed, it has been discovered here that in bridge amplification of CODEC fragments, mixed clusters are formed comprising the original CODEC library molecule which seeded the cluster and SI byproducts generated from one or both of the direct repeat sequences (FIG. 8K). Considering the log-linear nature of bridge amplification from a single “seeding” library molecule, the proportions of (i) CODEC molecule, to (ii) SI byproduct of “top” strand, to (iii) SI byproduct of “bottom” strand, could be skewed over several orders of magnitude. When NGS read primers are directed against end adapter regions, mixed fluorescence occurs for (i)- (iii) but it becomes challenging to discern which bases were truly present in top versus 464930-8958-8993.1114203-4000 (BI- 11308) bottom strand of the original DNA duplex from which the CODEC library molecule was derived (FIG. 8L).

[0166] The solution here is to place read primer binding sites in the linker region such that only CODEC fragments are sequenced. Yet, by nature of the linking process, segments 1 / 1’ and lb / lb’ of the CODEC adapter (FIG. 8H) will be present in both CODEC and SI byproducts (see FIG. 8J). Thus, to further ensure that SI byproducts will not be read, the NGS read primer binding sites were placed in the positions indicated in FIG. 8J, which originate from segments 2 and 3 of the adapter. This also means that the early cycles of each sequencing read will start in the end segments; and to ensure that these cycles are not wasted, they are used to encode sample indices and unique molecular identifiers for each DNA fragment. This has other unique advantages such as to mitigate index hopping as described in the next section, and encoding base diversity to improve cluster recognition and chastity filtration on the sequencer. The single-stranded segments also increase the product yield by introducing flexibility to the circularization process, which is otherwise limited by rigidity of double-stranded DNA. Most importantly, this solves the “mixed cluster” problem by ensuring that only CODEC molecules are sequenced in each forward and reverse read pair (FIG. 8M).(2) Preventing the misassignment of CODEC reads to the wrong samples

[0167] Another important feature of CODEC is index hopping suppression to prevent sample misassignment. This is particularly important when seeking to rely upon single CODEC reads to achieve duplex sequencing accuracy, as even just a small fraction of reads which are improperly assigned to the wrong samples could introduce large numbers of errors. The limitations of conventional indexing are tagging indices away from inserts and not tagging until PCR, which is the final step of sample preparation. Because indices are commonly placed towards the 5’ end of primers which target homologous regions of adapters, residual primers could easily ‘swap’ onto new library molecules and change the samples to which they are assigned. (The same could happen with partly extended library molecules, by way of PCR jumping.) To address this, CODEC indices were placed within the adapter complex itself, which enables attaching indices right next to inserts as soon as adapter ligation. Because reading an index and an insert is now seamless with a single read primer, there is much less chance of crosstalk among molecules during sequencing. Also, because CODEC requires insert 1 to match insert 2, any PCR jumping which occurs in the insert or linker 474930-8958-8993.1114203-4000 (BI- 11308) regions would be evident as it would create intermolecular byproducts with different insert 1 and insert 2 sequences. Of note, sufficient diversity was incorporated among CODEC indices so as to ensure proper cluster generation and chastity filtration, given that indices are read in the early cycles of sequencing, in this configuration. The index read cycles were also “repurposed” towards read 1 and read 2, so as not to “waste” cycles by reading indices at the start of each read. Reading indices inline also has the benefit of minimizing cluster crosstalk which has been shown to occur when index sequences are separately read apart from the inserts.Nucleic Acid Samples

[0168] In various aspects, the Methyl-CODEC sequencing methods for sequencing DNA involve obtaining samples of nucleic acid molecules for sequence. Nucleic acid generally is acquired from a sample or a subject. Target molecules for labeling and / or detection according to the methods of the invention include, but are not limited to, genetic and proteomic material, such as DNA, genomic DNA, RNA, expressed RNA and / or chromosome(s). Methods of the invention are applicable to DNA from whole cells or to portions of genetic or proteomic material obtained from one or more cells. Methods of the invention allow for DNA or RNA to be obtained from non-cellular sources, such as viruses. For a subject, the sample may be obtained in any clinically acceptable manner, and the nucleic acid templates are extracted from the sample by methods known in the art. Generally, nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Maniatis, et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp. 280-281, 1982), the contents of which are incorporated by reference herein in their entirety.

[0169] Nucleic acid templates include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). Nucleic acid templates can be synthetic or derived from naturally occurring sources. Nucleic acids may be obtained from any source or sample, whether biological, environmental, physical, or synthetic. In one embodiment, nucleic acid templates are isolated from a sample containing a variety of other components, such as proteins, lipids and nontemplate nucleic acids. Nucleic acid templates can be obtained from any cellular material, obtained from an animal, plant, bacterium, fungus, or any other cellular organism. Samples for use in the present invention include viruses, viral particles or preparations. Nucleic acid484930-8958-8993.1114203-4000 (BI- 11308) may also be acquired from a microorganism, such as a bacteria or fungus, from a sample, such as an environmental sample.

[0170] In the present invention, the target material is any nucleic acid, including DNA, RNA, cDNA, PNA, LNA and others that are contained within a sample. Nucleic acid molecules include deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA). Nucleic acid molecules can be synthetic or derived from naturally occurring sources. In one embodiment, nucleic acid molecules are isolated from a biological sample containing a variety of other components, such as proteins, lipids and non-template nucleic acids. Nucleic acid template molecules can be obtained from any cellular material, obtained from an animal, plant, bacterium, fungus, or any other cellular organism. In certain embodiments, the nucleic acid molecules are obtained from a single cell. Biological samples for use in the present invention include viral particles or preparations. Nucleic acid molecules can be obtained directly from an organism or from a biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue or body fluid specimen may be used as a source for nucleic acid for use in the invention. Nucleic acid molecules can also be isolated from cultured cells, such as a primary cell culture or a cell line. The cells or tissues from which template nucleic acids are obtained can be infected with a virus or other intracellular pathogen. In addition, nucleic acids can be obtained from non- cellular or non-tissue samples, such as viral samples, or environmental samples.

[0171] A sample can also be total RNA extracted from a biological specimen, a cDNA library, viral, or genomic DNA. In certain embodiments, the nucleic acid molecules are bound as to other target molecules such as proteins, enzymes, substrates, antibodies, binding agents, beads, small molecules, peptides, or any other molecule and serve as a surrogate for quantifying and / or detecting the target molecule. Generally, nucleic acid can be extracted from a biological sample by a variety of techniques such as those described by Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001). Nucleic acid molecules may be single-stranded, double-stranded, or double-stranded with single-stranded regions (for example, stem- and loop-structures). Proteins or portions of proteins (amino acid polymers) that can bind to high affinity binding moieties, such as antibodies or aptamers, are target molecules for oligonucleotide labeling, for example, in droplets.494930-8958-8993.1114203-4000 (BI- 11308)

[0172] Nucleic acid templates can be obtained directly from an organism or from a biological sample obtained from an organism, e.g., from blood, urine, cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue. In a particular embodiment, nucleic acid is obtained from fresh frozen plasma (FFP). In a particular embodiment, nucleic acid is obtained from formalin-fixed, paraffin-embedded (FFPE) tissues. Any tissue or body fluid specimen may be used as a source for nucleic acid for use in the invention. Nucleic acid templates can also be isolated from cultured cells, such as a primary cell culture or a cell line. The cells or tissues from which template nucleic acids are obtained can be infected with a virus or other intracellular pathogen. A sample can also be total RNA extracted from a biological specimen, a cDNA library, viral, or genomic DNA.

[0173] A biological sample may be homogenized or fractionated in the presence of a detergent or surfactant. The concentration of the detergent in the buffer may be about 0.05% to about 10.0%. The concentration of the detergent can be up to an amount where the detergent remains soluble in the solution. In a preferred embodiment, the concentration of the detergent is between 0.1% to about 2%. The detergent, particularly a mild one that is nondenaturing, can act to solubilize the sample. Detergents may be ionic or nonionic. Examples of nonionic detergents include triton, such as the Triton X series (Triton X-100 t-Oct-C6H4- (OCH2-CH2)xOH, x=9-10, Triton X-100R, Triton X-114 x=7-8), octyl glucoside, poly oxy ethylene(9)dodecyl ether, di gitonin, IGEPAL CA630 octylphenyl polyethylene glycol, n-octyl-beta-D-glucopyranoside (betaOG), n-dodecyl-beta, Tween 20 polyethylene glycol sorbitan monolaurate, Tween 80 polyethylene glycol sorbitan monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM), NP- 40 nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol n-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether (C14E06), octyl -beta-thioglucopyranoside (octyl thioglucoside, OTG), Emulgen, and polyoxyethylene 10 lauryl ether (C12E10). Examples of ionic detergents (anionic or cationic) include deoxycholate, sodium dodecyl sulfate (SDS), N- lauroylsarcosine, and cetyltrimethylammoniumbromide (CTAB). A zwitterionic reagent may also be used in the purification schemes of the present invention, such as Chaps, zwitterion 3-14, and 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulf-onate.

[0174] Lysis or homogenization solutions may further contain other agents, such as reducing agents. Examples of such reducing agents include dithiothreitol (DTT), beta.- mercaptoethanol, DTE, GSH, cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), or 504930-8958-8993.1114203-4000 (BI- 11308) salts of sulfurous acid. Once obtained, the nucleic acid is denatured by any method known in the art to produce single stranded nucleic acid templates and a pair of first and second oligonucleotides is hybridized to the single stranded nucleic acid template such that the first and second oligonucleotides flank a target region on the template.

[0175] In some embodiments, nucleic acids may be fragmented or broken into smaller nucleic acid fragments. Nucleic acids, including genomic nucleic acids, can be fragmented using any of a variety of methods, such as mechanical fragmenting, chemical fragmenting, and enzymatic fragmenting. Methods of nucleic acid fragmentation are known in the art and include, but are not limited to, DNase digestion, sonication, mechanical shearing, and the like (J. Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, 2. sup. nd Ed., Cold Spring Harbour Laboratory Press: New York, N.Y.; P. Tijssen, "Hybridization with Nucleic Acid Probes- Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II)", 1993, Elsevier; C. P. Ordahl et al., Nucleic Acids Res., 1976, 3: 2985-2999; P. J. Oefner et al., Nucleic Acids Res., 1996, 24: 3879-3889; Y. R. Thorstenson et al., Genome Res., 1998, 8: 848-855). U.S. Patent Publication 2005 / 0112590 provides a general overview of various methods of fragmenting.

[0176] Genomic nucleic acids can be fragmented into uniform fragments or randomly fragmented. In certain aspects, nucleic acids are fragmented to form fragments having a fragment length of about 5 kilobases or 100 kilobases. In a preferred embodiment, the genomic nucleic acid fragments can range from 1 kilobases to 20 kilobases. Preferred fragments can vary in size and have an average fragment length of about 10 kilobases. However, desired fragment length and ranges of fragment lengths can be adjusted depending on the type of nucleic acid targets one seeks to capture. The particular method of fragmenting is selected to achieve the desired fragment length. A few non-limiting examples are provided below.

[0177] Chemical fragmentation of genomic nucleic acids can be achieved using a number of different methods. For example, hydrolysis reactions including base and acid hydrolysis are common techniques used to fragment nucleic acid. Hydrolysis is facilitated by temperature increases, depending upon the desired extent of hydrolysis. Fragmentation can be accomplished by altering temperature and pH as described below. The benefit of pH-based hydrolysis for shearing is that it can result in single-stranded products. Additionally, temperature can be used with certain buffer systems (e.g. Tris) to temporarily shift the pH up 514930-8958-8993.1114203-4000 (BI- 11308) or down from neutral to accomplish the hydrolysis, then back to neutral for long-term storage etc. Both pH and temperature can be modulated to affect differing amounts of shearing (and therefore varying length distributions).

[0178] Chemical cleavage can also be specific. For example, selected nucleic acid molecules can be cleaved via alkylation, particularly phosphorothioate-modified nucleic acid molecules (see, e.g., K. A. Browne, "Metal ion-catalyzed nucleic Acid alkylation and fragmentation," J. Am. Chem. Soc. 124(27): 7950-7962 (2002)). Alkylation at the phosphorothioate modification renders the nucleic acid molecule susceptible to cleavage at the modification site. See I. G. Gut and S. Beck, "A procedure for selective DNA alkylation and detection by mass spectrometry," Nucl. Acids Res. 23(8): 1367-1373 (1995).

[0179] Methods of the invention also contemplate chemically shearing nucleic acids using the technique disclosed in Maxam-Gilbert Sequencing Method (Chemical or Cleavage Method), Proc. Natl. Acad. Sci. USA. 74:560-564. In that protocol, the genomic nucleic acid can be chemically cleaved by exposure to chemicals designed to fragment the nucleic acid at specific bases, such as preferential cleaving at guanine, at adenine, at cytosine and thymine, and at cytosine alone.

[0180] Mechanical shearing of nucleic acids into fragments can occur using any method known in the art. For example, fragmenting nucleic acids can be accomplished by hydroshearing, trituration through a needle, and sonication. See, for example, Quail, et al. (Nov 2010) DNA: Mechanical Breakage. In: eLS. John Wiley & Sons, Chichester.

[0181] The nucleic acid can also be sheared via nebulization, see (Roe, BA, Crabtree. JS and Khan, AS 1996); Sambrook & Russell, Cold Spring Harb Protoc 2006. Nebulizing involves collecting fragmented DNA from a mist created by forcing a nucleic acid solution through a small hole in a nebulizer. The size of the fragments obtained by nebulization is determined chiefly by the speed at which the DNA solution passes through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of the solution, and the temperature. The resulting DNA fragments are distributed over a narrow range of sizes (700-1330 bp). Shearing of nucleic acids can be accomplished by passing obtained nucleic acids through the narrow capillary or orifice (Oefiner et al., Nucleic Acids Res. 1996; Thorstenson et al., Genome Res. 1995). This technique is based on point-sink hydrodynamics that result when a nucleic acid sample is forced through a small hole by a syringe pump.524930-8958-8993.1114203-4000 (BI- 11308)

[0182] In HydroShearing (Genomic Solutions, Ann Arbor, Mich., USA), DNA in solution is passed through a tube with an abrupt contraction. As it approaches the contraction, the fluid accelerates to maintain the volumetric flow rate through the smaller area of the contraction. During this acceleration, drag forces stretch the DNA until it snaps. The DNA fragments until the pieces are too short for the shearing forces to break the chemical bonds. The flow rate of the fluid and the size of the contraction determine the final DNA fragment sizes.

[0183] Sonication is also used to fragment nucleic acids by subjecting the nucleic acid to brief periods of sonication, i.e., ultrasound energy. A method of shearing nucleic acids into fragments by sonification is described in U.S. Patent Publication 2009 / 0233814. In the method, a purified nucleic acid is obtained placed in a suspension having particles disposed within. The suspension of the sample and the particles are then sonicated into nucleic acid fragments.

[0184] Enzymatic fragmenting, also known as enzymatic cleavage, cuts nucleic acids into fragments using enzymes, such as endonucleases, exonucleases, ribozymes, and DNAzymes. Such enzymes are widely known and are available commercially, see Sambrook, J. Molecular Cloning: A Laboratory Manual, 3rd (2001) and Roberts RJ (January 1980). "Restriction and modification enzymes and their recognition sequences," Nucleic Acids Res. 8 (1): r63-r80. Varying enzymatic fragmenting techniques are well-known in the art, and such techniques are frequently used to fragment a nucleic acid for sequencing, for example, Alazard et al, 2002; Bentzley et al, 1998; Bentzley et al, 1996; Faulstich et al, 1997; Glover et al, 1995; Kirpekar et al, 1994; Owens et al, 1998; Pieles et al, 1993; Schuette et al, 1995; Smirnov et al, 1996; Wu & Aboleneen, 2001; Wu et al, 1998a.

[0185] The most common enzymes used to fragment nucleic acids are endonucleases. The endonucleases can be specific for either a double-stranded or a single stranded nucleic acid molecule. The cleavage of the nucleic acid molecule can occur randomly within the nucleic acid molecule or can cleave at specific sequences of the nucleic acid molecule. Specific fragmentation of the nucleic acid molecule can be accomplished using one or more enzymes in sequential reactions or contemporaneously.

[0186] Any of the above aspects and embodiments can be combined with any other aspect or embodiment as disclosed in the Summary, Drawings, and / or in the Detailed Description sections, including the below examples / embodiments.534930-8958-8993.1114203-4000 (BI- 11308)EXAMPLESExample 1 - Methyl-CODEC workflow

[0187] Methyl-CODEC enables simultaneous epigenetic and genetic sequencing with the accuracy of duplex sequencing by linking a methyl-converted strand with the opposite strand of native sequence in reverse complement. This is achieved by using conversion-resistant dCTP analogs in place of standard dCTPs during the strand displacement step of CODEC, followed by EM- seq conversion (FIG. 1A, FIG. 5A). Specifically, after end repair and adaptor ligation with the CODEC adaptor quadruplex — where standard dCTPs are replaced by conversion-resistant dCTPs in functional regions — strand displacing extension occurs in a mix of conversion-resistant dCTP variants, dATP, dGTP, and dTTP. These conversionresistant dCTPs are protected from being converted to Us and subsequently Ts by APOB EC with PCR amplification (FIG. 5B). This process allows the copied side of each original strand to remain intact after methylation conversion (referred to as the protected strand, in contrast to the original / converted strand), and creates two Methyl-CODEC linked products in opposite orientations, with each structure sufficient to form a duplex. This retains complete epigenetic and genetic information from both strands in the library.

[0188] A correct Methyl-CODEC products have one read of a pair from protected strand and the other read from converted strand, originating from opposing strands of each original DNA duplex (i.e., duplex reads) (FIG. 1A) , whereas Methyl CODEC byproducts may lack one strand or include strands from separate duplexes, similar to CODEC. To determine correct product reads, the protected strands were first aligned to the entire reference genome using BWA-mem. Next, the converted strands were aligned strictly to regions where protected strands were aligned, with an additional 500 bp padding, using custom BWA-SW without mismatch penalty for conversion induced C / T or G / A mismatches (FIG. IB & methods). Only alignments with a minimum alignment score of 30 for both strands were considered correct methyl-CODEC products; other fragments were classified as byproducts and kept for further analysis. A, C, G, T and 5mC calls were then made based on the deconvolution table (FIG. IB) from correct products in overlapping regions of the two strands and subject to fragment level and base level filtering, similar to CODEC (see Methods). Methylation calls were stored as Bismark-like tags for compatibility of downstream analysis. Applicant first544930-8958-8993.1114203-4000 (BI- 11308) confirmed that the Methyl-CODEC workflow can generate correct products using the human cell line NA12878. Using 4 different conversion-resistant analogs (5m-dCTP, carboxy-dCTP, hydroxy-dCTP and propargylamino-dCTP, FIG. 5B), 47.6%, 76.6%, 71.2% and 76.8% of the total reads, respectively, were confirmed as correct products, with the majority of byproducts (-95%) being intermolecular byproducts. Overall, hydroxy-dCTP was selected as the dCTP variant in Methyl-CODEC workflow due to its surprisingly superior protection against APOBEC among all cytosine analogs.Example 2: Methyl-CODEC matches EMseq WGMS and WGBS for methylation sequencing

[0189] Methyl-CODEC utilizes the same enzymatic conversion step as EM-seq, while WGBS remains a widely used alternative for methylation analysis. Therefore, methylation results from whole genome Methyl-CODEC were compared with Whole Genome Methylation Sequencing (WGMS) data from EM-seq and WGBS on the same cell line, NA12878, obtained from public repositories. For alignment of EM-seq and WGBS data, Bismark was used. First of all, Applicant found the genome-wide average cytosine methylation levels across three different contexts were similar (FIG. 2A). Additionally, per- CpG island methylation level from the Methyl-CODEC showed high concordance with EM- seq and WGBS, achieving Pearson correlation of 0.99 and 0.97 and strong linear fit with adjusted R20.97 and 0.95, respectively (FIG. 2B).

[0190] Unlike EM-seq and WGBS, Methyl-CODEC uniquely preserves the original DNA sequence for NGS alignment. To assess the impact of this on alignment, Applicant first removed duplicates and the simulated paired-end WGMS from Methyl-CODEC libraries using the converted strands of the correct Methyl-CODEC products and aligned them by Bismark. Applicant compared the concordance between the Methyl-CODEC alignments based on the original 4-letter DNA sequence, which is how Methyl-CODEC is configured, and the Bismark alignment based on the converted sequence (3-letters) as per WGBS or EM- Seq. After applying mapping quality thresholds for both methods, Applicant observed a sharp difference when using different Bismark alignment parameters (FIG. 6A). In particular, Bismark-local alignment mode significantly reduced the number of unaligned reads compared to the Bismark-default mode (11.3% unaligned vs 28.9% unaligned). However,554930-8958-8993.1114203-4000 (BI- 11308) this improvement came with a slight increase in the fraction of mis-placed correct-product reads, rising to 0.38% in local mode compared to 0.06% in default mode (FIG. 6A). These results suggest that Methyl-CODEC may enable more accurate read alignments for methylation sequencing, although its impact was limited to a minor subset of reads with incorrect alignments.

[0191] In addition, Methyl-CODEC can distinguish C>T mutations, including germline SNPs and rare mutations, from C to T conversions via enzymatic or chemical deamination, given its retention of the original DNA sequence. To explore this, Applicant prepared and sequenced three additional Methyl-CODEC libraries using Human HCT 116 DKO samples: fully unmethylated DNA, fully methylated DNA, and a 1 : 19 mix of these two. From the parent HCT116 cell line, around 590,281 germline mutations were detected, including indels, at CpG sites, with about 80% of these germline mutations being C>T / G>A homozygous and heterozygous SNPs (FIG. 6B). Applicant hypothesized that for EM-seq and WGBS, this could potentially result in false calling of hundreds of thousands of unmethylated CpG sites, which could lead to an appreciable underestimation of global methylation. Indeed, using the fully methylated genomes, 97.3% global CpG methylation with Methyl-CODEC was observed, compared to 95.8% (Bismark- default) and 92.4% (Bismark-local) global CpG methylation with simulated paired-end EM-seq WGMS using only the converted sequences (FIG. 6C). Applicant then calculated CpG methylation levels both within and outside the germline C>T / G>A sites. Methyl-CODEC consistently maintained global methylation levels in both cases (with 97.4% within germline C>T / G>A sites and 97.3% outside these sites, closely aligning with the global average of 97.3%). In contrast, simulated EM-seq WGMS aligned by Bismark-default achieved a similar methylation level (97.0%) to methyl-CODEC outside the germline C>T / G>A regions (FIG. 6D), but dropped significantly to 31.6% at germline C>T / G>A sites. (FIG. 6E). Of note, this shortfall of EM-Seq WGMS could be overcome by also performing whole-genome sequencing on the same sample, but this introduces added complexity whereas it is inherently a part of the Methyl-CODEC workflow. For the fully unmethylated genome, which contains low levels of DNA methylation (www fishersci.com / shop / products / hmn-hctl 16-dko-mth-dna-5ug- 20u / 50444311), all methods showed similar CpG methylation levels at 13.4%, 13.4%, and 13.1%, respectively. In all,564930-8958-8993.1114203-4000 (BI- 11308)Methyl-CODEC is superior to EM-seq and WGBS in distinguishing OT mutations from C to T conversions via enzymatic or chemical deamination.Example 3: Methyl-CODEC enables duplex sequencing with similarly high accuracy as CODEC

[0192] Duplex sequencing, as well as CODEC, rely on the principle that sequence information obtained from both DNA strands can distinguish genuine mutations from sequencing noise.

[0193] To determine whether duplex sequencing accuracy is retained for Methyl-CODEC, Applicant analyzed the NA12878 cell line for mutation detection (see Methods). This analysis yielded a raw deduplicated depth of 25x (duplex depth of 5x), with 41% of duplexes covered by just single read pairs of a single Methyl-CODEC products. Results confirmed that Methyl-CODEC products have the required accuracy across all base contexts, including C>T / G>A mutations mapped to the ‘G’ reference bases (FIG. 3A). This is achieved because the G nucleotide on the strand complementary to a C (referred to as the ‘G-strand’ in Bis- SNP21) remains unaffected by enzymatic methyl-conversion. On the other hand, the C>T / G>A mutations mapped to ‘C’ reference are prone to false detection, possibly due to 1) PCR or sequencing errors and / or 2) incomplete protection (FIG. 3B). It was hypothesized that impurities in commercially available dCTP analogs can lead to incomplete protection. Indeed, it was found that hydroxy-dCTP gave the best C>T accuracy (a 2-log reduction in error rate compared to 5m-dCTP) when ‘C’ is the reference (FIG. 7). The mutation frequency of OT was close to the raw Illumina sequencing error rate (IxlO-3), suggesting that incomplete protection was almost eliminated by using hydroxy-dCTP. On the other hand, propargylamino-dCTP yielded worse accuracy, and carboxy-dCTP yielded accuracy between that of 5m-dCTP and hydroxy-dCTP (FIG. 7). In addition, T>C mutations were also prone to false detection by PCR or sequencing errors due to unmethylated C being indistinguishable from a single T>C sequencing error on the protected strand (FIG. 3B), and it was observed that the T>C mutation rate was close to the raw Illumina sequencing error rate (FIG. 3 A). For all single base contexts, except for C>T and T>C, the residual SNV frequencies exhibited minimal or no strand bias, indicating all Methyl-CODEC products can be used for their detection with duplex sequencing accuracy. Meanwhile, for C>T and T>C, duplex sequencing accuracy is still attainable when the complementary mutation (e.g., G>A or A>G)574930-8958-8993.1114203-4000 (BI- 11308) is detected, which will be present on one of two Methyl-CODEC products from each DNA duplex.Example 4: Methyl-CODEC expands genome-wide mutation calling

[0194] The conventional single base substitution (SBS) alphabet, e.g., G>C, considers only mutations among the standard bases A, C, G, and T for both reference and alternative alleles. Methyl-CODEC extends this by distinguishing C or cytosine into non-methylated cytosine (umC) and 5- methylcytosine (5mC) as distinct alternative alleles, denoted as A>umC, G>umC, T>umC and A>5mC, G>5mC, T>5mC respectively (FIG. 3A). In NA12878 Methyl-CODEC data, Applicant found that 5 out of the 6 expanded mutation types have duplex level accuracy (FIG. 3A), with T>umC being an exception, which was dominated by sequencing errors that occurred on the protected strand (FIG. 3C). Observed frequencies for A>5mC and G>5mC and T>5mC were very low (between 10'9and 10'7), which aligns with expectations, as 5mC predominantly occurs at CpG dinucleotides which comprise only 1% of the genome.

[0195] Applicant then investigated the proportion of somatic mutations in NA12878 methyl- CODEC that result in the formation of novel cytosine (7.9%) and CpG dinucleotides (2.3%, FIG. 4A), after removing C>T and T>umC due to their sub-duplex-sequencing accuracy. In analyzing the trinucleotide contexts of the 148 somatic mutations creating 5mC and 656 somatic mutations creating umC, Applicant observed that most of the novel 5mC mutations occurred at newly formed CpG sites, particularly for T>5mC and A>5mC, whereas most of the novel umC mutations formed at non-CpG sites (FIG. 4B), consistent with established data and models indicating that methylation predominantly occurs at CpG sites in humans. Interestingly, for G>5mC mutations, a significant portion (17 / 35) did not occur at newly formed CpG sites, suggesting a large fraction of G>5mC mutations may not be genuine. As methylation is assessed from only a single strand, it is possible there is a greater room for error unless both Methyl CODEC products are recovered, in which case duplex accuracy for methylation analysis could in theory be possible too.

[0196] Methyl-CODEC is a simple yet powerful adaption of CODEC that enables simultaneous methylation and duplex sequencing using single read pairs. It swaps conversion-resistant dCTP analogs for CODEC strand linking and adds an enzymatic methyl conversion step. While prior methods have been developed for combined genetic and584930-8958-8993.1114203-4000 (BI- 11308) epigenetic analysis, Methyl-CODEC is the first to achieve both genetic duplex sequencing accuracy and single-base methylation resolution using single read pairs at the whole genome level.

[0197] To demonstrate this, Applicant prepared and sequenced Methyl-CODEC samples using NA12878 cell line, comparing Methyl-CODEC genetic sequencing accuracy to standard CODEC and its methylation sequencing performance to EM-seq WGMS and WGBS on the same cell line. The results showed that Methyl-CODEC achieves duplex-level accuracy in all single nucleotide contexts using single read pairs. For methylation sequencing, Methyl-CODEC demonstrated strong concordance with widely used techniques like WGBS or EM-seq, while it uniquely preserves the original DNA sequence for improved NGS alignment and C>T mutation detection. Using commercially available methylation control genomes (HCT116), Applicant found Methyl-CODEC uniquely improved methylation detection at CpG sites (around 590,000 sites) overlapping germline C>T / G>A mutations. Methyl-CODEC also expands genome-wide mutation calling by distinguishing C into umC and 5mC as distinct alternative alleles. This enabled us to discover genome-wide, low- frequency co-occurring mutations and methylation at single bases in NA12878. Those novel 5mC were found to be enriched in newly formed CpG contexts compared to non CpG contexts, suggesting that most of these novel 5mC sites are genuine and could reveal new biological processes. Methyl-CODEC could be useful in many areas such as for cancer and minimal residual disease detection using liquid biopsies.

[0198] Methyl-CODEC generates two duplex copies per original duplex, but recovering both in a whole genome library is rare; however, targeted capture libraries may improve this. This could help overcome current limitations in sequencing accuracy for C>T and T>umC detection, as well as other residual, including end-repair errors. Methyl-CODEC, like CODEC, is also conceptually compatible with targeted sequencing workflows. By preserving a native strand sequence, it stands to streamline and mitigate potential biases associated with hybrid capture following bisulfite (or enzymatic methyl-conversion) sequencing.

[0199] Methyl-CODEC has the potential to enable a wide range of clinical applications. In oncology, for example, it can integrate tumor-specific epigenetic and genetic markers to enhance comprehensive cancer detection and management, thereby advancing precision oncology. Duplex sequencing accuracy is pivotal for sensitive detection of minimal residual disease detection from blood and Methyl-CODEC uniquely preserves this accuracy. By 594930-8958-8993.1114203-4000 (BI- 11308) capturing epigenetic and genetic modifications in cfDNA, Methyl-CODEC could improve liquid biopsy -based cancer screening. For example, while mutations on cfDNA can indicate the presence of cancer, epigenetic patterns detected on the same molecules can help identify the tissue of origin. This capability can guide targeted imaging or biopsies to confirm the presence and extent of cancer prior to initiating therapy. Additionally, Methyl-CODEC can aid in the classification and selection of treatment strategies for cancers of unknown primary (CUP), a particularly challenging group to treat, by combining mutation detection with methylation pattern analysis. Beyond oncology, Methyl-CODEC could have applications in prenatal testing and transplantation, where it can integrate epigenetic and genetic data to identify the tissue of origin of DNA molecules derived from the fetus or tissue donor32. Methyl-CODEC, by capturing epigenetic and genetic data from each DNA molecule, could also be applied to other biological areas, such as detecting allele-specific methylation which is crucial for localizing regulatory sequence polymorphisms and assessing biological aging.

[0200] In conclusion, by enabling simultaneous methylation and duplex sequencing using single read pairs, Methyl-CODEC opens promising avenues for future applications in precision medicine, oncology, and beyond.Methods

[0201] Samples and oligonucleotides: Universal methylated and non-methylated standard DNAs of HCT 116 DKO samples were purchased from ZYMO Research (Catalog no. D5014). Genomic DNA of NA12878 cell line was obtained from Coriell Institute of Medical Research (Catalog no. GM12878). DNA fragmentation was performed by a Covaris ultrasonicator to achieve a mean fragment size of 150 bp. The fragmented DNA was purified via AMPure (Beckman Coulter), followed by low TE buffer elution. 20ng of fragmented DNA was used as input for library preparation. To prepare a 95% non-methylated library, fragmented non-methylated standard DNA was mixed with fragmented methylated standard DNA in a 19: 1 ratio. Methyl-CODEC adapter oligonucleotides containing methylated- Cytosine, instead of unmodified Cytosine, were ordered from Integrated DNA Technologies (IDT) with polyacrylamide gel electrophoresis (PAGE) purification. Three different sets of adapter oligonucleotides with distinct indices were purchased (Supplementary Table 1). The Methyl-CODEC adapter quadruplex was formed by adding 5 pM of each oligonucleotide to604930-8958-8993.1114203-4000 (BI- 11308) low TE buffer and 100 mM NaCl in a total volume of 40 pl. The mixture was then heated at 85 °C for 3 min, gradually cooled to 20 °C, and incubated at room temperature for 12 h.Methyl-CODEC library preparation

[0202] End-repair / dA-tailing and ligation. NEBNext Ultra II DNA Library Prep Kit for Illumina (Catalog no. E7645) was used according to the manufacturer’s protocol (New England Biolab). 10-20 ng input DNA was eluted in low TE to a final volume of 50 pl. This was combined with 7 pl of End Prep reaction buffer and 3 pl of End Prep Enzyme Mix, followed by incubation at 20 °C for 30 min and then at 65 °C for another 30 min. Subsequently, DNA purification was performed using 1.8X AMPure XP beads according to the manufacturer’s protocol (Beckman Coulter), followed by elution in 60 pl of low TE buffer. Adaptor Ligation was performed by adding 30 pl of NEBNext Ultra II Ligation Master Mix, 1 pl of Ligation Enhancer, and 3 pl of 5 '-deadenylase (NEB, Catalog no.M033 IS) to the 60 pl end-prep product. Instead of using Illumina adaptors, 2.5 pl of 500 nM freshly diluted Methyl-CODEC quadruplex adaptor was used, and the incubation time was increased to 1 hour at 20°C. Purification with AMPure XP beads was carried out using 0.9X beads, followed by final elution in 40 pl of low TE buffer. Subsequently, 1.5 pl of USER (NEB, catalog no. M5505) was added, followed by incubation at 37 °C for 30 min.

[0203] Phi29 strand displacing extension. For the strand displacement step, Phi29 DNA polymerase (NEB, Catalog no. M0269L) was applied by adding the following to 41.5 pl of ligated DNA from the previous step, followed by incubation at 30 °C for 20 min: 10 pl of 10X buffer, 1 pl of polymerase, 47 pl of nuclease-free water, and 2 pl of 10 mM customized dNTPs, including dATP, dTTP, dGTP, and conversion-resistant dCTP analogues, evenly. Four different conversion-resistant dCTPs were used, including 5-methyl-dCTP (NEB, Catalog no. N0356S), 5-Carboxy-dCTP (TriLink, Catalog no. N-2063), 5-Hydroxymethyl- dCTP (Jena Bioscience, Catalog no. NU-932S), and 5-Propargylamino-dCTP (Jena Bioscience, Catalog no. NU-809-1). The Phi29 extended product was purified using 0.75X AMPure XP beads, followed by final elution in 28 pl of nuclease-free water to prepare for the enzymatic methyl-conversion step.

[0204] Enzymatic methyl-conversion. NEBNext Methyl-seq Conversion Module (NEB, Catalog no. E7125S) was used for DNA conversion based on the manufacturer’s protocol. The protocol consists of two main steps: TET2 Oxidation / BGT glucosylation and APOBEC Deamination. The former is done by adding the following to 28 pl Phi29 extended product: 614930-8958-8993.1114203-4000 (BI- 11308)10 pl TET2 buffer, 1 pl Oxidation Supplement, 1 pl Oxidation enhancer (BGT), 1 DTT, 4 pl TET2 enzyme, and 5 pl diluted (1 : 1250 ratio) Fell sulfate hexahydrate. The oxidation step happens at 37 °C for 1 h followed by adding 1 pl Stop Reagent for 30 min at 37 °C. Next, DNA was purified by 0.75X AMPure XP beads and final elution in 16 pl nuclease free water. The latter step, APOBEC Deamination, is initiated by DNA denaturation by adding 4 pl of formamide to 16 pl DNA and incubating at 85 °C for 10 min. Afterwards, the 20 pl denatured DNA was immediately transferred on ice followed by adding 68 pl nuclease free water, 10 pl APOBEC buffer, 1 pl APOBEC, and 1 pl BSA and incubation for 3 h at 37 °C. Finally, the converted DNA was cleaned up by 0.75X AMPure XP beads, eluted in 16 pl nuclease free water.

[0205] Library amplification. Methyl-CODEC library amplification was performed by adding 25 pl Q5U Hot Start High-Fidelity DNA Polymerase (NEB, Catalog no. M0515) and 5 pl KAPA Library Amplification Primer Mix (Roche, Catalog no. KK2623) to 20 pl Methyl CODEC library molecules. PCR was run by Mastercycler X50 Eppendorf with the following program: Initial denaturation for 30 sec, then 8-10 cycles of 98 °C for 20 sec, 60 °C for 30 seconds, 65 °C for 2 min followed by final extension at 65 °C for 5 min. After PCR, the libraries were twice purified by 0.65X AMPure XP beads, consecutively, with 20 pl final elution. The libraries were quantified and qualified by Qubit dsDNA High Sensitivity kit (Invitrogen, Catalog no. Q33230) and Agilent High Sensitivity DNA Kit (Catalog no. 5067- 4626).

[0206] Sequencing: The 4 Methyl-CODEC NA12878 samples were sequenced using NovaSeq S4 with the index length of 0 and the read length 169, whereas the 3 Methyl-CODEC HCT116 DKO samples were sequenced using NovaSeq X with the index length of 8 and the read length of 161. The 4 Methyl-CODEC NA12878 samples had 930M (hydroxy-C), 166M (5mC), 999M (carboxy-C) and 419M (propargylamino-C) read pairs. And the 3 Methyl- CODEC HCT116 yield 834M (methylated control), 820M (unmethylated control) and 772M read pairs (mixed one).Methyl-CODEC analysis workflow

[0207] Methyl-CODEC analysis workflow is based on CODEC workflow and shares many components. In brief, Methyl-CODEC uses the same demultiplexing and adapter trimming steps as CODEC.624930-8958-8993.1114203-4000 (BI- 11308)

[0208] i) Confirming read structure: After adapter trimming, Methyl-CODEC includes an additional step to determine whether a read pair has the correct structure (i.e., one read is from converted strand, and the other is from the protected strand) and to identify its specific structure (i.e., product 1 vs product 2). This is achieved using a two-step algorithm. In the first step, if one read has a fraction of G less than 5% and the other has a fraction of G greater than 5%, the former is marked as the converted strand / read and latter as the protected strand / read. If both reads have a fraction of G either all above 5% or all below 5%, the second step is applied. In the second step, overlap alignment is performed, aligning the suffix of the first read to the prefix of the reverse complement of the second read using custom alignment parameters (e.g., no mismatch penalty for conversion induced C / T and G / A mismatches and high penalties for gaps). The C / T and G / A mismatches in the overlap region are counted. If the number of C / T mismatches is greater, the read pair is identified as product 1 (the first read is the protected, and the second is converted). Conversely, if the number of G / A mismatches are larger, the read pair is identified as product 2 (the first read converted while the second read is protected). If no overlap alignment is found, the read pair is marked as a byproduct. The first step accelerates the process and accommodates cases where the two reads have little or no overlap.

[0209] ii) Alignment of the protected reads: Methyl-CODEC utilizes standard alignment algorithms without requiring methylation specific aligners. After confirming the read structure (i.e., product 1 vs product 2), Methyl-CODEC uses standard BWA-mem16to align the protected read to the entire reference genome (HG38 in this study). If the alignment is unsuccessful, the read-pair is marked as a byproduct. For successfully aligned reads, the reference sequence of the alignment is extracted, including 500 base pairs as padding.

[0210] iii) Alignment of converted reads. The converted reads are aligned to the local reference sequence obtained from the protected strand alignment using BWA-SW (a version of Smith-Waterman algorithm). Custom alignment parameters are applied here as well, avoiding penalties for conversion-induced mismatches (C / T mismatches for product 1 and G / A mismatches for product 2). Reads with an alignment score below 30 are marked as byproducts.

[0211] iv) Deduplication: For all Methyl-CODEC product reads, Picard MarkDuplicates is used to mark PCR duplicates using UMIs, start and stop positions of the aligned paired-end reads. Among all PCR-duplicated read pairs for a single duplex, only the one with the best sum of quality scores was used for down-stream analysis in order to confirm that a single read pair634930-8958-8993.1114203-4000 (BI- 11308) is sufficient to achieve duplex accuracy. However, generating a consensus from duplicates may further improve accuracy.

[0212] v) Determining methylation status and cytosine contexts. After obtaining deduplicated paired-end alignments, the methylation statuses of cytosines in the overlapped regions are determined. For product 1, the Cs in the protected strands are first scanned. A methylated C or a non-methyl C is identified when the corresponding base on the converted strand is C or T, respectively, providing both bases pass the quality score threshold (Q30). The same process applies to product 2 but focuses on G in the protected strand and G or A in the converted strand. In both situations, the 12 base pairs from the fragment ends are ignored. These methyl-resolved cytosines are then annotated based on their contexts (CpG, CHG or CHH) in a reference-independent manner. The context is called only if the bases next to a C (product 1) or G (product 2) from the two strands agree (accounting for conversion induced C / T, G / A mismatches) and either both meet the quality score threshold (Q30) or match the reference base. Cytosines in the CpG context are annotated with a Bismark-like symbol Z (methylated) or z (unmethylated). CHG and CHH contexts are determined based on the further adjacent bases. A BAM file is generated for each correctly identified read pair, storing methylation status and context information.

[0213] All steps (i-iv) are encapsulated in a single executable file. Alignments are performed in memory using direct calls to the BWA API in C++ code to minimize IO compute time.

[0214] Methylation extraction: After generating BAM alignment with Bismark-like tags, fragment-level and locus-level methylation information is extracted using bismark methylation extractor, which also calculates sample-level methylation fractions for CpG, CHG, and CHH contexts. Methylation fractions for CpG islands were calculated by intersecting the locus-level methylation with CpG island coordinates.

[0215] Processing EM-seq WGMS and WGBS. A 20-ng input EM-seq library (repl) and a 50-ng input WGBS library (repl) were downloaded from the EM-seq paper4. Reads were then aligned using Bismark (default mode), and methylation information was extracted with bismark methylation extractor, ignoring the first 12 bases from read 2 (parameters: —ignore 2 — ignore_3 prime 2 — ignore_r2 12 — ignore_3prime_r2 2) to adjust for M-bias.

[0216] Simulating paired-end EM-seq WGMS from Methyl-CODEC for Comparison. Paired-end EM-seq WGMS data were simulated from Methyl-CODEC products by in-silico converting the protected strand (converting C to T for product 1 and G to A for product 2). The 644930-8958-8993.1114203-4000 (BI- 11308) simulated paired-end WGMS data were aligned using Bismark in two modes (default vs local). The local mode allows soft-clipping at the ends, while default mode enforces end-to-end alignment. NA12878 and three HCT116 samples were simulated. In NA12878, the Methyl- CODEC alignment was compared with Bismark alignments. In three simulated HCT116 data, methylation results were extracted from the original converted strands, excluding the first and last 12 bps in the reads, and then compared with Methyl-CODEC results.

[0217] Extract HCT116 methylation levels in CpG sites with and without germline mutations. Germline mutations in the HCT116 parent cell line were identified using GATK HaplotypeCaller. Germline mutations (SNPs and Indels) overlapping CpG sites in the HG38 genome were further filtered to include only C>T and G>A SNPs. Methylation levels were calculated for CpGs sites overlapping C>T and G>A SNPs and for CpGs that were unaffected by germline mutations, i.e., CpGs at least 3 bps away from any germline mutations.

[0218] Somatic mutation detection. Methyl-CODEC uses the CODECsuite to call somatic mutations from single read pairs, with modifications to account for methylation conversion: i) Mutations are called if a base C from a protected strand is paired with T from the converted strand (product 1) or G from the protected strand is paired with A from the converted strand (product 2). ii) The conversion-induced mismatches (C / T and G / A) are excluded from all filters. 3) Soft-clipping below a certain length is allowed at the 5' end (default is 3bp, adjustable via command-line options). 4) Alignment score filter is turned off for converted strands, though mapping quality filter is still applied. To calculate residual SNV frequencies by mutation contexts in NA12878, mutations detected in product 2 were reverse complemented (e.g., OT became G>A) and combined with product 1 mutations (without reverse complementing). For the denominator in the residual SNV frequencies, Applicant complemented the bases detected in product 2 (e.g., C became G and G became C) and combined with the bases detected in product 1.Further Embodiments

[0219] Features described above as well as those claimed below may be combined in various ways without departing from the scope thereof. This disclosure further provides the following non-limiting combinations:

[0220] Embodiment 1. A method of preparing a DNA sample for methylation sequencing, comprising:654930-8958-8993.1114203-4000 (BI- 11308)(a) providing a sequencing adapter having: a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second singlestrand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule664930-8958-8993.1114203-4000 (BI- 11308) comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5- propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

[0221] Embodiment 2. The method of embodiment 1, further comprising:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0222] Embodiment 3. The method of embodiment 1 or embodiment 2, the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double-stranded DNA molecule.

[0223] Embodiment 4. The method of any one of embodiments 1-3, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

[0224] Embodiment 5. The method of any one of embodiments 1-4, wherein the DNA sample comprises cfDNA.

[0225] Embodiment 6. The method of any one of embodiments 1-5, wherein the DNA sample is from a diseased subject.

[0226] Embodiment 7. The method of any one of embodiments 1-6, wherein the diseased subject suffers from a proliferative disease or a tumor.674930-8958-8993.1114203-4000 (BI- 11308)

[0227] Embodiment 8. A method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter comprising at least ten (10) regions (RO 1 -RIO) in the following configuration:wherein ’ —1represents bonding, wherein R01, R02, and R03 comprise a first oligonucleotide, wherein R04 and R05 comprise a second oligonucleotide, wherein R06 and R07 comprise a third oligonucleotide, wherein R08, R09, and RIO comprise a fourth oligonucleotide, wherein R01 and R06 are annealed to one another, wherein R03 and R08 are annealed to one another, wherein R05 and RIO are annealed to one another, wherein R02 and R07 are not annealed to one another, and wherein R04 and R09 are not annealed to one another; wherein R02 comprises a single- stranded linker, afirst unique molecular identifier (UMI), and a first read primer site, and wherein R09 comprises a single-stranded linker, a second UMI, and a second read primer site, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;684930-8958-8993.1114203-4000 (BI- 11308)(b) ligating the sequencing adapter to a dsDNA duplex as follows: ligating the 5' end of R01 to the 3' end of a first strand of the dsDNA duplex; ligating the 3' end of R05 to the 5' end of the first strand of the dsDNA duplex; ligating the 5' end of RIO to the 3' end of a second strand of the dsDNA duplex; and ligating the 3' end of R06 to the 5' end of the second strand of the dsDNA duplex; thereby forming a partially circularized DNA molecule comprising the target DNA molecule and the sequencing adapter;(c) synthesizing first and second single-strand DNA molecules by extending the free 3' end of R03 and 3' end of R08 on the sequencing adapter each using the opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5- propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

[0228] Embodiment 9. The method of embodiment 8, further comprising:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized694930-8958-8993.1114203-4000 (BI- 11308) double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0229] Embodiment 10. The method of embodiment 8 or embodiment 9, the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double-stranded DNA molecule.

[0230] Embodiment 11. The method of any one of embodiments 8-10, wherein:(1) R01 comprises a first adapter;(2) R03 comprises a first sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase;(3) R04 comprises a free 5' end comprising a first next-generation sequencing (NGS) adapter sequence;(4) R05 comprises a third adapter and a first sample index;(5) R06 comprises a second adapter and a second sample index;(6) R07 comprises a free 5' end comprising a second next-generation sequencing (NGS) adapter sequence;(7) R08 comprises a second sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; and / or(8) R10 comprises a fourth adapter, optionally wherein the first sequence and the second sequence, further comprise the same or different primer binding sites, and optionally wherein the first primer site and the second primer site are oriented to initiate sequencing by addition in opposing directions.

[0231] Embodiment 12. The method of any one of embodiments 9-11, wherein the ligating of step (b) comprises adding ligase.704930-8958-8993.1114203-4000 (BI- 11308)

[0232] Embodiment 13. The method of any one of embodiments 9-12, wherein the synthesizing of step (c) comprises contacting the circular double-stranded DNA intermediate with a polymerase.

[0233] Embodiment 14. The method of any one of embodiments 9-13, wherein the polymerase is a DNA-dependent DNA polymerase.

[0234] Embodiment 15. The method of any one of embodiments 9-14, wherein the polymerase has a strand- displacement activity.

[0235] Embodiment 16. The method of any one of embodiments 9-15, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

[0236] Embodiment 17. The method of any one of embodiments 9-16, wherein the DNA sample comprises cfDNA.

[0237] Embodiment 18. The method of any one of embodiments 9-17, wherein the DNA sample is from a diseased subject.

[0238] Embodiment 19. The method of any one of embodiments 9-18, wherein the diseased subject suffers from a proliferative disease or a tumor.

[0239] Embodiment 20. A method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having: a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter comprises unmethylated cytosine;714930-8958-8993.1114203-4000 (BI- 11308)(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second single-strand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double-stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and standard dATP, dGTP, dCTP, and dTTP deoxynucleotides.

[0240] Embodiment 21. The method of embodiment 20, further comprising:(d) deaminating methylated cytosines to uracil or dihydro-uracil;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double-stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

[0241] Embodiment 22. The method of embodiment 21, methylated cytosines are deaminated by a CpG specific deaminase selected from CseDaOl, MGYPDaO6, LbDaO2, CrDaOl, MGYPDa829, LbsDaOl, or combinations thereof.

[0242] Embodiment 23. The method of embodiment 21, methylated cytosines are deaminated by a TET-assisted pyridine borane treatment.

[0243] Embodiment 24. The method of any one of embodiments 20-23, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.724930-8958-8993.1114203-4000 (BI- 11308)

[0244] Embodiment 25. The method of any one of embodiments 20-24, wherein the DNA sample comprises cell-free DNA (cfDNA).

[0245] Embodiment 26. The method of any one of embodiments 20-25, wherein the DNA sample is from a diseased subject.

[0246] Embodiment 27. The method of any one of embodiments 20-26, wherein the diseased subject suffers from a proliferative disease or a tumor.EQUIVALENTS AND SCOPE

[0247] In the articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Embodiments or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0248] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claims that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or734930-8958-8993.1114203-4000 (BI- 11308) sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0249] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the embodiments. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any embodiment, for any reason, whether or not related to the existence of prior art.

[0250] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended embodiments. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following embodiments.744930-8958-8993.1

Claims

114203-4000 (BI- 11308)CLAIMSWhat is claimed is:

1. A method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having: a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second singlestrand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule,754930-8958-8993.1114203-4000 (BI- 11308) wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5- propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

2. The method of claim 1, further comprising:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized doublestranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

3. The method of claim 1 or claim 2, wherein the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double- stranded DNA molecule.

4. The method of any one of claims 1-3, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.764930-8958-8993.1114203-4000 (BI- 11308)5. The method of any one of claims 1-4, wherein the DNA sample comprises cell-free DNA (cfDNA).

6. The method of any one of claims 1-5, wherein the DNA sample is from a diseased subject.

7. The method of any one of claims 1-6, wherein the diseased subject suffers from a proliferative disease or a tumor.

8. A method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter comprising at least ten (10) regions (RO 1 -RIO) in the following configuration:wherein ’ —1represents bonding, wherein R01, R02, and R03 comprise a first oligonucleotide, wherein R04 and R05 comprise a second oligonucleotide, wherein R06 and R07 comprise a third oligonucleotide, wherein R08, R09, and RIO comprise a fourth oligonucleotide, wherein R01 and R06 are annealed to one another, wherein R03 and R08 are annealed to one another,774930-8958-8993.1114203-4000 (BI- 11308) wherein R05 and RIO are annealed to one another, wherein R02 and R07 are not annealed to one another, and wherein R04 and R09 are not annealed to one another; wherein R02 comprises a single- stranded linker, afirst unique molecular identifier (UMI), and a first read primer site, and wherein R09 comprises a single-stranded linker, a second UMI, and a second read primer site, and wherein the sequencing adapter is modified to contain 5-hydroxy cytosine, 5- carboxy cytosine or 5-propargylaminocytosine in place of unmethylated cytosine;(b) ligating the sequencing adapter to a dsDNA duplex as follows: ligating the 5' end of R01 to the 3' end of afirst strand of the dsDNA duplex; ligating the 3' end of R05 to the 5' end of the first strand of the dsDNA duplex; ligating the 5' end of RIO to the 3' end of a second strand of the dsDNA duplex; and ligating the 3' end of R06 to the 5' end of the second strand of the dsDNA duplex; thereby forming a partially circularized DNA molecule comprising the target DNA molecule and the sequencing adapter;(c) synthesizing first and second single-strand DNA molecules by extending the free 3' end of R03 and 3' end of R08 on the sequencing adapter each using the opposite strand of the partially circularized DNA molecule as a template, thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule784930-8958-8993.1114203-4000 (BI- 11308) comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and 5-hydroxy-dCTP, 5-carboxy-dCTP or 5-propargylamino-dCTP along with standard dATP, dGTP and dTTP deoxynucleotides.

9. The method of claim 8, further comprising:(d) deaminating unmethylated cytosines to uracils;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double-stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

10. The method of claim 8 or claim 9, wherein the method further comprises:(g) detecting a C to T, a T to C, an A to G or a G to A mutation from the sequence of the top strand of the linearized double-stranded DNA molecule or from the sequence of the bottom strand of the linearized double-stranded DNA molecule.

11. The method of any one of claims 8- 10, wherein:(1) R01 comprises a first adapter;(2) R03 comprises a first sequence at or near the 3' end capable of primingDNA synthesis by a DNA-dependent DNA polymerase;(3) R04 comprises a free 5' end comprising a first next-generation sequencing (NGS) adapter sequence;(4) R05 comprises a third adapter and a first sample index;(5) R06 comprises a second adapter and a second sample index;(6) R07 comprises a free 5' end comprising a second next-generation794930-8958-8993.1114203-4000 (BI- 11308) sequencing (NGS) adapter sequence;(7) R08 comprises a second sequence at or near the 3' end capable of priming DNA synthesis by a DNA-dependent DNA polymerase; and / or(8) RIO comprises a fourth adapter, optionally wherein the first sequence and the second sequence, further comprise the same or different primer binding sites, and optionally wherein the first primer site and the second primer site are oriented to initiate sequencing by addition in opposing directions.

12. The method of any one of claims 8-11, wherein the ligating of step (b) comprises adding ligase.

13. The method of any one of claims 9-12, wherein the synthesizing of step (c) comprises contacting the circular double-stranded DNA intermediate with a polymerase.

14. The method of any one of claims 9-13, wherein the polymerase is a DNA- dependent DNA polymerase.

15. The method of any one of claims 9-14, wherein the polymerase has a stranddisplacement activity.

16. The method of any one of claims 9-15, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.

17. The method of any one of claims 9-16, wherein the DNA sample comprises cell-free DNA (cfDNA).

18. The method of any one of claims 9-17, wherein the DNA sample is from a804930-8958-8993.1114203-4000 (BI- 11308) diseased subject.

19. The method of any one of claims 9-18, wherein the diseased subject suffers from a proliferative disease or a tumor.

20. A method of preparing a DNA sample for methylation sequencing, comprising:(a) providing a sequencing adapter having: a first end, a second end and a central portion positioned between the first end and the second end, wherein the first end comprises a first duplex comprising a first oligonucleotide annealed to a second oligonucleotide, wherein the second end comprises a second duplex comprising a third oligonucleotide annealed to a fourth oligonucleotide, and wherein the second oligonucleotide and the fourth oligonucleotide are annealed to one another over a complementary region to form a third duplex that is positioned in the central portion, wherein the sequencing adapter further comprises a pair of read primer binding sites on either side of the third duplex in single stranded regions, and wherein the sequencing adapter comprises unmethylated cytosine;(b) ligating the first end and the second end of the sequencing adapter to an original DNA duplex having a top strand and an opposing bottom strand, thereby forming a partially circularized DNA molecule comprising the original DNA duplex and the sequencing adapter; and(c) synthesizing a first single-strand DNA molecule and a second singlestrand DNA molecule by extending the free 3' ends on the sequencing adapter each using an opposite strand of the partially circularized DNA molecule as a template,814930-8958-8993.1114203-4000 (BI- 11308) thereby forming a linearized double-stranded DNA molecule, wherein the first strand of the linearized double- stranded DNA molecule comprises a concatemer of the top strand of the original DNA duplex and the reverse complement sequence of the bottom strand of the original DNA duplex, and the second strand of the linearized double-stranded DNA molecule comprises a concatemer of the bottom strand of the original DNA duplex and the reverse complement of the top strand of the original DNA duplex, and wherein the synthesizing step comprises contacting the free 3' ends with a DNA polymerase and standard dATP, dGTP, dCTP and dTTP deoxynucleotides.

21. The method of claim 20, further comprising:(d) deaminating methylated cytosines to uracil or dihydro-uracil;(e) determining the sequence of the top strand of the original DNA duplex and the bottom strand of the original DNA duplex by next generation sequencing of the top strand of the linearized double- stranded DNA molecule and / or the bottom strand of the linearized double-stranded DNA molecule; and(f) inferring methylation positions in the top strand and / or the bottom strand of the original DNA duplex from the sequences determined in (e).

22. The method of claim 21, wherein the methylated cytosines are deaminated by a CpG specific deaminase selected from CseDaOl, MGYPDaO6, LbDaO2, CrDaOl, MGYPDa829, LbsDaOl, or combinations thereof.

23. The method of claim 21, wherein the methylated cytosines are deaminated by a TET-assisted pyridine borane treatment.

24. The method of any one of claims 20-23, wherein the DNA sample is obtained from blood, liver, kidney, brain, heart, skin, lung, colon, or pancreas.824930-8958-8993.1114203-4000 (BI- 11308)25. The method of any one of claims 20-24, wherein the DNA sample comprises cell-free DNA (cfDNA).

26. The method of any one of claims 20-25, wherein the DNA sample is from a diseased subject.

27. The method of any one of claims 20-26, wherein the diseased subject suffers from a proliferative disease or a tumor.834930-8958-8993.1

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